Humanitariannet antibody and method thereof, polynucleotide (options), hybridoma cell line (options)

 

(57) Abstract:

The invention is a monoclonal antibody that specifically immunoreactive with protein of HIV-1 gpl20 or gpl60 and characterized by the ability to neutralize in vitro infection of H9 cells by live strains of HIV-1. Antibodies are allocated hybridoma cell lines. The pharmaceutical compositions of this invention will be used in the treatment of people infected with HIV-1, using passive immunization. 10 S. and 6 C.p. f-crystals, 30 Il, 5 PL.

Background of the invention

The present invention relates generally to materials and methods for the prevention and treatment of infections caused by human immunodeficiency virus (HIV-1). More specifically, the invention relates to monoclonal antibodies that are suitable for passive immunization susceptible or HIV-1-infected animals, especially humans.

Infectious process caused by HIV-1 in vivo has recently been the subject of review articles McCune, Cell 64, pp.351-363 (1991). Briefly, HIV-1 infects several cell lines, such as T cells, monocytes/macrophages and neurons that Express the CD4 receptor. Since the vast majority of CD4+in the body are "resting" etki CD4+include transcriptionally inactive virus. Stimulation of the immune system of infected animals, including active immunization can lead to polyclonal activation and signal for resting CD4+go to S phase of the cell cycle. Can replicate cells then actively produce virus particles, causing widespread infection. Taking into account this negative effect of stimulation of the immune system of the infected HIV-1 animal, it is possible that the most effective method of prevention and treatment of HIV-1 infection is passive immunization, i.e. the introduction of anti-HIV-1 antibodies susceptible or infected animal.

Jackson et al., Lancet, 2, pp. 647-652 (1988) reported that a single injection of anti-HIV-1 antibodies in the form of plasma sick people suffering from development of acquired immunodeficiency syndrome (AIDS, a syndrome of progressive degradation of the immune system associated with the HIV-1), temporarily led to a decrease in symptoms, short-term increase in the number of T-lymphocytes, reducing the incidence of infections caused by conditionally pathogenic microorganisms and to reduce the extent to which HIV-1 was able to stand out from the plasma or lymphocytes of the patients. Cm. also the antibodies, specifically reactive with HIV-1 prior to infection of an animal by HIV-1 led to the fact that chimpanzees did not manifest signs of viral infection. These studies show that antibodies capable of neutralizing HIV-1, can be useful for the prevention/treatment of HIV-1 infection.

The main glycoprotein shell HIV-1 gp 120, associated with the cellular CD4 receptor and facilitates the introduction of the virus. With the development of neutralizing antibodies is associated with a number of epitopes of the glycoprotein. But et al., Science, 239, pp. 1021-1023 (1988) reported that amino acids 254 - 274 gp 120 cause the generation of polyclonal antisera capable of group-specific neutralizing three different strains of HIV-1. Conformationally dependent epitopes, make not the primary amino acid sequence, gp 120 is also included in the induction of the synthesis of antibodies that neutralize different strains of the virus, according Haigwood et al., Vaccines 90, pp. 313-320 (1990) and Ho et al., J. Virol, 65(1), pp 489-493 (1991). The so-called "principal neutralizing determinant (PND) gp 120 of HIV-1 localized at V3a loop of gp 120. Cm. Puthey et al., Science, 234, pp 1392-1395 (1986), Pusche et al., Proc. Natl. Acad. Sci. USA, 85, pp. 3198-3202 (1988), Coudsmit et al., Proc. Natl. Acad. Sci. USA, 85, pp. 4478-4482 (1988), Palker et al., Proc. Natl. Acad. Sci USA, 85, pp. 1932-1936 (1988), Holley et al., Froc. Natl. Acad. Sci. USA, I links between cysteine residues flanking domain. Loop V3HIV-1MNfor example, is formed by a disulfide bond between cysteine residues at positions 320 and 336 gp 120.

Recombinant and synthetic protein fragments, including the number of amino acid residues of the loop V3from different HIV isolates was reported to induce the formation of an isolate and typespecification neutralizing antibodies in rodents, according to Lasky et al., Science, 233, pp. 209-212 (1986), Palker et al., above Matsushita et al., J. Virol., 62, pp. 2107-2114 (1988), Javaherian et al., Proc., Natl. Acad. Sci. USA 86, pp 6768-6772 (1989). More recent studies (Puthey et al., above and La Posa et al., Science 249, pp. 932-935 (1990) showed that the structure - turn loop V3is the site recognized by isolate-specific antibodies. Scott et al., Proc. Natl. Acad. Sci. USA, 87, pp. 8597-8601 (1990) report that PND can also induce the formation of typespecific antibodies in humans. Hypervariability PND can explain typespecification neutralizing activity generated by the epitope.

Several studies suggest that antibodies raised against recombinant gp 120, purified gp 120 or synthetic peptides from the region V3can neutralize different isolates of HIV-1. Javarian et al. , Science, 250 pp. 1590-1593 (1990) and Weiss et al., Nat received from rabbits, accordingly immunized with a peptide corresponding to the PND from MN insulators and recombinant gp 120, obtained from isolate IIIB. Cm. also Haynes et al., US Letters Patent 5019387.

Akerblom et al., AIDS, 4, pp. 953-960 (1990) describes the preparations of monoclonal antibodies that neutralize IIIBand eleven primary isolates of HIV-1. Cm. also the Patent Cooperation Treaty (PCT) Publication No. WO 91/11198 of Wahren et al., published on August 8, 1991 Homology strains of primary isolates Akerblom not defined, however, eleven isolates can also be IIIB. Durda et al., AIDS Res. Hum. Retrov., 6, pp. 1115-1123 (1990) reported monoclonal antibodies that block the formation of syncytium cells infected as MN, and IIIBbut do not neutralize the infectivity of MN that are installed using LAV capture immunoassay" ("LAV capture immunoassay"), the analysis is intended to give results that correlate with the activity of the reverse transcriptase. A patent application for the patent cooperation Treaty N WO 90/15078 of Scott et al., published December 13, 1990, describes monoclonal antibodies that inhibit the formation of syncytium cells infected with vaccinia virus expressing PND, isolates MN or MN such". Using standard issledovalis" no action will neutralize multiple strains of live HIV-1. Cm. also PCT Publication Nos. WO 88/09181, WO 90/12868, WO 91/09625 of Tanox Biosystems, Inc., published December 1, 1988; November 1, 1990 and July 11, 1991, respectively, PCT Publication N WO 91/19797 new York University, published on 26 December 1991, and Liou et al., J. Immunol., 143 (12), pp. 3967-3975 (1989).

The above publication shows that monoclonal antibodies that react with HIV PND-1, received to date, show different levels of group reactivity, but may not have broad neutralizing activity. Various examples of type - and group-specific reactivity shown in these studies, may be associated with the amino acid sequence and conformation of the area of the loop of gp 120.

Several studies suggest that the CD4 receptor may not represent the only cell receptor responsible for the invasion of the virus. The results of these studies increase the likelihood that the introduction described here previously, antibodies that block infection of CD4+patient can provide only limited protection against HIV-1 infection. Cheng. Mayer et al., Proc. Natl. Acad. Sci USA, 84, pp. 3526-3539 (1987) reported that infection with HIV-1 glial cells, including another receptor, but not nocity mediated by receptor endocytosis and enhance viral replication. Similar-dependent antibodies increased infection was described by Halsted et al., Nature, 265, pp. 739-741 (1977), Peiris et al., Narure, 289, pp. 189-191 (1981) and Schlesihger et al., J. Immunol., 127, pp. 659-665 (1981),

Previous work has shown that certain animal viruses inactivated by complement, especially Clg, through antibody-dependent mechanism. Cm. Weiss, in Molecular Biology of Tumor Viruses, RNA Tumor Viruses, Wliss et al. , Eds. , Cold Spring Harbor Laboratory, New York, pp. 1219-1220 (1982), Welsh et al., Virology, 74, pp. 432-440 (1976), Bartholomew et al., J. Exp. Med., 147, pp. 844-853 (1978), Cooper et al., J. Exp. Med., 144, pp. 970-984 (1976) and Sherwin et al., Int. J. Cancer, 21, pp. 6-11 (1978). While Banapour et al., Virology. 152, pp. 268-271 (1986) describe the preparations of normal serum, which have no effect on the density of HIV-1 or its ability to infect mononuclear cells of peripheral blood, Spear et al., J. Virol. , 64 (12), pp. 5869-5873 (1990) report that HIV-1 treated with a combination of complement and whole serum from patients seropositive for HIV-1, shows reduced infectivity.

Thus remains a need for new drugs, monoclonal antibodies (including, for example, murine antibodies, "humanized" antibodies and immunologically active fragments of antibodies that are specifically immunoreactive with HIV-1. Ideally, such antibodies should characterize SSA using standard research on reverse transcriptase, p24, MT-2 and the formation of a syncytium, including appropriate cultured cells of the host (for example, cells H9). In view of the intended use for passive immunization of infected and uninfected patients, such monoclonal antibodies should optimally be able to participate in (i.e., the mediating) the complement-according to viralize particles of HIV-1 and antibody-dependent cytolysis of cells infected with HIV-1.

Summary of invention

The present invention is a monoclonal antibody that specifically reagents with that part of the protein gp 120 or gp 160 of HIV-1, which includes the amino acid sequence of picindian-glycine-arginine (G-P-G-R), shown in the LAST ID No. 1, and characterized by their ability to neutralize infection of H9 cells in culture strains MN and IIIBliving HIV-1, as determined through research on reverse transcriptase, p24, MT-2 and the formation of the syncytium. The products of this invention may also be characterized by its ability to mediate complement-dependent viralis particles of HIV-1 and/or antibody-dependent cellular cytotoxicity of HIV-1-infected cells.

Monoclonal antibodies of the present invention MoE is, the blood). Monoclonal antibodies of the present invention, preferably IgG antibodies, are also particularly suitable for use in anti-HIV-1 treatment of animals, especially humans, susceptible to or infected with HIV-1. Immunologically effective amount of a monoclonal antibody are introduced to a patient infected by HIV-1, or at risk of infection with this virus to create a passive immunity to HIV-1 infection and is preferably carried out in the presence of complement-dependent viralis particles of HIV-1 and/or antibody-dependent cytotoxicity for HIV-1-infected cells in a patient.

Chimeric or "humanized" antibodies (including CDR-transplanted antibody), antibody fragments, and especially bespecifically antibodies on the basis of the claimed monoclonal antibodies included in the consideration of the present invention as recombinant relevant to antibody products produced in prokaryotic and eukaryotic cells. For example, fragments of antibodies, such as Fab and F(ab')2fragments can produce in the culture of host cells such as E. coli, yeast, insect cells and mammalian cells on the basis of definition information about the structure (sequence) glacie makes possible the collection of CDR-transplanted antibody. In addition, chimeric antibodies (e.g., mouse/human antibodies) can be obtained by using the transformed cells of the mouse myeloma or hybrid cells, and bespecifically antibodies can be produced hybrid hybridoma cells. Special consideration shall be antibodies which consist essentially of the variable regions of human antibodies comprising the amino acid sequence of at least one complementarity determining region of an antibody, characterized by the ability to specifically bind with the amino acid sequence of gp 120 or gp 160 of HIV-1 consisting essentially of the sequence shown in the sequence of TH ID No. 1, and the ability to neutralize in vitro infection of H9 cells by live strains MN and IIIBHIV-1 when determining reverse transcriptase, p24, MT-2 and the formation of the syncytium. Subject to review DNA sequences encoding such antibodies, the cells of the host, producing such antibodies, and recombinant methods for producing such antibodies.

Also in consideration of the present invention includes the application, when the anti-HIV-1 treatment combination products of the present invention and drugability include complement, antibodies that are associated with different neutralizing and neitralizatsii areas proteins of HIV-1 and chemical agents, such as AZT.

As described in the following detailed description, the monoclonal antibodies of the present invention was obtained by immunization of a suitable host live HIV-1, thus presenting gp120 in its natural conformation.

Specifically illustrate the present invention murine monoclonal antibodies (designated NM-01), produced by the hybridoma line cells HB 10726, which was obtained deposited with American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland 20852, 9 April 1991 and marked ATSS, receipt N HB 10726, and humanized variants of the antibody NM-01, denoted by NM-01 HuVH/HuVK, MN-01 HuVH/HuVKF, NM-01 HuVH/HuVK, NM-01 HuVHS/HuVK, NM-0,1 HuVHS/HuVKF and N-01 HuVHM/HuVKF produced by the cell lines, which are accepted for storage by the European Collection of Animal Cell Cultures (ESAS) August 20, 1993, PHLS Centre for Applied Microbiology &Research, Porten Down, Salisbury, Creat Britain SP4 PJG and were marked ESAS receipt NN 93082022, 93082019, 93082020, 93082023, 93082018 and 93082021 respectively.

Numerous aspects and advantages of the present invention will be apparent upon consideration of the illustrative examples and descriptions of practical implementation of this autoradiogram uninfected H9 cells, proteins of HIV-1MNand subjected to Western blot turns with monoclonal antibodies of this invention and immune sera from seropositive AIDS patients.

Fig. 2 graphically represents the results of determination of the immunoreactivity of the antibodies of this invention with peptides corresponding to the region of the loop V3from different strains of HIV-1.

Fig. 3 is bar graph showing the effect of the peptides corresponding to the region of the loop V3on the binding of the antibody of this invention and two other antibodies anti-HIV gp120.

Fig. 4A-4C, 5, 6A through 6B, 7A through 7B, 8, 9A, 9B graphically present the results of screening studies on reverse transcriptase, p24, MT-2 and the formation of a syncytium, respectively, of a monoclonal antibody of this invention for their ability to neutralize infection of H9 cells by live strains of HIV-1.

Fig. 10 graphically represents the results of studies to determine the peptide blockade of neutralization of infectivity by antibodies of this invention.

Fig. 11A, 11B, 12A-12F and 13A-13F are electron micrographs of particles of HIV-1 that were treated with a combination of monoclonal antibodies of this invention and complement.

th, respectively, monoclonal antibodies of this invention, NM-01, with the amino acid sequences of light and heavy chains of three different anti-HIV-1 monoclonal antibodies.

Fig. 16 and 17 represent the alignment of amino acid sequences of the variable regions of light and heavy chains, respectively, mouse monoclonal antibody NM-01) with amino acid sequences of light and heavy chains of humanized antibody NM-01 of this invention, indicated HuVH/HuVKF.

Fig. 18, 19, 20, 21 and 22 graphically present the results of validation studies on reverse transcriptase, p24, MT-2 and the formation of a syncytium, respectively, of the biological activity of chimeric and humanized antibodies of this invention.

Examples

The following examples illustrate the practical implementation of this invention, when receiving a cell line of hybridoma HB 10726, separation from her monoclonal antibodies immunoreactive with the protein gp120 (or its predecessors gp160 HIV-1 as well as with peptides comprising the amino acid sequence G-P-G-R presented in TH ID No. 1, determination of the characteristics of such monoclonal antibodies.

More specifically, example 1 is intended for demat relation to the mapping of viral atipta, recognized by the antibody NM-01. Example 3 describes the characteristics of reactivity of monoclonal antibodies with different isolates of HIV-1. Example 4 involves checking antibody NM-01 on the ability to neutralize infection of H9 cells of different living strains of HIV-1, as demonstrated by the analysis of the inverse transcriptase and p24. Example 5 is devoted to the further testing of antibodies for their ability to neutralize the infectivity of living isolates of HIV-1, which illustrates a determination of MT-2 and formation of the syncytium. Example 6 relates to a peptide blocking properties neutralize the infectivity of HIV-1 monoclonal antibody NM-01. Example 7 describes the analysis of the ability of monoclonal antibody NM-01 to mediate complement-dependent lysis of HIV-1. Example 8 relates to the determination of the validity of the combination of monoclonal antibody NM-01 and complement to infection with HIV-1 susceptible cells in culture. Example 9 describes the DNA and deduced amino acid sequences of variable regions of heavy and light chains of monoclonal antibody NM-01. Example 10 relates to the production of chimeric or humanized variants of monoclonal antibody NM-01 and the determination of the immunological and biological activity.

Example 1.

A. Purification of live HIV-1MN< / BR>
300 ml culture of H9 cells infected with HIV-1MN, centrifuged at 1500 rpm for 5 minutes to precipitate the cells. Virus-containing supernatant was removed and retained, while the sediment re-centrifuged at 2100 rpm for 20 minutes. The second supernatant was collected and combined with the first and the supernatant was subjected to ultracentrifugation in a SW 27 rotor at 25000 rpm for 90 minutes at 4oC to precipitate virus particles. The resulting supernatant was discarded. Sediment viruses resuspendable in about 10 ml of buffer TOE (100 mm NaCl, 10 mm Tris-HCl, pH of 7.7, 1 mm EDTA). Prepared ultracentrifuge tube containing the bottom layer of 10 ml of the TRE with 50% sucrose, the middle layer of 10 ml of the TRE with 25% sucrose and the top layer of 10 ml sample of the virus, and centrifuged in an ultracentrifuge at 25000 rpm at 4oC for 90 minutes. The virus was deposited in the form of a white stripe between the layers of the TRE with sucrose and was going Pasteur pipette. The viruses were added twenty ml of the TRE/15 mm EDTA (100 mm NaCl, 10 mm Tris-HCl, pH 7,7, 15 mm EDTA) and a sample of the virus is purified by live HIV-1NM.

C. Immunization and getting hybridoma

To immunize each of the three two-month mice Balb/ C by intraperitoneal injection used 100 µg live virus HIV-INM. Secondary injection of 30 μg of virus to mice did after 3 weeks and after 3 weeks again, 100 μg of viral drug. Mice were scored 3 days after the second reimmunization and received cell line of hybridoma by fusion of splenocytes with cells RZ-H-Ag8-U1 (ATSS CRL 1597). Line hybridoma cells were obtained from spleens of mice immunized with chronically infected H9 cells (10 mice), acutely infected H9 cells (9 mice) and membranes of infected H9 cells (3 mice). Chronically infected H9 cells are cells after 2-3 weeks after infection, with values in the study of the reverse transcriptase () from 100 000 to 150 000 pulses/min, while acutely infected H9 cells are cells after 10-12 days after infection, with values FROM 200 000 to 250 000 pulses/min

Line hybridoma cells were obtained in the following way.

A mixture of spleen cells from immunized mice was centrifuged at 800 g for 5 minutes. The supernatant was aspirated from the sediment cells and relax the road to mix the tip of the pipette for 15 seconds and repeat). The mixture was stirred additionally for a minute by the end of the same pipette, without breaking the lumps of cells. Within 1 minute in the same way (0.25 ml every 15 seconds) then was added one ml of incomplete medium" (RPMI 1640 (JRH Biosciences), supplemented with 25 mm HEPES (Sigma Co.), 10 000 U/ml penicillin and 10,000 mg/ml streptomycin) and 1 ml was added for a further 1 minute. Then 7 ml of incomplete medium was mixed for 2-3 minutes (1 ml every 20 seconds) that resulted in the suspension with small clumps of cells. The final suspension was centrifuged at 500 g in a clinical centrifuge for 5 minutes and supernatant was removed. Sediment resuspendable rotation of the tube (not usuaria or papeterie the solution up and down) in "complete medium" ("incomplete environment, which is described above supplemented with 15% fetal calf serum (FBS) to a concentration of 2106cells per ml of medium. Then 0.1 ml of this suspension (2105cells only) per cell was filled 96-yacheechnye Board. Board incubated at 37oC in an atmosphere with 7% CO2. The day the merger was considered day 0.

C. Selection in the NAT and initial screening of hybridomas

Twenty-four hours after confluence (day 1) in each well was added 0.1 ml of a NAT environment (10-4M gipoksantina, 510the Yali 0.1 ml of fresh medium NAT. On days 2 through 5 cells apparently contain only dead cells. Hybridoma begin to appear between day 5 and 10. Hybridoma were easily visible in the form colonies strongly refracting cells surrounded by cell remnants.

D. Screening hybridomas

For the selection of hybridoma supernatant was used in several studies. Hybridoma, secreting antibodies reactive with HIV-1, originally identified by selecting membranes obtained from uninfected and infected MN H9 cells using EL ISA with supernatant cultures hybrid. This initial selection was followed by selection on immunofluorescence and radioimmunoassay studies in addition to the EL ISA data on the binding of the antibody with live infected cells.

The cell membrane for EL ISA were obtained from infected or uninfected H9 cells. Cells suspended in 250 mm sucrose/Tris-HCl buffer at a pH of 7.4, containing 1 mm EDTA. The suspension is homogenized in a Dounce homogenizer placed in an ice bath, until the discovery of living cells with the exception on Trypanosoma blue. The mixture was centrifuged for 2 minutes at 50 g. The resulting precipitate is re-homogenized who was multiserial in the same buffer and centrifuged for 20 minutes and sediment resuspendable in 7 ml original buffer 250 mm sucrose-EDTA. This solution was then layered on top of the buffer 2 M sucrose/10 mm Tris-HCl containing 1 mm EDTA and centrifuged for 1 hour at 80 000 g. The result is a fluffy white intermediate surface, which was collected and resuspendable 250 mm sucrose buffer. The protein content was determined by BCA method (Pierce Chemical Company). The suspension was divided into equal samples and stored at -70oC.

For EL ISA in 96-akaichou charge was added to the cell membrane at a concentration of 400 ng/cell, and dried overnight at 25oC. the Board was washed with 0.5% Triton Xphosphate-buffered saline (PBS), blocked with 5% fetal bovine serum (FBS)/PBS and again washed. The hybrid supernatant (40 μl) was diluted in 50 μl PBS was added to the cells, leaving overnight at 4oC. After washing cells were added to rabbit antimachine IgG (H+L) conjugated with horseradish peroxidase (HRP) (Zymed) for 2 hours at 25oC. Cells were washed with 0.5% Triton X/PBS and then incubated in the presence of ABTS (Bio-Rad substrate kit) for 20 minutes before monitoring the optical density at 405 and 650 nm.

Supernanny hybrid derived from CL and, was taken on a positive reaction on the membrane of uninfected cells and membranes of infected cells at EL ISA, indicating that antibodies produced by hybridomas, are not HIV-1-specific. From 1039 hybrid derived from spleen cells of mice immunized with the membranes of infected cells, 5 of them supernatants strongly reacted with the membrane of infected cells and very weakly reacted with membranes of uninfected cells. Was carried out Western blotting with supernatants from these hybrid cell lines, and found that three of these produced monoclonal antibodies was associated with p55 by HIV-1, one was associated with p55 and p24 of HIV-1, and the last did not give the bands at Western-blotting (data not shown). Results EL ISA presented in table 1 in terms of ratios of the values obtained for the membranes of infected cells in comparison with the membranes of uninfected cells.

One thousand one hundred eighty-seven hybrids were obtained from spleen cells of mice immunized with live HIV-1MN. Four lines hybridoma cells were selected for further screening based on the results of EL ISA, showing that antibodies in four supernatant greatly exceed pernatty four hybridomas were cloned by serial dilution method and produced the selection to radioimmunoassay (RIA). Rabbit anti - mouse IgG labeled with125I (R M IgG125I) was purified on a column of Sephadex G-50 (NEN-DuPont). Uninfected H9 cells or H9 cells (7,5105cells in 150 ál) infected with HIV-1MN, was placed in 15 ml tubes. Fifty μl of supernatant from each hybridoma was added to each tube containing uninfected and infected cells, and the mixture incubated overnight at 4oC. Cells were washed 2 times in 2 ml of PBS/50% tween-20with shaking between washings. Fifty ál R M IgG125I (750 000 pulses/min) in PBS/5% FBS was added and the mixture is again incubated overnight at 4oC. After incubation, the cells 3 times washed with PBS/50% tween-20oC. one Hundred μl of PBS/5% Triton-Xadded to disinfect the cells and 100 μl of 1 M NaOH was added to facilitate the transfer of the labels to the containers for scintillation. The samples were calculated, and the results of the RIA are presented in table 1, as the ratio of the values of the pulse/min, obtained for infected cells in comparison with values for uninfected cells.

Then four lines hybridoma cells were selected by immunofluorescence (IFA). Two ml or uninfected or HIV-1-infected H9 cells (approximately 1106cells/ml) or 10 ml PBS by filling the tubes, stirring, centrifugation at 100 rpm for 5 minutes and sucking all of the supernatant, in addition to about 100 μl, leaving the "milk" of the cell suspension. When working under the hood with laminar flow 51 mm glass slides with 10 cells (Cel I Line Association) was coated with the suspension of the cells by filling each cell to the brim and then retracting the suspension back into the pipette tip. Covered by the suspension subject slides were allowed to air dry and then were fixed in methanol at room temperature for 10 minutes. The supernatant from each of the four hybridomas were tested undiluted and at a titer of 1:50 (supernatant diluted in 0.02% skimmed milk) for reactivity with drugs uninfected and infected cells on a slide. In each cell slides were added fifteen μl of undiluted or diluted supernatants. Slides were incubated at 37oC for 30 minutes and immersed in PBS with shaking for 5 minutes. The slide is then quickly washed with distilled water and dried in air under the hood with laminar flow. Sixteen μl of fragment F(ab)2(H+L)cosito-antimelanoma IgG (Cappel Biomedical), diluted 1: 80 0,02% was taken molecolari in PBS. Slides were washed in 0.01% solution of Evans blue in PBS for 5 seconds and 2 times were rinsed with distilled water. Slides were viewed on immunofluorescence, and the results of the analysis are presented in table 2, and mouse IgG (MIgG), antibody 5C6 (anti-IIIB) and the supernatant frac. 5 (hybridoma obtained from spleens of mice immunized with the membranes of infected cells) serve as control antibodies.

Hybridoma cell line HB 10726 was selected as the most promising in relation to the antibodies on the basis of the RIA and immunofluorescence. This cell line did not possess the highest degree of binding in EL ISA, but as the results of the RIA and immunofluorescence are linking with live infected cells, whereas EL ISA represents the binding with the dried membranes of cells, the data of the RIA are more important. This cell line was twice subclinically and produced her monoclonal antibodies marked NM-01. Mice intraperitoneally standard method was introduced this cell line and monoclonal antibodies from ascitic fluid was concentrated by using purification on affinity column of protein A. (Pierce). Using specific antisera (Bi O-Rad) was what was used in the subsequent examples.

Example 2.

To characterize viral epitope recognized by the monoclonal antibody NM-01, were first examined using Western blot analysis of the reactivity of the antibody with purified proteins of the virion MN and IIIBand then using EL ISA reactivity with overlapping peptides corresponding to the amino acid sequence region loops V3gp120 of HIV-1.

A. Western blot analysis

The virion MN and IIIBpeeled from supernatant culture infected H9 cells were destroyed in 1.3% sodium dodecyl sulfate (LTOs)/3% - mercaptoethanol and then subjected to electrophoresis in 0.1% LTOs-10% polyacrylamide gel. After transferring the proteins to nitrocellulose paper strips were incubated overnight with a monoclonal antibody NM-01 in blocking buffer (0.02 M Tris-HCl, pH of 7.4, 0.1 M NaCl, 0,05% normal goat serum and 5% nonfat dry milk) at 4oC and then washed in 0.02 M Tris-HCl, pH of 7.4, 0.1 M NaCl and 0.3% tween. The strips are then incubated with biotinylated goat anti-mouse IgG (Zymed) for 1 hour, washed and allowed to react with125I-streptavidin (Amersham, Arlington Heights, IL) for an additional hour at 4oC, the reactivity of monoclonal ant is, the rich strip of gel 1 and 3 contained the membrane of uninfected H9 cells; lane 4 contained the membrane with HIV-1MNthe H9 cells; lanes 2 and 5 contain the virus HIV-1MN; and lanes 6 and 7 contain a virus Antibody NM-01 reacted with proteins in the bands 1, 2 and 6, whereas the serum of the patient is seropositive for HIV-1 reacted with proteins in lanes 3-5 and 7.

Monoclonal antibody NM-01 showed reactivity with viral proteins MN and IIIBhaving an approximate molecular weight of 120 kD, but did not react with any other viral antigens, indicating that the antibodies recognize an epitope of gp120.

For comparison, a monoclonal antibody F58/H3 and P4/D10, described by Wahren et al. PCT Publication N 91/11198 were obtained them ECACC (inflow NN 90011607 and 90011608 respectively) and were tested for binding to recombinant gp120 of HIV-1MN(Agmed Inc., Bedford, MA), recombinant gp120 (DuPont-NEN, Boston, MA), natural gp120 of HIV-1MNand natural gp120 Western-blotting along with monoclonal antibody NM-01. Western blot was performed essentially as described above, except that rabbit anti-mouse secondary antibodies were used in colorimetric study for determination of antibody binding. Monoclonal an. monoclonalny antibody F58/H3 and P4/D10, however, reacted only with natural and recombinant gp120 gp120 derived from

B. epitope Mapping using EL ISA

To identify specific gp120 epitope recognized by antibody NM-01, antibodies was checked by EL ISA on reactivity with overlapping peptides corresponding to the region of the loop V3gp120. Peptides synthesized using a Multiple Peptide Systems, San Diego, CA, corresponded to amino acids 302-316, 312-326 and 322-336 gp120 of HIV-1MN.

Three peptide (250 ng/50 μl of 0.1 M borate buffer, pH 8.0 per cell) were incubated overnight at 37oC motherboard Immulon 2 (Dynatech). Fees were washed in PBS and blocked PBS/0.1% tweenwith 0.1% bovine serum albumin (BSA) for 1 hour at room temperature. The blocking agent was removed, and different amounts of antibody NM-01 or mouse IgG (MIgG), diluted to 100 μl of medium HAT, was added to the Board. The antibodies were allowed to react for 2 hours at room temperature. Then the Board 10 times washed with tap water. Conjugated with horseradish peroxidase rabbit anti-mouse secondary antibody diluted to 1:1000, brought in PBS/0.05% tween/0.5% BSA and added to 100 μl of the cell. Board the ABTS (Bio-Rad) was added for 20 minutes, and the pay was analyzed at 650 nm. TH ID N 2-4 show the amino acid sequences of peptides, and table 3 presents the results of studies using overlapping peptides, and antibodies MIgG and environment HAT were negative controls.

While not found the reactivity on the basis of monoclonal antibodies NV-01 with peptides corresponding to amino acids 302-316 or 322-336 loop V3observed the binding of the antibody with the peptide representing amino acids 312-326. The control antibody, mouse IgG was not associated with peptides.

Example 3.

The demonstration that monoclonal antibody NM-01 contact area of the loop V3gp120 of HIV-1MNprompted further studies to determine the degree of reactivity with other isolates of HIV-1. The antibodies were tested using EL ISA on reactivity with peptides corresponding to the region of the loop V3HIV-1 isolates IIIB, RF, CDC4, NY/5, Z6, Z2 and EL I. Amino acid sequences of the peptides are shown below in table 4 and in the sequence listing, as LAST ID N 5-12, respectively.

Peptides (250 ng/0.1 M borate buffer, pH 8.0, synthesized American Biotechnologies, Cambridge, MA) were incubated overnight at 4oC and then incubated with monoclonal antibody NM-01 for 1 hour at 37oC. After washing with tap water Board incubated with conjugated with horseradish peroxidase rabbit against mouse secondary antibodies for 1 hour at 25oC and then with ABTS substrate (Bio-Rad) for 20 minutes. Reactivity was determined by monitoring the optical density at 650-405 nm. The results of the study are presented in Fig. 2.

Monoclonal antibody NM-01 reacted with peptides loops of MP (shaded circle), IIIB(empty circle), RF (empty triangle) and CDC4 (filled triangle) isolates. Antibody binding to peptides IIIB, RF and CDC4 was comparable to the binding obtained with peptide MN. Antibodies also showed a lower affinity for peptide NY/5 (stars). Monoclonal antibody NM-01 also falsely reactive with RF-like peptide shown in TH ID N 13. Conversely, the reactivity was weak, if present, loop peptides from Z6 (filled square), Z2 (inverted empty triangle) and EL 1 (empty square) of the isolates. These results indicate that monoclonal antibody NM-01 recognize, in particular, the epitope loop V3gp120 (HIV-1 isolates with aminocyclo also used for reactivity with peptides loop V3MN, IIIB, RF-like, CDC4, NY/5, Z2, Z6 and EL 1. In contrast to the monoclonal antibody NM-01 as monoclonal antibody F58/H3 and P4/D10 reacted only with peptide IIIBand to a lesser extent with RF-like peptide, shown in the LAST ID No. 14.

Other monoclonal antibodies against gp120 of HIV-1, monoclonal antibody BAT123 described by Liou et al. above, as the reactive loop peptides, V3MN-like and IIIB-like strains, and non-reactive with the peptide RF-like (TH ID N 13) (see Fig. 5A on page 3972 work Liou et. al., above). This reported reactivity different from the reactivity of monoclonal antibody NM-01, as described in the preceding paragraph. While antibody NM-01 and BAT123 communicate relatively well with IIIBpeptide, it will take about pyatidesyatiletnie increasing the concentration BAT 123 to obtain the binding of peptide MN, which is similar to the binding of NM-01. In addition, the NM-01 reacts with peptide RF, shown in table 4 and PEFC ID No. 7, and RF-like peptide, shown in the LAST ID No. 14, while BAT123 is not associated with RF-like peptide TH ID N 13 even at concentrations of antibodies 10000 µg/ml.

In competitive analysis, the binding of monoclonal antibody NM-01, F58/H3 and P4/D10 definition is IRIQRGPG (peptide 1), RIQRGPGR (peptide N 2), IQRGPGRA (peptide N 3), ORGPGRAF (peptide N 4), RGPGRAFV (peptide N 5) and GPGRAFVT (peptide N-6). The research was carried out as follows. 100 μl of recombinant gp120 IIIB(0,5 µg/ml in PBS) was coated charge Measurement 4 (Dynatech), and incubated at room temperature overnight. Then the card was blocked with 250 μl of blocking buffer (5% normal rabbit serum in PBS) for 1 hour at 37oC. Monoclonal antibody NM-01, F58/H3 and P4/D10 was diluted to a concentration of 10 μg/ml blocking buffer, and each peptide of the six peptides loop IIIBwas diluted to a concentration of 100 μg/ml of blocking buffer. Then each of the antibodies was separately mixed with each peptide 1:1 by volume with a final concentration of antibody 5 μg/ml and the concentration of peptide 50 μg/ml of a Mixture of antibodies and peptides were incubated at room temperature for 40 minutes and then transferred to the cells (100 μl/cell) in a locked, covered with gp120 fee for research. Control cells contained 5 μg/ml antibody and did not contain peptide. Fees were incubated for 40 minutes at 37oC and then washed four times with detergent buffer (0.005% tween-20 in PBS). Linked to horseradish peroxidase (HRP) rabbit anti-mouse antibodies (100 μl/cell) using the 37oC. Then charge again washed and showed, using 100 μl/cell TMB (tetramethylbenzidine). The demonstration was stopped by 100 μl/cell H2SO4(0,36 N) and the charge was read at 450 nm - 650 nm.

The results of the research competition is shown in Fig. 3. In this study, peptide 4 was the strongest inhibitor of the binding of monoclonal antibody NM-01) with recombinant gp120 IIIBwhile peptides 3 and 4 were the strongest inhibitors of the binding of the monoclonal antibody F58/H3 and peptide 2 was the strongest inhibitor of binding of the monoclonal antibody P4/D10.

Example 4.

Monoclonal antibody NM-01 was tested for the ability to neutralize infection of H9 cells by live strains MN, IIIBand RF, which was estimated by determining the reverse transcriptase and strains MN and IIIBHIV-1 was assessed by studies on p24.

Analysis of reverse transcriptase and p24

Dilution of monoclonal antibody NM-01 incubated with 40 TCID50MN or 100 TCID50IIIBlive virus in 96-cell boards for 1.5 hours at 37oC monoclonal antibodies 0,5 (AIDS Reserch and Reference Reagent Program Catalog National Institute of Allergy and Indectious Diseases) was used as a put is. the ATEM in each cell was added H9 cells (2.5 to 104and Board were incubated for one hour at 37oC. Then the suspension of H9 cells were diluted in RPMI 1640/15% FBS, and incubated in 24-cell Board at 37oC. Production of virus was determined by analysis of the reverse transcriptase (FROM) performed on day 7, as described by Poiesz et al. , Proc. Natl. Acad. Sci. USA. 77, pp. 7415-7419 (1980) and p24 analysis performed on day 5 (Dupont HIV-1 p24 Core Profile EL ISA). The results of these two studies is shown in Fig. 4A through 4B, and 5, respectively.

Monoclonal antibody NM-01 (filled circles in Fig. 4A) completely neutralized the infectivity of live virus MN, which are identified by the study FROM, at concentrations of 10-100 μg/ml in Addition, the use of antibodies at concentrations of < 1 μg/ml resulted in inhibition of infectivity of the virus (ID50up to 50%. These results contrasted with the absence of detectable neutralizing monoclonal antibodies to 0.5 (empty circles, Fig. 4A). Monoclonal antibody NM-01 also neutralized live virus IIIBif ID50approximately 0.1 μg/ml (Fig. 4B). Monoclonal antibodies neutralized with 0.5 IIIBa bit more effectively than monoclonal antibody NM-01 (Fig. 4B). Similar results were obtained for MN intitle NM-01 also suppressed live virus when RF ID50approximately 0.05 μg/ml (Fig. 4C).

These data show that monoclonal antibody NM-01 neutralize the infectivity of at least three different strains of HIV-1.

The ability of a monoclonal antibody F58/H3 and P4/D10 to neutralize infection of H9 cells by live strains MN and IIIBHIV-1 was also evaluated according to the definition of reverse transcriptase and research on p24 as described above for monoclonal antibody NM-01. The results are shown in Fig. 6A through 6B and 7A by 7B. In the study from again found that the monoclonal antibody NM-01 fully neutralize the infectivity of live virus MN at concentrations of 10-100 μg/ml, and the use of antibodies at a concentration of < 1 μg/ml resulted in a 50% reduction in the infectivity of the virus (ID50(see empty circles in Fig. 6A). These results contrasted with the absence of detectable neutralizing monoclonal antibody F58/H3 and P4/D10 (see the filled and empty triangles in Fig. 6A). In the study on using live virus IIIBmonoclonal antibody NM-01 neutralized the virus when ID50approximately 0.1 μg/ml (empty circles in Fig. 6B). Monoclonal antibody F58/H3 and P4/D10 neutralized IIIBless effectively, what you have been received for three monoclonal antibodies using MN and IIIBHIV-1 study on p24 (see Fig. 7A and 7B).

Example 5.

The study of neutralizing the infectivity of live HIV-1, which was demonstrated by the analysis of the reverse transcriptase and p24 were extended with studies on the action of monoclonal antibody NM-01 analyses on MT-2 with the use of live viruses MN and IIIBand syncytium formation by use of live viruses MN, IIIBand RF.

A. Analysis of MT-2

Study with MT-2 was performed as described by Richman, AIDS Research and Reference Realent Program, Courier N 90-01, pp. 6-9 (1990) with some modifications. Live viruses MN and IIIBincubated with dilutions of monoclonal antibody NM-01 for 1.5 hours at 4oC in 96-cell boards. Cells MT-2 (8 of 105) was added to the cells and fees were incubated for 3 days at 37oC. was Then carried out research on the reduction of MTT staining by Mosmann, J. Immunol. Meth., 65, pp 55 to 63 (1983) and Pauwels et al., J. Virol. Meth. , 20, pp. 55 to 63 (1983) to determine cell viability. The results of the study with MT-2 confirm the results of studies, and p24 and shown in Fig. 8, and empty circles represent values for IIIB(100 TCID50) and the filled circles represent the value for MN (40 TCID50).

MES is l, respectively.

C. Determining the formation of syncytium

Study of the suppression of the binding is a modification of the method previously described in Johnson and Walker, Eds., Techniques in HIV-1 Research, Stockton Press, New York, NY, pp. 92-97, (1990). Briefly, H9 cells chronically incisione or MN, or IIIBvirus, incubated with dilutions of monoclonal antibody NM-01 for 1 hour at 37oC. C8166 Cells were then added into each well and incubated for 2 hours at 37oC. Syncytia more than three limfozitah cells in diameter was counted and compared with sincetime obtained from control and infected H9 cells processed in the absence of antibodies. The results of the study of the formation of syncytium also confirmed by the results of studies on OT and p24 and shown in Fig. 9A, and empty circles represent values for IIIB(100 TCID50)and the filled circles represent values for MN (40 TCID50). Monoclonal antibody NM-01 inhibited the formation of syncytium infected MN H9 cells with ID50equal to 2 μg/ml and infected IIIBthe H9 cells with ID50equal to 3 µg/ml.

The corresponding results of suppressing the formation of syncytium presents for monoclonal antibodies Wat 123 is kami, infected MN, reported that 25 mcg VAT suppress 51%, while 25 μg NM-01 inhibit approximately 85% of syncytium formation infected IIIBcells VAT reportedly suppresses 77,8% 25 µg Wat 123, as is also reported to inhibit the formation of syncytium infected RF cells by 51%. Monoclonal antibody NM-01 also inhibit the formation of syncytium infected RF cells (see Fig. 9B). In the study described in the preceding paragraph, the concentration of 25 µg NM-01 inhibits approximately 59% of syncytium formation infected RF cells. Monoclonal antibody NM-01 inhibited the formation of syncytium infected RF cell ID50equal to 4 µg/ml.

Taken together, the research results reverse transcriptase, p24, MT-2 and the formation of a syncytium of examples 4 and 5 show that the monoclonal antibody NM-01 neutralize binding and infectivity of different strains of HIV-1 at concentrations less than 10 µg/ml.

Example 6.

To confirm that the monoclonal antibody NM-01 block the infectivity MN and IIIBHIV-1 by binding to a part of the V3loops of gp120, peptides loop V3had the ability to block neutralizing antibodies.

3strains MN, IIIBand Z6 (sequences of the peptides are given in table 4) for 30 minutes at 37oC before the addition of 100 TCID50live virus IIIB. Then H9 cells were added for 1 hour and activities were determined FROM after growing cells in complete medium for 7 days as described in example 4. The results of the study are presented in Fig. 10.

While monoclonal antibody NM-01 fully neutralized the infectivity IIIBat the lowest concentrations of the peptide, this effect is progressively blocked by preincubation with increasing concentrations of peptide loops MN (filled circles) and IIIB(empty circles). When similar concentrations of a peptide corresponding to the loop V3strain Z6 (filled diamonds), which has no amino acid sequence that is recognized by monoclonal antibody NM-01 registered, the effect was absent. These results indicate that monoclonal antibody NM-01 block the infectivity of HIV-1 by reaction with a specific part of the domain V3gp120.

Example 7.

There have also been studies to determine whether the monoclonal antibody NM-01 to activate metabolic pathways whom the TKA.

Lysis of HIV-1 monoclonal antibody NM-01 and complement

The H9 cells infected with the strain IIIBHIV-1, washed in the environment to determine the cytotoxicity (Cedarlane Lab. Ltd.). Cells resuspendable in the environment to determine the cytotoxicity or in the absence or in the presence of 40 μg/ml of monoclonal antibody NM-01. After incubation for 2 hours at 4oC was added rabbit complement (Low-tox-MA; Cedarlane Lab. Ltd.) in a dilution of 1: 6. Cell suspension was incubated at 4oC for 20 minutes and then for 45 minutes at 37oC. the Cells twice were fixed with 2% glutaraldehyde (0.1 M phosphate buffer and 1% osmium tetroxide) 0.1 M phosphate buffer. After placing in the epoxy resin was placed thin slices and double-stained with uranylacetate and lead citrate. Fig. 11A through 11B, 12A through 12F and 13A through 13F are typical electron micrographs of thin sections.

Some rabbit serum (Fig. 11B) and a monoclonal antibody NM-01 - never had a noticeable effect on the morphology of HIV-1. Exposure of HIV-1 with a monoclonal antibody NM-01 and complement was associated with the emergence of numerous viral particles with damaged membranes and the loss of electron dense core (Fig. 11A). Typical Prearalie 10% of the virions were intact or had partially broken outer shell. Higher magnification revealed that the destruction of HIV-1 occurs by direct lysis, as shown in the series of microphotographs of lysis Mature and incomplete viral particles in Fig. 12A through 12F and 13A through 13F, respectively.

Example 8.

The combination of monoclonal antibody NM-01 and complement were then subjected to analysis to determine its impact on the infectivity of HIV-1.

The definition of infective doses for tissue culture

Infected H9 cells were twice washed in the environment to determine the cytotoxicity (Cedarlane Lab. Ltd.) and then resuspendable in the environment to determine the cytotoxicity containing 2 μg/ml of monoclonal antibody NM-01 or control IgG2b. After incubation at 4oC for 2 hours, the samples were divided into equal parts and added or rabbit complement, or inactivated by heating with rabbit serum (Cedarlane Lab. Ltd.) in a dilution of 1: 6. Cells were incubated at 4oC for 20 minutes and then at 37oC for 45 minutes, washed with medium, resuspendable in the environment of 50% FBS/RPMI 1640 and shook. The supernatant or viral isolate was diluted tenfold and then serially diluted 2 times before adding 25 μl of H9 cells(1 to 105/25 μl). After incubation in techeniya infection was determined after 6 days by research on reverse transcriptase. The infective dose for tissue culture for 50% of the samples of H9 cells (TCID50) was determined by the dilution that gave 50% infection. Table 5 presents the results of experiments.

While monoclonal antibody NM-01, by themselves, capable of neutralizing the infectivity processing of monoclonal antibody NM-01, together with the complement reduced the infectivity more than 10 times. A similar effect was also observed when it was applied human complement (human serum) with monoclonal antibody NM-01. These results indicate that exposure of HIV-1 with a monoclonal antibody NM-01 and complement associated with a significant reduction in viral infectivity, and additionally confirm the role of mediated NM-01-dependent complement viralize in the treatment of HIV-1. Cm. also Nakamura et al. AIDS RESEARCH AND HYMAN RETROVIRUSES.

9(7), pp. 619-626 (1993), which are hereby incorporated by reference.

Example 9.

The DNA sequence of variable regions of the heavy and light chains of the monoclonal antibodies were cloned by the PCR using cDNA produced from cytoplasmic PHK hybrid HB 10726 as a matrix. DNA variable regions each then suryanelli regions of the heavy and light chains NM-01 presented at TH ID NN 15 and 16 and PEFC ID NN 17 and 18, respectively. Nucleotides 1-21 and 334-363 TH ID NN 15 corresponds to the PCR primers used for amplification of sequences of the light chain and nucleotide 1-27 and 385-402 TH ID N 17 correspond to the PCR primers used for amplification of sequences of the heavy chain NM-01. Reserveerimine variable regions of monoclonal antibody NM-01 led to the sequences presented in TH ID NN 19 and 20 and PEFC ID NN 21 and 22, which are DNA and deduced amino acid sequence of the variable region of the heavy chain and the DNA and deduced amino acid sequence of the variable region of the light chain, respectively. Amino acid sequence of the variable region of the light chain (MC), as installed, is the most homologous Kabat murine Kappa subgroup III, the amino acid sequence of the variable region of the light chain (VH), as installed, is a member of subgroup IA Kabat murine heavy chains.

The first 120 residues of the amino acid sequences of variable regions of heavy (PEFC ID NN 20) and light (PEFC ID NN 22) chains NM-01 also shown in Fig. 14 and 15, respectively, and the enclosed amino acids are the scopes that define complementarity (C whom you respect CDR, shown in the earlier International patent application N PCT/WO92/07111 to bring them into line with the definitions of Kabat CDR et al., Sequences of Proteins of Immunolgical Interest, 5 th Edition, US Department of Health and Human Services, Government Printing. Office (1991).

In Fig. 14 and 15, each amino acid sequence of the heavy or light chain compared with the corresponding amino acid sequence (LAST ID N: 23 and 24) variable regions of the heavy and light chains of monoclonal antibodies VAT, which reported Liou et al., above, and with the corresponding amino acid sequences (PEFC ID NN 25 and 26) variable regions of the heavy and light chains of a monoclonal antibody F58/H3 and P4/D10 obtained from ECACC. Amino acid sequences of variable regions of a monoclonal antibody F58/H3 and P4/D10, as found, are identical.

Variable region of the heavy chain NM-01 differs from that of VAT forty six amino acids out of the total of one hundred and twenty amino acids. Variable regions of light chains of these two antibodies differ in twenty-three amino acids. It is important that the three CDRs in the heavy chain (V-H) NM-01 molecule from about 41% to 90% differ in sequence from those in the BAT-123, while posledovatelnostj heavy chains NM-01 differs from that F58/H3 and P4/D10 hundred three amino acids from the total number of one hundred and twenty, while variable region chains differ by three amino acids. The three CDRs in the heavy chain (V-H) molecules NM-01 differ by about 86% to 100% sequence from those F58/H3 and P4/D10, while the sequences of the three CDRs in the light chain (V-L) vary from 13% to 19%.

Analysis of the primary structure of NM-01 in comparison with the primary structure of BAT123, F58/H3 and P4/D10, therefore, establishes that the NM-01 is a new antibody.

Example 10.

On the basis of information about the DNA sequence presented in TH ID NN : 19 and 21 were obtained chimeric and humanized/modified variants of the antibody NM-01. To obtain chimeric variant NM-01 methods were applied Orlandi et al., Proc.Natl. Acad. Sci. USA, 86, pp. 3833-3837 (1989). Humanized variants were obtained by methods similar to the methods transplantirovali CDR Tempest et. al., BIO/TECHNOLOCY, 9, pp. 266-271, (1991) and Piechmann et al., Nature, 322, pp. 323-327 (1988).

A. Obtaining chimeric antibodies

Variable regions of the NM-01 cloned in two stages in the expression vectors mammals, to be able to obtain chimeric antibodies with murine variable regions of a human. First of all, completely sequenced VH or VK amplified from clones M13mp19/mp19 NM-01 described in example 9, ipoe to transfer the resulting amplificatory fragment in the vector M13VHPCRI or M13VKPCRI (Orlandi et al., above).

Thus the variable region was placed behind a promoter and signal peptide gene in the correct orientation for stitching in gene constant region. In the second stage of the insertion of M13, including the sequence encoding the promoter, signal peptide and variable region (VH or VK), cut from RF DNA and cloned into the expression vector mammals, respectively containing, as appropriate, the gene of human IgGl (vector pSV-gpt) or constant region of the Kappa (vector pSV-hyg).

Plasmids encoding chimeric light and heavy chains NM-01 then contracyclical in VB2/0 myeloma cells in rats (ATCC CRL 1662), which were then sorted on the presence contentstripecolourtwo gene (gpt), detectable expression vector of the heavy chain. The supernatant was checked for the presence of human IgG, and cells secreting antibodies, were multiplied. Chimeric antibodies were labeled NM-01 MuVH/MuVK.

B. Obtaining humanized antibodies

CDR-transplantation was performed by site-directed mutagenesis of samples of variable regions of human rights. The genes for the variable region of the person selected for CDR-transplantation CDR NM-01, were NEWH VH (Saul et al., L. Biol. Chem. , 253, pp. 585-597 (1978) and RVH) included four murine amino acid residue before the first CDR in positions 27-30 with Fig. 16 and mouse arginine at position 73 in Fig. 16 (position 71 in Kabat). Four residue before the first CDR, although not hypervariable been shown to affect hypervariable loop conformation by Chothia et al., J. Mol. Biol., 196, pp. 901-917 (1987). The residue at position 71 in Kabat, as shown, is placed between the CDR loops 1 and 2, and is important in determining the conformation of CDR 2 (Tramontano et al., J. Mol. Biol., 215, pp. 175-182 (1990)).

Received two variants HuVK NM-01, including the option with the CDR-grafting (HuVK) and the option with the CDR-grafting (HuVKF) with murine phenylalanine at position 75 in Fig. 17. The side chain of the amino acids in this position, as was shown to affect the conformation of CDR 1 (Chothia et al., above), and the inclusion of the murine residue had a positive effect on the binding capacity of other humanized antibodies. Cm. for example, Foote et al., J. Mol. Biol., 224, pp. 487-499 (1992).

DNA and derived amino acid sequence of HuVH, HuVK and HuVKF NM-01, respectively, shown in TH ID N 27 and 28, 29 and 30, 31 and 32. Humanized variable regions of the NM-01 were obtained from M13 phage containing the gene variable regions of the heavy or light chain, as follows.

The M13 phage containing the genes for the variable regions of the heavy or light chains, were grown in E. coli RZ1032 (dut ung) to Polygonaceae, which sanatorium matrix M13 in the forward direction from the inserted DNA. Then mutagenesis oligonucleotides encoding the murine residues were senatoriable matrix in 20 μl of 40 mm Tris-HCl pH 7.5, 20 mm MgCl2, 50 mm NaCl by heating to 80oC for 5 min and slow cooling to room temperature.

For the initial PCR reaction variable regions of the heavy chain used mutagenesis oligonucleotides:

VH oligo CDR 1 (LETTER ID # 33)

5' CTGTCTCACC CAGTGCCAGC AATAACTACT

ACTTGTGATG GAGAAGCCA ACAC 3'

moreover, the oligonucleotide is ortogonality DNA that encodes amino acids (amino acids 24-41 of TH ID N 28 and sequence HuVH in Fig. 16), and the underlined amino acids are the murine residues introduced into the matrix sequence variable regions;

VH Oligo CDR 2 (POST ID No. 34)

5' CATTGTCACT CTGCTTTTGA TGGATGGACT ATAGCTATT

AACCTTCAT AACATATGCG TCC(A/C)ATCCAC TCAAGA 3'

moreover, the oligonucleotide is ortogonality DNA that encodes the amino acid L EW(1/M) (amino acids 47-71 TH ID N 28 and sequence HuVH in Fig. 16) and the underlined amino acids are the murine residues introduced into the matrix sequence variable regions, and

VH oligo CDR 3 (LETTER ID # 35)

5' CCAGTA yuusha amino acids (amino acids 94-III TH ID N 28 and sequence HuVH in Fig. 16) and the underlined amino acids are the murine residues introduced into the matrix sequence variable regions.

Human matrix used for mutagenesis of the variable region of the light chain, actually encodes the structural areas that were related but not identical to REI and the reaction mutagenesis eliminate these inconsistencies (using undescribed nucleotides) as well as the introduction of CDR NM-01. The only difference that you want to discuss here especially, is in position 71 of the matrix, which encodes phenylalanine residue that is not in the sequence REI. This remnant was preserved in HuVKF NM-01 (see amino acid 75 HuVKF in Fig. 17), but was changed for the remainder of REI in HuVK NM-01 (see amino acid 75 HuVKF in Fig. 17) using the oligonucleotide REI Y71, the sequence of which is presented below.

VK oligo REI Y71 (LETTER ID # 36)

5' ATGGTGAAGG TGTAGTCGGT ACCGC 3'

moreover, the oligonucleotide is ortogonality DNA that encodes amino acids GTDYTFT (amino acids 72-78 TH ID N 32 and the sequence HuVK in Fig. 17). This primer was not involved in the reaction mutagenesis, which produced HuVKF NM-01.

For both variable regions of light chains HuVK and HuVKF murine CDR 1 NM-01 and matumi CDR 2 and 3 required a change matrix CDR 2 and 3, and methanediamine the oligonucleotides used here, were

VK oligo CDR 2 (PEFC ID N 37)

5' AGGTTGGATG CAACGTAGAT CAGCAG 3'

moreover, the oligonucleotide is ortogonality DNA that encodes amino acids (amino acids 50-57 TH ID N 30 and the sequence HuVK in Fig. 17), and the underlined amino acids are the murine residues introduced into the matrix sequence variable regions, and

VK oligo CDR 3 (PEFC ID N 38)

5' CCGAACGTGA GCGGATCTTC ATTATTTTGC TGGCAGTA 3'

moreover, the oligonucleotide is ortogonality DNA that encodes amino acids (amino acids 91-102 TH ID N 30 and the sequence HuVK in Fig. 17), and the underlined amino acids are the murine residues introduced into the matrix sequence variable regions.

To produce HuVH NM-01, VN oligo CDR 1, CDR 2 and CDR 3 has senatoriable in the human matrix NEWH. To produce HuVK NM-01, VK oligo REI Y71, VK oligo VK CDR2 and oligo CDR 3 has senatoriable in human DNA templates. To get HuVKF NM-01 senatoriable VK oligo CDR 2 and VK oligo CDR 3. Then in the same buffer was added dATP, dCTP, dGTP and dTTP to a final concentration of 250 μm, DTT was added to 7 mm ATP was added to 1 mm and added to 0.5 units of T7 DNA polymerase (United States Biochemical, Cleveland, OH) and 0.5 tournament 1 hour and then the DNA precipitated with ethanol. To produce single-stranded gap of the parent matrix circuit, the DNA was dissolved in 50 μl of 60 mm Tris-HCl pH 8.0, 1 mm DTT, 0.1 mg/ml BSA containing 1 unit of uracil-DNA glycosylase (Boehringer Mannheim, Kewis, Sussex, UK) and incubated at 37oC for 1 hour before being added NaOH to 0.2 M, and the incubation continued at room temperature for 5 minutes. DNA again precipitated with ethanol to remove fragmented parental DNA. Mutant DNA was then dissolved in 20 μl of TE, and insert the variable region amplified by the PCR using forward and reverse M13 primers. Reaction PCR mixture contained 2 μl of mutant DNA, 0.5 µm of each primer, 250 μm each dATP, dCTP, dGTP and dTTP, 10 mm Tris HCl pH to 8.3, 50 mm KCl, 1.5 mm MgCl2, 0.01% tween-20, 0.01% of gelatin, of 0.01% NP40 and 2 units of termales (Thermalase) (IBI, Canbridge, UK) in 50 μl. Amplification was achieved using 15 cycles of 94oC, 30 seconds, 50oC 30 seconds, 72oC, 1 minute, finishing with 72oC, 5 minutes. Produced DNA cloned with M13 mp19 in the form of a Hind III-BamHI fragments and specific clones sequenced. At first there were only obtained partial mutants with murine CDR 1 and 3 for the heavy chain. To obtain mutants with murine CDR 2, the above reaction was repeated using partially mutirovannye and characteristic clones sequenced.

Fragments of Hind III-BamHI encoding correct HuVH, HuVK and HVKF NM-01 then respectively cloned into the expression vectors before coding sequence of human IgGI (vector pSV-gpt) or Kappa (vector pSV-hyg) gene constant region as appropriate. The resulting vectors were toelectroproportional in YB2/0 or NSO cells (ECACC 85110503 to obtain a cell line producing a fully humanized antibody NM-01 (HuVH/HuVK) produced by cell line YB2/0, deposited as ECACC 93082022, HuVH/HuVKF produced by the cell line deposited as ECACC 93082019) or they were electroporative separately together with vectors encoding the corresponding heavy or light chain of chimeric NM-01, as described above, to obtain a cell line producing smeshanno-combined (mix & match) antibodies, where one of the chains was chimeric (e.g., MuVH/HuVKF). Antibodies were purified by affinity chromatography on agarose with protein A.

Four other variants of humanized antibody NM-01 were obtained by the methods described above. First HuVHM/HuVK (produced by the cell line YB2/0 ECACC 93082020) and the second HuVHM/HuVKF (produced by the cell line YB2/0, deposited as 93082021) include the methionine at position 48 HuVH. A third option HuVHS/HuVK (radio is her cell YB2/0, deposited as ECACC 93082018) include serine at position 93 HuVH. These humanized antibodies had similar properties bind to the antigen with those antibody NM-01 HuVH/HuVK or NM-01 HuVH/HuKF, depending on the light chain. Properties of binding antigen NM-01 HuVH/HuVK and NM-01HuVH/HuVKF described below.

C. Activity of chimeric and humanized antibodies

The binding of the humanized antibody NM-01) with gp120 was evaluated in comparison with the binding of murine NM-01 in competitive research. The Board was covered with recombinant gp120 (American Biotechnologies Inc. , Cambridge, MA) (5 ng/cell) and blocked with 5% normal goat serum (Life Technologies). Breeding (10-1000 ng/100 ál) of humanized antibody NM-01, chimeric antibody NM-01, mouse antibody NM-01 or negative control humanized antibodies were added to cells and fees were incubated at 37oC for 30 minutes. Added biotinylated mouse antibody NM-01 (500 ng/50 μl PBS per cell), and incubation continued for 1 hour. Fees were washed in PBS - 0.05% tween-20. Added HRPO-streptavidin (Sera-Lab Limited, Crawley Down, Sussek, UK; 40 ng/100 μl PBS per cell), and Board were incubated for 30 minutes. The Board then washed and incubated in the presence of o-phenyldiamine for 5 minutes or until retweets color. Paknam, as a murine antibody NM-01 at blocking the binding of labeled mouse antibody NM-01 c gp120, whereas humanized NM-01 antibody HuVH/HuVKF were about four times more active than mouse antibodies. Chimeric antibody NM-01 were less active than the murine antibody NM-01.

Chimeric and humanized antibody NM-01 also assessed the activity of neutralizing HIV-1 using analysis, p24 and syncytium formation. Studies performed essentially as described in the preceding examples was as follows.

In the analysis ON antibodies were serially diluted in medium RPM1 1640 with 15% fetal bovine serum. Breeding antibodies were incubated with 100 doses of infectious up to 50% tissue culture (TCID50) virus MN or IIIBin 96-aceing boards for 2 hours at 4oC. Then, each cell was added H9 cells (2,5105cells) and cost were incubated for one hour at 37oC. Suspension of H9 cells were then diluted in 2 ml of RPMI medium 1640/15% fetal bovine serum and incubated in 24-cell Board at 37oC. the Products of the virus were determined using studies FROM on the 7th day. The results of the study are presented in Fig. 18 (MN) and 19 (IIIB).

In the study on p24 CL is I. The presence of p24 antigen in the supernatant tissue culture was then quantified using profiled for p24 core of HIV-I enzyme-linked immunosorbent assay (ELISA), using methods described by the manufacturer (DuPont-NEN). Briefly, the complex antigen-antibody were determined using conjugate with horseradish peroxidase (HRP). The final product was quantitatively determined by the intensity of yellow colour, which is directly proportional to the amount of captured antigen p24 core of HIV-I. Manifestation of staining was recorded at 450 nm, using a reading device for the blade to EL ISA, and the results of the study are presented in Fig. 20 (MN) and 21 (IIIB), and monoclonal antibodies 2990.7 served as negative control.

And finally, the study of syncytium H9 cells chronically infected by any virus, MN, incubated with dilutions of monoclonal antibody NM-01 for one hour at 37oC. was Then added to the cells from the indicator cell line S (3104cells/cell) and cost incubated additionally for 2 to 12 hours at 37oC. Syncytia more than three limfozitah cell diameter was calculated and compared with data obtained for kontrolny.7 serve as a negative control.

The results of three studies show that the humanized antibody NM-01 HuVH/HuVKF were equally effective or more effective than murine NM-01 monoclonal antibodies neutralising isolates MN and IIIBHIV-I.

Although the present invention has been described in terms of preferred implementations, it is clear that changes and improvements will take place in the mind of the professionals. So have in mind that the accompanying form of the invention includes all such equivalent modifications that are included in the scope of the invention that is claimed.

The sequence listing is provided at the end of opisaniya

1. Humanitariannet antibody containing essentially variable region light and heavy chains of human antibodies, characterized in, that can specifically bind with the amino acid sequence of the protein gp120 or gp 160 HIV1, representing, essentially, a sequence of G-P-G-R, and is able to neutralize in vitro infection of H9 cells by live strains MP and IIIINIV-1 in the analysis of reverse transcriptase and the formation of entities, and these variable regions contain at least one region, complementarity determining (CDR) of murine monoclone the body under item 1, characterized in that an antibody NM-01 HuVH/HuVKF produced by hybridoma cell line ESAS N 93082019.

3. Humanitariannet antibody under item 1, characterized in that an antibody NM-01 Hu VH/HuVK produced by hybridoma cell line ESAS N 93082022.

4. The antibody under item 1, characterized in that an antibody NM-01 Hu VH/HuVK produced by hybridoma cell line ESAS N 93082020.

5. The antibody under item 1, characterized in that an antibody NM-01 Hu VH/HuVKF produced by hybridoma line ESAS N 93082021.

6. The antibody under item 1, characterized in that an antibody NM-01 Hu VHS/HuVK produced by hybridoma cell line ESAS N 93082023.

7. The antibody under item 1, characterized in that an antibody NM-01 Hu VHS/HuVKF produced by hybridoma cell line ESAS N 93082018.

8. Polynucleotide encoding the antibody NM-01 Hu VH/HuVK produced by hybridoma cell line ESAS 93082022, and a heavy chain of the indicated antibody NM-01 Hu VH/HuVK has the amino acid sequence:

DIQMTQSPSS LSASVGDRVT ITCRASESVD SYGNSFMHWY QQTPGKAPKL LIYVASNLES GVPSRFSGSG SGTDFTFTIS SLQPEDIATY YCQQNNEDPL TFGOGTKLOI T

9. Polynucleotide encoding the antibody NM-01 Hu VH/HuVKF produced by hybridoma cell line ESAS 93082019, THE RASESVD SYGNSFMHWY QQTPGKAPKL LIYVASNLES GVPSRFSGSG SGTDYTFTIS SLQPEDIATY YCQQNNEDPL TFGQGTKLQI T

10. Hybridoma cell line ESAS 93082022 encoding the antibody NM-01 Hu VH/HuVK.

11. Hybridoma cell line ESAS 93082019 producing antibody NM-01 Hu VH/HuVKF.

12. Hybridoma cell line ESAS 93082020 producing antibody NM-01 Hu VHM/HuVK.

13. Hybridoma cell line ESAS 93082021 producing antibody NM-01 Hu VHM/HuVKF.

14. Hybridoma cell line ESAS 93082023 producing antibody NM-01 Hu VHS/HuVK.

15. Hybridoma cell line ESAS 93082018 producing antibody NM-01 Hu VHS/HuVKF.

16. The method of obtaining gumanitarnogo antibodies on PP.1 - 7, providing for (a) obtaining a paste by combining at least one region complementarity determining (CDR) of murine monoclonal antibodies with variable area gumanitarnogo antibodies, b) introduction of the insert in the expression vector, C) obtaining hybridoma cell lines by transforming cells of a myeloma expression vector, g) cultivation of the hybridoma cell line in a suitable nutrient medium, d) isolation gumanitarnogo antibodies on PP.1 - 7 of cells hybridoma or from the environment.

 

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