Antibody against ccr5

FIELD: medicine, molecular biology, antibodies.

SUBSTANCE: invention relates to an antibody raised against CCR5 and comprising: (i) two light chains wherein each light chain comprises product of plasmid expression and designated as pVK:HuPRO140-VK (ATCC - PTA-4097), and (ii) two heavy chains wherein each heavy chain comprises product of plasmid expression and designated as pVg4:HuPRO140 HG2-VH (ATCC - PTA-4098), or plasmid designated as pVg4:HuPRO140 (mut B+D+I)-VH (ATCC - PTA-4099), or fragment of such antibody binding with CCR5 on a human cell surface. Invention relates to nucleic acid encoding light and heavy chains of antibody, expression vector, cell-host transformed with at least one vector, and a method for preparing antibody. Antibody is used as an active component in composition used for inhibition of infection of cells CD4 + HIV-1, and to a pharmaceutical composition used in treatment of a patient with HIV-1 infection. Also, invention relates to antibody conjugate against CCR5 and its using. Use of antibodies provides enhancing effectiveness of prophylaxis and treatment of HIV-1 infection.

EFFECT: valuable medicinal properties of antibody.

31 cl, 23 dwg, 3 ex

 

This application is a partial continuation of the application for the grant of a U.S. patent serial No. 10/081128, filed February 22, 2002, the contents of which are incorporated by reference into this application, and claims the priority of the specified application.

Throughout this application various publications are marked with Arabic numerals. Full references for these publications can be found at the end of this description immediately before the claims. The description of these publications is incorporated by reference into this application to more fully describe the field to which this invention relates.

Background of the invention

The human immunodeficiency virus type 1 (HIV-1) for penetration into the target cell causes the fusion of the membranes of the virus and cells (8, 15, 66). The first high-affinity interaction between the virion and the cell surface is a binding glycoprotein gp120 on the viral surface antigen CD4(13, 30, 41, 42). This in turn induces conformational changes in gp120 that allows him to interact with one of several chemokines receptors(4, 5, 21, 36). Receptor CC-chemokines CCR5 is the principal coreceptor for genotype to macrophages (R5) strains and plays a key role in sexual HIV transfer-1 (4, 5, 21, 36). Genotype to the lines of T-cells (X4) viruses to penetrate into cells Misha and use CXCR4 and usually, but not always, appear in the course of the disease or later as the result of the multiplication of the virus in tissue culture(4, 5, 21, 36). Some primary isolates of HIV-1 have double trapnest (R5X4), as they can use both forms, though not always with equal efficiency (11, 57). Studies of mutagenesis, paired with a resolution crystal structure of the protein bark gp120 showed that the coreceptor binding site on gp120 contains several conservative residues(32, 53, 65).

It is shown that the tyrosine residues and residues with a negative charge in the N-terminal domain (Nt) CCR5 is essential for the binding of gp120 with the receptor and to merge with HIV-1 and HIV penetration-1 (6, 18, 20, 22, 28, 31, 52, 54). Residues in the extracellular loops (ECL) 1-3 CCR5 was irrelevant to coreceptor functions, however, cross-domain configuration CCR5 was necessary to maintain optimal fusion and penetration of the virus (24). This led to the conclusion that either gp120 interacts with diffuse surface on the ECL, or that communication with the remnants in ECL Nt support in a functional conformation. Studies with the chimeric used and monoclonal antibodies against CCR5 also showed the importance of the extracellular loops for penetration of the virus(5, 54, 64).

Molecules that specifically bind to the CCR5 and CXCR4 and blocking interaction is with their ligands, represent a powerful tool for further studies of the structural/functional relationships can be used. Classification of such compounds may also contribute to the development of effective therapeutic agents targeting mediated by the used stages of an infection. Inhibitors coreceptor functions CCR5 or CXCR4, identified to date, vary in nature and include low molecular weight compounds, peptides, chemokines and their derivatives and monoclonal antibodies (mAb). The mechanisms of action of low molecular weight compounds that block the penetration by inhibiting the function of CXCR4 as coreceptor, it is not clear(17, 49, 55, 68). One such inhibitor, low molecular weight anionic compound AMD3100, in order to inhibit the penetration of the virus depends on residues in ECL2 and the fourth transmembrane domain (TM) CXCR4, but does not clear whether it is through breaking the binding of gp120 with CXCR4 or through a violation stages after binding, leading to fusion of the membranes (16, 34, 55). So far not reported low molecular weight compounds, specifically blocking mediated CCR5 penetration of HIV-1. Chemokines mediate the inhibition of entry of HIV-1, at least two different mechanisms: blocking vzaimode istia gp120/co-receptor and internalization of the complex chemokine/receptor (3, 26, 59, 63). Option AOP-RANTES also inhibits recirculation of CCR5 cell surface (40, 56). Such options as RANTES 9-68 and Met-RANTES only prevent the interaction of gp120/CCR5 and not cupressinum CCR5 (67). Variants of SDF-1 probably act by blocking mediated CXCR4 virus entry via a similar mechanism (12, 27, 39). Only one mAb against CXCR4, 12G5, characterized by its antiviral properties. It was reported that the effectiveness of inhibiting the penetration of the virus through 12G5 depends on the cells and isolate (43, 58). This mAb binds to CXCR4 with ECL2, but the mechanism by which it inhibits the penetration, unknown (7). Some characterized to date mAb against CCR5 effectively prevent the penetration of HIV-1 (28, 64). Interestingly, mAb, epitopes for which lie in the Nt-domain of CCR5, containing the binding site of gp120, inhibit the fusion of virus and virus entry is less effective than mAb 2D7, the epitope for which lies in ECL2. 2D7 is also an antagonist of the actions of CC-chemokines (64).

Have isolated and characterized a panel of six murine mAb, designated PA8, PA9, PA10, PA11, PA12 and PA14. All six mAb specifically associated with CCR5+cells, but with different efficiencies, depending on the cell type. Research on the mapping of the epitopes identified residues important for the binding of mAb, as well as provide awile information about the installation and the interactions of the extracellular domains of CCR5. All mAb inhibited fusion and penetration of HIV-1, but the correlation between the ability of the mAb to inhibit fusion and penetration and its ability to inhibit the binding of gp120/sCD4 with CCR5+cells were not observed.

The invention

This invention relates to an antibody against CCR5, containing (i) two light chains, each light chain contains the expression product of the plasmid designated pVK:HuPRO140-VK (inventory number in ATCC - PTA-4097), and (ii) two heavy chains, each heavy chain contains the expression product or the plasmid designated pVg4:HuPRO140 HG2-VH (inventory number in ATCC - PTA-4098)or a plasmid designated pVg4:HuPRO140 (mut B+D+I)-VH (inventory number in ATCC - PTA-4099), or a fragment of such antibody, binding to CCR5 on the surface of human cells.

This invention also relates to an antibody against CCR5, which contains two light chains, where each chain contains consecutive amino acids, amino acid sequence listed in SEQ ID No. 6, and two heavy chains, each heavy chain contains the sequence of amino acids, amino acid sequence listed in SEQ ID No. 9.

This invention also relates to an antibody against CCR5, which contains two light chains, where each chain contains consecutive amino acids, amino acid sequence listed in SEQ ID No. 6, and d is e heavy chain, where each heavy chain contains the sequence of amino acids, amino acid sequence listed in SEQ ID No. 12.

This invention also relates to an isolated nucleic acid that encodes a polypeptide containing consecutive amino acids, amino acid sequence listed in SEQ ID No. 6. In a specific implementation of the nucleic acid contains the sequence specified in SEQ ID No. 5.

This invention also relates to an isolated nucleic acid that encodes a polypeptide containing consecutive amino acids, amino acid sequence listed in SEQ ID No. 9. In a specific implementation of the nucleic acid contains the sequence specified in SEQ ID No. 8.

This invention also relates to an isolated nucleic acid that encodes a polypeptide containing consecutive amino acids, amino acid sequence listed in SEQ ID No. 12. In a specific implementation of the nucleic acid contains the sequence specified in SEQ ID No. 11.

This invention also relates to compositions containing at least one antibody against CCR5 or fragment, as described above, together with a carrier.

This invention also relates to compositions containing antibody against CCR5 or its fragment with Pris is United to him in substance, such as a radioisotope, a toxin, a polyethylene glycol, a cytotoxic agent and/or detected tag.

This invention also relates to a method of inhibiting infection of CD4+cells, comprising contacting CD4+cells with the antibody containing (i) two light chains, each light chain contains the expression product of the plasmid designated pVK:HuPRO140-VK (inventory number in ATCC - PTA-4097), and (ii) two heavy chains, each heavy chain contains the expression product or the plasmid designated pVg4:HuPRO140 HG2-VH (inventory number in ATCC - PTA-4098)or a plasmid designated pVg4:HuPRO140 (mut B+D+I)-VH (inventory number in ATCC - PTA-4099), or a fragment of such antibody, binding to CCR5 on the surface of CD4+cells, in such quantities and under such conditions that the fusion of HIV-1 or HIV infected-1 cells with CD4+the cell was depressed, thus inhibiting infection of CD4+-cells of HIV-1.

This invention also relates to a method for treatment of the affected HIV-1 subject comprising administration to the subject is effective for the treatment of HIV-1 doses of antibodies against CCR5, containing (i) two light chains, each light chain contains the expression product of the plasmid designated pVK:HuPRO140-VK (inventory number in ATCC - PTA-4097), and (ii) two heavy chains, each heavy chain contains the expression product or plasmid, designated pV4:HuPRO140 HG2-VH (inventory number in ATCC - PTA-4098)or a plasmid designated pVg4:HuPRO140 (mut B+D+I)-VH (inventory number in ATCC - PTA-4099), or a fragment of such antibody, binding to CCR5 on the surface of human cells, under conditions effective for the treatment of infected HIV-1 entity.

This invention also relates to a method of preventing a subject from infection with HIV-1, including an introduction to the subject is effective to prevent infection, HIV-1 dose of antibodies against CCR5, containing (i) two light chains, each light chain contains the expression product of the plasmid designated pVK:HuPRO140-VK (inventory number in ATCC - PTA-4097), and (ii) two heavy chains, each heavy chain contains the expression product or the plasmid designated pVg4:HuPRO140 HG2-VH (inventory number in ATCC - PTA-4098)or the plasmid designated pVg4:HuPRO140 (mut B+D+I)-VH (inventory number in ATCC - PTA-4099), or a fragment of such antibody, binding to CCR5 on the surface of human cells, under conditions effective for the prevention of infection of the subject's HIV-1.

This invention also relates to conjugate antibodies against CCR5, including antibody against CCR5, containing (i) two light chains, each light chain contains the expression product of the plasmid designated pVK:HuPRO140-VK (inventory number in ATCC - PTA-4097), and (ii) two heavy chains, each heavy chain contains the expression product or the plasmid designated pVg4:HuPRO140 HG2-VH (Inventa the hydrated number ATCC - PTA-4098)or a plasmid designated pVg4:HuPRO140 (mut B+D+I)-VH (inventory number in ATCC - PTA-4099), or a fragment of such antibody, binding to CCR5 on the surface of human cells, conjugated to at least one polymer.

This invention also relates to a method of inhibiting the infection of CCR5+-cells of HIV-1, including an introduction to the subject with the risk of infection with HIV-1 described above conjugate in the amount and under conditions effective to inhibit infection of CCR5+cells of the subject of HIV-1.

This invention also relates to a method for treatment of infection of HIV-1 in a subject, comprising the introduction of the above-described conjugate infected HIV-1 to a subject in an amount and under conditions effective for the treatment of infection by HIV-1 in the subject.

This invention also relates to transformed cell host containing at least two vectors, where at least one vector contains a nucleic acid sequence encoding a heavy chain antibodies against CCR5, and at least one vector contains a nucleic acid sequence encoding a light chain antibodies against CCR5, where the antibody against CCR5 contains two heavy chain with the amino acid sequence shown in SEQ ID No. 9, and two light chain with the amino acid sequence shown in SEQ ID No. 6.

This image is the buy also relates to transformed cell host, contains at least two vectors, where at least one vector contains a nucleic acid sequence encoding a heavy chain antibodies against CCR5, and at least one vector contains a nucleic acid sequence encoding a light chain antibodies against CCR5, where the antibody against CCR5 contains two heavy chain with the amino acid sequence shown in SEQ ID No. 12, and two light chain with the amino acid sequence shown in SEQ ID No. 6.

This invention also relates to a vector containing a nucleic acid sequence encoding a heavy chain antibodies against CCR5, where heavy chain contains the amino acid sequence indicated in SEQ ID No. 9.

This invention also relates to a vector containing a nucleic acid sequence encoding a heavy chain antibodies against CCR5, where heavy chain contains the amino acid sequence indicated in SEQ ID No. 12.

This invention also relates to a method for obtaining antibodies against CCR5, comprising culturing the host cell containing (i) a plasmid designated pVK:HuPRO140-VK (inventory number in ATCC - PTA-4097), and (ii) or a plasmid designated pVg4:HuPRO140 HG2-VH (inventory number in ATCC - PTA-4098)or a plasmid designated pVg4:HuPRO140 (mut B+D+I)-VH (inventory number in ATCC - PTA-4099), under conditions that allow the related products antibodies contains two light chains encoded by the plasmid designated pVK:HuPRO140-VK (inventory number in ATCC - PTA-4097), and two heavy chains encoded or by the plasmid designated pVg4:HuPRO140 HG2-VH (inventory number in ATCC - PTA-4098)or by the plasmid designated pVg4:HuPRO140 (mut B+D+I)-VH (inventory number in ATCC - PTA-4099), so that the thus formed antibody against CCR5.

This invention also relates to a method for obtaining antibodies against CCR5, which includes a) transforming the host cell (i) a plasmid designated pVK:HuPRO140-VK (inventory number in ATCC - PTA-4097), and (ii) or by the plasmid designated pVg4:HuPRO140 HG2-VH (inventory number in ATCC - PTA-4098)or by the plasmid designated pVg4:HuPRO140 (mut B+D+I)-VH (inventory number in ATCC - PTA-4099), and (b) culturing the transformed cell host under conditions that allow production of the antibody comprising two light chain encoded by the plasmid designated pVK:HuPRO140-VK (inventory number in ATCC - PTA-4097), and two heavy chains encoded or by the plasmid designated pVg4:HuPRO140 HG2-VH (inventory number in ATCC - PTA-4098)or by the plasmid designated pVg4:HuPRO140 (mut B+D+I)-VH (inventory number in ATCC - PTA-4099), so that the thus formed antibody against CCR5.

This invention also relates to a kit for use in a method of producing antibodies against CCR5. The kit includes (a) a vector containing a nucleic acid sequence encoding a light chain which antibodies against CCR5, where light chain contains the amino acid sequence indicated in SEQ ID No. 6, and (b) a vector containing a nucleic acid sequence encoding a heavy chain antibodies against CCR5, where heavy chain contains the amino acid sequence indicated in SEQ ID No. 9, or a vector containing a nucleic acid sequence encoding a heavy chain antibodies against CCR5, where heavy chain contains the amino acid sequence indicated in SEQ ID No. 12.

Brief description of drawings:

Figure 1:

Binding of monoclonal antibodies against CCR5 with CCR5+cells

To detect the expression of CCR5 protein on the surface CCR5+-L1.2 cells and have just highlighted, stimulated by PHA/IL-2 PBMC was used flow cytometry. Cells were incubated with saturating concentrations of each mAb, which was identified PE labeled reporter antibody against mouse IgG. Presents the results of a typical experiment. The results for each mAb is expressed as the average fluorescence intensity (m.f.i.), and as % of collected cells. As PA8-PA12 and PA14 all represent the subclass IgG1, m.f.i. comparable directly. 2D7 is an IgG2a.

Figure 2:

The CI values for different combinations of mAb and viral inhibitors:

For various combinations of inhibitors of virus entry p is bodily experiments, similar to the experiments described in the caption to figure 7. mAb against CCR5 tested in combination with each other, CC-chemokines and CD4-IgG2, which inhibit adherence of HIV-1 to the target cell. The range of concentrations PA11 and PA12 was 0-250 μg/ml; the concentration range 2D7 and PA14 were 0-25 µg/ml; range of concentrations of RANTES was 0-250 μg/ml; the concentration range of CD4-IgG2 was 0-25 µg/ml concentrations of separate funds or their mixtures required for obtaining 50% and 90% inhibition of fusion or penetration, and quantitatively compared using conditions known as combination index (CI).

Figure 3:

Values IC50for inhibition of the fusion of cells, penetration of the virus and the binding of gp120/sCD4 mAb against CCR5:

For comparison purposes, the authors of the present invention summarized values IC50obtained in different tests, where tested mAb against CCR5. The values of the IC50calculated only for those mAb, which are able to inhibit >90% confluence, penetration or binding.

Figure 4:

Mapping epitopes of mAb against CCR5:

To assess the binding of mAb with a mutant CCR5 proteins labeled at the C-end of the peptide HA, applied the Protocol staining of the two dyes. HeLa cells expressing a point mutant CCR5 were incubated with saturating concentrations of each Ab followed by detection of PE labeled antibodies against mouse IgG. The expression of the receptor on the cell surface was measured by dual staining of cells labeled with FITC mAb against HA. Four of the grid correspond to the four extracellular domains of CCR5. The first line of each grid indicates the amino acid sequence corresponding to the extracellular domain of CCR5 (SEQ ID nos 1-4). Binding of mAb against CCR5 with alanine mutant at each of residues expressed as a percentage of binding to CCR5 wild-type, as described in "materials and methods".

Figure 5:

Inhibition of the mobilization of calcium in CCR5+cells mAb against CCR5:

In CCR5+-L1.2 cells were injected Indo-1AM and consistently stimulated mAb against CCR5 or PBS, followed RANTES (a). Changes in fluorescence were measured by an, and the curve of the monitoring are presented from a typical experiment. Inhibition of release of calcium by PA14 and 2D7 were tested for a wide range of mAb concentrations (b). The results are presented as % inhibition of the release of calcium = [1- (relative fluorescence in the presence of mAb ÷ relative fluorescence in the absence of mAb)] × 100% and represent average values of three independent experiments.

Figure 6:

Inhibition coreceptor functions through CCR5 mAb against CCR5:

Inhibition merge cells mAb against CCR5 tested in the analysis of RET (a). To see the C cells HeLa-Env JR-FL+and PM1 labeled with F18 and R18, respectively, were added 0-250 μg/ml PA8-PA12 or 0-25 µg/ml PA14 or 2D7. Fluorescence in RET was measured after 4 hours of incubation. The results represent average values from three independent experiments and are presented as % inhibition merge = [1-(% RET in the presence of mAb ÷ % RET in the absence of mAb)] × 100%. Inhibition of penetration of HIV-1 by mAb against CCR5 tested in a single round-based replication luciferase analysis of penetration (b). Cells U87-CD4+CCR5+infected bearing shell JR-FL reporter virus NLluc+env+in the presence of 0-250 μg/ml PA8-PA12 or 0-25 µg/ml PA14 or 2D7. Luciferase activity (relative light units, r.l.u.) was measured in cell lysates 72 hours after infection. The results of typical experiments and are presented as % inhibition of penetration = [1- (r.l.u. in the presence of mAb ÷ r.l.u. in the absence of mAb)] × 100%. Binding of biotinylated [b] gp120, sCD4 and complexes of b-gp120-CD4 with CCR5+-L1.2 cells (c). Strong binding was observed when originating from virus R5 HIV-1JR-FLgp120 formed a complex with equimolar amounts of sCD4. In the absence of sCD4 or originating from virus HIV-1LAIX4 gp120 binding was observed. Of all the curves subtracted background binding to CCR5+-L1.2 cells. Inhibition of St. the statements gp120/sCD4 with CCR5 +-L1.2 cells tested in the presence of different concentrations of each antibody (d). The cells were pre-incubated in 96-well tablets with mAb against CCR5, followed by incubation with saturating concentrations of biotinylated gp120/sCD4. In conclusion, we measured the binding of PE labeled streptavidin with cells using the device for reading the fluorescence in tablets. The results of typical experiments and are presented as % inhibition of binding of gp120/sCD4 = [1- (m.f.i. in the presence of mAb ÷ m.f.i. in the absence of mAb)] × 100%.

Figure 7:

Synergistic inhibition of fusion of cells by PA12 and 2D7:

For mAb used separately or in combination, the obtained curves dose-response. To a mixture of cells HeLa-EnvJR-FL+and PM1 labeled R18 and F18, respectively, were added 0-50 µg/ml PA12, 0-25 µg/ml 2D7 or their combination in the ratio of 2:1. Fluorescence in RET was measured after 4 hours of incubation. The results are presented as % inhibition of mergers and represent average values from three independent experiments. Data were analyzed using the principle of the median effect, which can be written as

f=1/[1+(K/c)m] (1)

where f represents the fraction of affected/inhibited, c is the concentration, K is the concentration of funds, necessary is to obtain the median effect and m is an empirical coefficient that describes the shape of the curve dose-response. Equation (1) represents a generalized form of the equations describing enzyme kinetics Michaelis-Menten, adsorption isotherms the Langmuir and ionization equilibrium Henderson-Hasselbalch, for which m=1. In this case, K is equivalent to the value of the IC50. K and m were determined by approximation by points of curves dose-response, and equation (1) was changed to ensure that the calculation of c for a given f. The most relevant parameters for K and c are of 8.8 μg/ml and 0.54 for PA12, of 0.36 µg/ml and 0.68 for 2D7 and 0.11 μg/ml and 1.1 for their combination. These curves are shown and indicate an acceptable degree of agreement between experiment and theory.

Figure 8:

This figure indicated the amino acid sequence of the variable region of the light chain gumanitarnogo variant mouse antibodies against CCR5 PA14 (SEQ ID No. 6) and the nucleic acid sequence encoding it (SEQ ID No. 5), according to this invention. SEQ ID No. 7 identifies the region in SEQ ID No. 5, encoding the amino acid sequence indicated in SEQ ID No. 6. This variable region light chain is present in the antibody identified in the present description as PRO140 # 1 and # 2. Define complementarity region ("CDR") are underlined.

Figure 9:

This figure indicated the Ana amino acid sequence of the first variable region of the heavy chain gumanitarnogo variant mouse antibodies against CCR5 PA14 (SEQ ID No. 9) and the sequence of the nucleic acid, encoding it (SEQ ID No. 8), according to this invention. SEQ ID No. 10 identifies the region in SEQ ID No. 8 encoding amino acid sequence indicated in SEQ ID No. 9. This variable region of heavy chain present in the antibody identified in the present description as PRO140 No. 2. CDRs are underlined.

Figure 10:

This figure indicated amino acid sequence of the second variable region of the heavy chain gumanitarnogo variant mouse antibodies against CCR5 PA14 (SEQ ID No. 12) and the nucleic acid sequence encoding it (SEQ ID No. 11), according to this invention. SEQ ID No. 13 identifies the region in SEQ ID No. 11, encoding the amino acid sequence indicated in SEQ ID No. 12. This variable region of heavy chain present in the antibody identified in the present description as PRO140 No. 1. CDRs are underlined.

Figure 11:

Single dose gumanitarnogo antibodies against CCR5 effectively reduces viral loadin vivo:

The SCID mice restored normal human PBMC and infected HIV-1JR-CSF. When reaching the stationary state of the virus in animals subjected to a single exposure to 1 milligram intraperitoneal dose gumanitarnogo antibodies against CCR5 (PRO140) or izotopicheskogo control antibodies and watched plasma HIV RNA (Roche Amplicor Assay).

Figure 12:

Prolonged suppression of viral load:

The SCID mice restored normal human PBMC and infected HIV-1JR-CSF. When reaching the stationary state of the animal virus every three days was subjected to 0.1 mg intraperitoneal doses gumanitarnogo antibodies against CCR5 (PRO140) and watched plasma HIV RNA (Roche Amplicor Assay).

Figure 13:

Demonstrates that the application obtained by the invention are antibodies against CCR5 (PRO140) reduction of lymphocytes were not observed.

Figure 14:

Humanitariannet antibody against CCR5 (PRO140) effectively blocks mediated CCR5 merge the cell with the HIV-1-cell.

Mouse antibody against CCR5 humanitarianly with the method of grafting complementarity determining regions (CDR) and substitutions in the frame. Humanized antibodies against CCR5 (PRO140 No. 1 and PRO140 No. 2) expressed in cells of Sp2/0 was purified by chromatography with protein A and tested for the ability to block mediated replication env HIV-1JR-FLthe merging of the membranes as described (Litwin, et al., J. Virol., 70:6437, 1996).

Figure 15:

Humanitariannet antibody against CCR5 (PRO140) provide effective, independent of the subtype inhibition of HIV-1.

Antibodies against CCR5 (PRO140 No. 1 and No. 2) according to this invention were tested for their ability to block replica of the human HIV-1 wild-type in mononuclear cells of peripheral blood (PBMC), as described (Trkola et al., J. Virol., 72:396, 1998). The degree of virus replication was measured by assessing the content of p24 antigen in supernatant 7-day PBMC cultures.

Figure 16:

This figure presents a map of the plasmid pVK-HuPRO140 that encodes a variable region light chain, represented in the figure 8, and a constant region of human Kappa, as described in Co et al., J. Immunol., 148:1149, 1992.

Figure 17:

This figure presents a map of the plasmid pVg4-HuPRO140 HG2 that encodes a variable region of the heavy chain, presented on figure 9, and a constant region of a human heavy chain human CH1, hinge section, IgG4 CH2 and CH3, as described by He et al., J. Immunol., 160:1029 (1998).

Figure 18:

This figure presents a map of the plasmid pVg4-HuPRO140 (mut B+D+I)that encodes a variable region of the heavy chain, presented on figure 10, and a constant region of a human heavy chain, CH1, hinge section, CH2 and CH3 of human IgG4, as described by He et al., J. Immunol., 160:1029 (1998).

Figure 19:

Hu PRO140 blocks HIV-1, but not the signal transmission RANTES

Antibody PRO140 according to this invention were tested for their ability to block induced RANTES mobilization of calcium in CCR5+-L1.2 cells (Olson, et al., J. Virol., 72:396, 1998). This figure shows that humanitariannet antibody against CCR5 (PRO140) blocks HIV-1, but not transmission the signal RANTES.

Detailed description of the invention

The plasmid, designated as HuPRO140-VK, HuPRO140 (mut+B+D+I)-VH and HuPRO140 HG2-VH, which figures 16, 18 and 17 designated as pVK-HuPRO140, pVg4-HuPRO140 (mut B+D+I) and pVg4-HuPRO140 HG2 respectively, were deposited in the American type culture collection (ATSS), Manassas, Va., U.S.A. 20108 22 February 2002 under inventory numbers ATCC PTA 4097, PTA 4099 and PTA 4098 respectively. Data deposition carried out in accordance with the terms of the Budapest agreement on the international recognition of the Deposit of microorganisms for the purpose of the procedure for granting patents (Budapest Treaty).

This invention relates to a composition for inhibiting infection with HIV-1 containing at least two compounds in having a synergetic effect for the inhibition of infection of HIV-1 numbers, where at least one of the two connections prevents productive interaction between HIV-1 and the receptor for merging with HIV-1.

As used in the present description, "composition" means a mixture. Songs include as non-limiting examples of compositions suitable for oral, rectal, intravaginal, local, nasal, ophthalmic, or parenteral administration to a subject. As used in the present description "parenteral" includes, as non-limiting examples subcutaneous, intravenous, inside the muscle or intrasternal injection or infusion.

As used in the present description, "HIV-1" means the human immunodeficiency virus type 1. HIV-1 includes, as non-limiting examples of extracellular viral particles and forms of HIV-1 found in infected HIV-1 cells.

As used in the present description, "infection with HIV-1" means the introduction of the genetic information of HIV-1 in the target cell, for example, by fusion of the membrane of target cells with HIV-1 or cells carrying the glycoprotein shell of HIV-1. Cell-target may be a cell in the organism of the subject. In a preferred implementation of the cell as the target is a cell of the human body.

As used in the present description, "inhibition of infection of HIV-1" means reducing the number of HIV-1 genetic information introduced into a population of target cells, compared with the number that could be entered without a specified composition.

As used in the present description, "connection" means a molecule, including as non-limiting examples of peptides, polypeptides and other organic and inorganic molecules, and combinations thereof.

As used in the present description, "having a synergetic effect" means that the joint effect of the compounds when used in combination is higher than the sum of their effects when used separately.

As used in this description is, "productive interaction" means that the interaction of HIV-1 coreceptor for HIV-1 can lead to the merger of the specified HIV-1 or cells carrying the glycoprotein shell of HIV-1, and bearing the receptor membrane.

As used in the present description, "prevents productive interaction" means that the degree of interaction is lower than the level that would have occurred without this connection. Interaction can be prevented by masking or modifying the interacting regions on the receptor or HIV-1 or by changing the expression, aggregation, conformation or state Association coreceptor.

As used in the present description, "the receptor for the merger with HIV-1" means the cellular receptor mediating the fusion of expressing the receptor of target cells and HIV-1 or cells carrying the glycoprotein shell of HIV-1. The can be used to merge with HIV-1 include as non-limiting examples of CCR5, CXCR4 and other receptors chemokines.

This invention also relates to inhibiting the fusion of HIV-1 or cells carrying the glycoprotein shell of the HIV-1 cell-targeted compositions containing at least two compounds in quantities having a synergetic effect for inhibiting the fusion of HIV-1 or cells carrying the glycoprotein shell of the HIV-1 cell-target, where, by at least one of the compounds prevents productive interaction between HIV-1 and the receptor for merging with HIV-1.

As used in the present description, "merger" means a connection or Association of lipid bilayer membranes, which in mammalian cells or viruses, like HIV-1. This process differs from the accession of HIV-1 to target cells. Adherence is mediated by the binding of the external glycoprotein of HIV-1 with human CD4 receptor, which is not a coreceptor for the mail merge.

As used in the present description, "inhibits" means that the amount compared to the amount that could have taken place without the composition is reduced.

As used in the present description, "cell-target" means a cell that can be infected HIV-1 infected or HIV-1 cell or merge with them.

As used in this description, "chemokine" refers to the cytokine that can stimulate the movement of leukocytes. They can be classified or cys-cys, or cys-X-cys, depending on whether two aminobenzene residue cysteine directly next to each other or separated by one amino acid. They include as non-limiting examples of RANTES, MIP-1α, MIP-1β, SDF-1 or another chemokine that blocks infection by HIV-1.

In one implementation of the above compositions is the second receptor is a receptor for chemokines. In the preferred implementation of the above compositions the receptor for chemokines is a CCR5 or CXCR4. It is known that some other chemokine and related receptors function as used for HIV and include as non-limiting examples of CCR2, CCR3, CCR8, STRL33, GPR-15, CX3CR1 and APJ (69).

As used in the present description, "the receptor for chemokines" means a representative of a homologous family of seven times permeating the membrane of the cell surface proteins that bind chemokines.

As used in the present description, "CCR5 is a receptor for chemokines, linking representatives of the C-C group, chemokines, and amino acid sequence which contains the amino acid sequence represented under inventory number 1705896 in GenBank, and related polymorphic variants.

As used in the present description, "CXCR4 is a receptor for chemokines, linking representatives of the C-X-C-groups chemokines, and amino acid sequence which contains the amino acid sequence represented under inventory number 400654 in GenBank, and related polymorphic variants.

In one implementation of the above compositions, at least one of the compounds is a ones molecule. In one implementation ones molecule Ave is dstanley bellanue connection AMD3100. (16).

As used in the present description, "ones molecule" means a molecule that is not entirely composed of a linear sequence of amino acids connected by peptide bonds. However, the ones molecule can contain one or more peptide bonds.

In one implementation of the above compositions, at least one of the compounds is an antibody. In one implementation, the antibody is a monoclonal antibody. In another implementation, the antibody is an antibody against the receptor for chemokines. In one implementation, the antibody is an antibody against CXCR4. In an additional implementation antibody against CXCR4 is a 12G5. (43). In a preferred implementation, the antibody is an antibody against CCR5. Antibody against CCR5 includes, as non-limiting examples PA8, PA9, PA10, PA11, PA12, PA14 and 2D7. In this composition of the compounds are in a suitable ratio. The ratio is in the range from 1:1 to 1000:1.

Monoclonal antibodies PA8, PA9, PA10, PA11, PA12 and PA14 deposited in accordance with the conditions and requirements of the Budapest agreement on the international recognition of the Deposit of microorganisms for the purposes of patenting in the American type culture collection (ATCC), 10801 University Boulevard, Manassas, Virginia 20110-2209 2 de is of u 1998 under the following inventory numbers: ATCC number HB-12605 (PA8), No. HB-12606 (PA9), No. HB-12607 (PA10), No. HB-12608 (P11), No. HB - 12609 (PA12), No. HB-12610 (PA14).

In another implementation of the above compositions of two or more compounds are antibodies. In one implementation of the invention antibodies include as non-limiting examples PA8, PA9, PA10, PA11, PA12, PA14 and 2D7. In this composition antibodies are in a suitable ratio. The ratio is in the range from 1:1 to 50:1.

As used in the present description, "antibody" means containing two heavy chains and two light chain immunoglobulin molecule that recognizes the antigen. Immunoglobulin molecule can be from any of the known classes, including as non-limiting examples of IgA, secretory IgA, IgG and IgM. The IgG subclass is also well known to specialists in this field and includes, as non-limiting examples of human IgGl, IgG2, IgG3 and IgG4. As an example, he includes both naturally occurring and not occurring antibodies. Specifically, "antibody" includes polyclonal and monoclonal antibodies and their monovalent and bivalent fragments. In addition, the "antibody" includes chimeric antibodies, synthetic antibodies, single-chain antibodies, and fragments thereof. Optionally, the antibody can be marked detektivami marker. Detected markers include, for example, radioactive or f is uorescent markers. The antibody can be a human antibody and non-human antibody. Non-human antibody can be humanize recombinant ways to reduce its immunogenicity in humans. Methods of humanizing antibodies known to specialists in this field.

As used in the present description, a "monoclonal antibody"also referred to as mAb, used to describe molecules, antibodies, whose primary sequence substantially identical and which have the same antigenic specificity. Monoclonal antibodies can be obtained by hybridoma, recombinant, transgenic and other well-known specialist in this field means.

As used herein, "antibody against receptor for chemokines" means an antibody that can recognize and bind to the epitope on the receptor for chemokines. As used herein, "antibody against CCR5" means a monoclonal antibody that can recognize and bind to the epitope on the receptor chemokines CCR5.

As used in the present description, "the appropriate proportion" means the mass or molar ratio in which the compounds have a synergistic effect.

In one implementation of the above compositions, at least one compound is a chemokine or manufacturers who are deficient in the chemokine. Chemokines include as non-limiting examples of RANTES, MIP-1α, MIP-1β, SDF-1, or combinations thereof. In this composition, the compounds are suitable ratio. Derived chemokines include as non-limiting examples of Met-RANTES, AOP-RANTES, RANTES 9-68 or combinations thereof.

As used in the present description, "derived chemokine" means a chemically modified chemokine. Chemical modifications include as non-limiting examples of amino acid substitutions, additions or deletions, additions ones compounds or oxidation. The person skilled in the art is able to obtain such derivatives.

In another implementation of the above compositions, at least one compound is an antibody and at least one compound is a chemokine or derived chemokine. In this composition of the compounds are in a suitable ratio. The ratio is in the range from 100:1 to 1000:1.

In another implementation of the above compositions, at least one connection associated with the gp41 subunit of the glycoprotein shell of HIV-1. In one implementation, at least one compound is a peptide inhibitor of entry of HIV-1 T-20 (70).

In another implementation of the above compounds, at least one of the compounds ing viruet the accession of HIV-1 to the target cell. In one implementation, at least one connection associated with CD4. In one implementation, at least one connection is a glycoprotein membrane of HIV-1. In one implementation, at least one compound is an antibody against CD4. In one implementation, at least one connection associated with the glycoprotein shell of HIV-1. In one implementation, at least one compound is an antibody to the glycoprotein shell of HIV-1. In one implementation, at least one connection represents based on the CD4 protein. In one implementation, at least one connection is a CD4-IgG2.

In another implementation of the above compositions, at least one compound is an antibody and at least one connection associated with the glycoprotein shell of HIV-1. In one implementation, the join is based on the CD4 protein. In one implementation, the connection is a CD4-IgG2. In this composition of the compounds are in a suitable ratio. The ratio is in the range from 1:1 to 10:1.

As used in the present description, "accession" means a process mediated by binding of the glycoprotein shell of the HIV-1 with human CD4 receptor, which is not a coreceptor for the mail merge.

As used in the present description, "CD4" means Mature, natural, associated with the membrane of the CD4 protein containing a cytoplasmic domain, a hydrophobic transmembrane domain and extracellular domain to bind to the glycoprotein shell of the HIV-1 gp120.

As used in the present description, "the glycoprotein shell of the HIV-1 is encoded by the HIV-1 protein containing surface protein gp120, a transmembrane protein gp41 and their oligomers and precursors.

As used in the present description, "based on the CD4 protein" means any protein that contains at least one sequence of amino acid residues corresponding to the portion of the CD4 sequence, which is necessary for formation of a complex with CD4 glycoprotein shell of the HIV-1 gp120.

As used in the present description, "CD4-IgG2" means heterotetrameric hybrid protein of human IgG2 and CD4 expressing the encoded by the vector deposited under inventory numbers ATCC 75193 and 75194.

In one implementation of the above compositions, at least one of the compounds contains a polypeptide that bind to CCR5 epitope. In one implementation epitope located at the N end of one of the three areas extracellular loops, or combinations thereof. In one implementation epitope located at the N-end. The epitope may contain N13 and Y15 at N-end. The epitope may contain Q4 at the end. In another implementation, the epitope includes residues at the N-end and the second extracellular loop. The epitope may contain D2, Y3, Q4, S7, P8 and N13 to the N-end and Y176 and T177 in the second extracellular loop. The epitope may contain D2, Y3, Q4, P8 and N13 to the N-end and Y176 and T177 in the second extracellular loop. The epitope may contain D2 N-end and R168 and Y176 in the second extracellular loop. In one implementation, the epitope is located in the second extracellular loop. The epitope may contain Q170 and K171 in the second extracellular loop. The epitope may contain Q170 and E172 in the second extracellular loop.

As used in the present description, throughout to designate a particular amino acid is used, the following standard abbreviations:

A=ala=alanineR=arg=arginine
N=asn=asparagineD=asp=aspartic acid
C=cys=cysteineQ=gln=glutamine
E=glu=glutamic acidG=gly=glycine
H=his=histidineI=ile=isoleucine
L=leu=leucineK=lys=lysine
M=met=methionineF=phe=phenylalanine
P=pro=ProlineS=ser=serine
T=thr=threonineW=trp=tryptophan
Y=tyr=tyrosineV=val=valine

As used in the present description, "polypeptide" means two or more amino acids linked by peptide bond.

As used in the present description, an "epitope" means a portion of a molecule or molecules forming the surface for binding of antibodies or other compounds. The epitope may contain contiguous or noncontiguous amino acids, carbohydrates, or other ones group or specific oligomer surface.

As used in the present description, the "N-end" means an amino acid sequence covering the initial methionine and the first transmembrane region.

As used in the present description, "the second extracellular loop" means an amino acid sequence covering the fourth and fifth transmembrane region and presents on the surface.

In one implementation of the above compositions, at least one of the compounds contains a light chain antibodies. In another implementation of the above compositions, at least one of the compounds contains a heavy chain antibodies. In another implementation of the above compositions, at least one of the compounds contains Fab-fragment antibodies. In another implementation of the above compositions, at least one of the compounds contains a variable domain of the antibody. In another implementation antibody poluchaut the form of antibodies from a single polypeptide, or "single chain", which contains the variable domains of the heavy and light chain, a genetically linked by an intermediate sequence of amino acids. In another implementation of the above compositions, at least one of the compounds contains one or more parts of the CDRs of the antibody.

As used in the present description, "heavy chain" means a larger polypeptide molecule antibody single variable domain (VH) and three or four constant domains (CH1, CH2, CH3 and CH4) or their fragments.

As used in the present description, "light chain" means little more polypeptide molecules of the antibody single variable domain (VL) and one constant domain (CL) or their fragments.

As used in the present description, "Fab" means the monovalent antigen binding fragment of immunoglobulin, consisting of one light chain and part of the heavy chain. You can get short papain cleavage or by recombinant methods.

As used in the present description, "F(ab')2" means a bivalent antigen binding fragment of immunoglobulin, consisting of both light chain and part of the two heavy chains. You can get short-splitting pepsin, or by recombinant methods.

As used in the present description, "CDR" or "defining complementary to the TB area" means vyisokopribyilnoy the amino acid sequence of the variable domain of the antibody.

This invention relates to the above compositions and a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers are well known to specialists in this field. Such pharmaceutically acceptable carriers may include as non-limiting examples of aqueous or non-aqueous solutions, suspensions and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters, such as etiloleat. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, salt and buffer environment. Parenteral carriers include sodium chloride, dextrose and ringer, dextrose and sodium chloride, ringer's lactate or fatty oil. Intravenous carriers include liquid and nutritional supplements, supplements electrolytes, such as additives, based on dextrose ringer, etc. can Also contain preservatives and other additives, such as, for example, antimicrobials, antioxidants, chelating means, inert gases, etc.

This invention relates to a method of treatment of the affected HIV-1 subject, which includes an introduction to the subject an effective dose of the above compositions.

As used in the present description, "subject" means Liu is th animal or artificially modified animal, capable of Contracting HIV. Artificially modified animal includes, as non-limiting examples of SCID mice with human immune systems. Animals include as non-limiting examples of mice, rats, dogs, Guinea pigs, ferrets, rabbits and primates. In the preferred implementation of the subject is a human.

As used in the present description, the "treatment" means or slowing, stopping or reversing the development of disorders caused by HIV-1. In a preferred implementation of the "treatment" means the treatment of development until the violation is corrected. As used in the present description, the "treatment" also means a decrease in the number of viral infections, reducing the number of infectious viral particles, reducing the number of virus-infected cells or relief of symptoms associated with HIV-1.

As used in the present description, "the affected HIV-1" means that the subject is present, at least one cell, infected with HIV-1.

As used in the present description, "introduction" can be implemented or performed with the use of any well-known specialists in the field of methods. The methods may include intravenous, intramuscular or subcutaneous methods.

The dose of the composition of the invention varies depending on subject and depending on the specific item is kinemage route of administration. The dose can be in the range from 0.1 to 100,000 μg/kg Based on the composition, the dose can be delivered continuously, for example by continuous infusion, or at periodic intervals. For example, when one or more separate cases. The person skilled in the art without undue experimentation can determine the desired time intervals for multiple doses of a specific composition.

As used in the present description, the "effective dose" means a sufficient amount of or for the treatment of the subject, or to prevent contamination of the subject of HIV-1. The person skilled in the art can conduct simple experiments by titration to determine the number needed to treat of the subject.

This invention relates to a method for preventing infection of a subject's HIV-1, including an introduction to the subject an effective dose of the above compositions.

As used in the present description, "infection with HIV-1" means infection with HIV-1, the genetic information which is replicated in the cells of the host and/or embedded in them.

This invention relates to a monoclonal antibody against CCR5. The antibody includes, as non-limiting examples of the following: PA8 (inventory number ATCC HB-12605), PA9 (inventory number ATCC HB-12606), PA10 (inventory number ATCC HB-12607), PA11 (inventory is th number ATCC HB-12608), PA12 (inventory number ATCC HB-12609) and PA14 (inventory number ATCC HB-12610).

This invention relates to humanized forms of the above antibodies.

As used in the present description, "humanitarianly" refers to antibodies in which some, most or all of the amino acids outside the CDR regions replaced by the corresponding amino acids derived from human immunoglobulin molecules. In one implementation of humanized forms of antibodies, some, most or all of the amino acids outside the CDR regions replaced with amino acids derived from human immunoglobulin molecules, but some, most or all of the amino acids in one or more CDR regions are not changed. Small additions, deletions, insertions, substitutions or amino acid modifications are permissible as long as they do not deprive the antibodies ability to bind to this antigen. Suitable human immunoglobulin molecules may include molecules IgGl, IgG2, IgG3, IgG4, IgA, and IgM. "Humanitariannet" antibody can save similar to the original antibody antigenic specificity, ie, according to the present invention, the ability to bind to CCR5.

Professionals in this field know how to get humanized antibodies according to this invention. Various publications, some of which are included as references in the application, also describe how to obtain the humanized antibodies. For example, described in U.S. patent No. 4816567 (71) the methods include obtaining a chimeric antibody with a variable region of one antibody and a constant region of another antibody.

In U.S. patent No. 5225539 (72) described another approach to obtain gumanitarnogo antibodies. This patent describes the use of recombinant DNA technology to obtain gumanitarnogo antibodies, where the CDR variable regions of a single immunoglobulin replace the CDR of an immunoglobulin with a different specificity so that humanitariannet antibody can recognize a desirable goal, but it is a meaningful way may not recognize the human immune system. Specifically, for CDR grafting in the frame used site-specific mutagenesis.

Other approaches for humanization of antibodies is described in U.S. patent No. 5585089 (73) and 5693761 (74) and WO 90/07861, describes methods for obtaining humanized antibodies. They have one or more CDR and possibly additional amino acids from the donor immunoglobulin and a frame region of acceptrules human immunoglobulin. In these patents describes a method of increasing the affinity of antibodies to the desired antigen. Some amino acids in the frame are chosen so that they were the same amino acid as the amino acids at the same positions is provided in the donor, and not in the acceptor. Specifically, these patents describes the obtaining of gumanitarnogo antibodies that bind to the receptor, by combining the CDRs of mouse monoclonal antibodies with the frame and constant regions of a human immunoglobulin. The human skeleton can be selected to maximize homology with the murine sequence. To identify amino acids in the framework region, possibly interacting with a CDR or a specific antigen, it is possible to apply a computer model, and then at these positions to create gumanitarnogo antibodies can be used murine amino acids.

In the above patents 5585089, and 5693761, and WO 90/07861 (75) also offered four possible criteria that can be applied when designing humanized antibodies. The first proposal was that for the acceptor to apply the framework of specific human immunoglobulin as possible homologous donor immunoglobulin for humanization, or apply a consensus framework from many human antibodies. The second proposal was that if the amino acid in the framework of the human immunoglobulin is unusual, as the amino acid donor in this position typical for sequences in humans, it is possible to choose the amino acid donor, and not the acceptor Third proposal was in positions immediately adjacent to the 3 CDR in the chain gumanitarnogo antibodies, you can select the amino acid donor, and not the acceptor. The fourth proposal was the use of amino acid donor in such positions of the frame, in which predicted that amino acids in the three-dimensional model of antibodies atom of the side chain is in the 3RD from the CDR, and predicted that it is able to interact with the CDR. The above methods are presented only to illustrate some of the ways that you can apply a specialist in this field for obtaining humanized antibodies. The affinity and/or specificity of binding gumanitarnogo antibody can be increased by application of the methods of directed evolution as described by Wu et al. (1999) J. Mol. Biol. 284:151 and U.S. patent No. 6165793, 6365408 and 6413774.

In one implementation of the invention humanitariannet form antibody contains a variable amino acid sequence of the light chain as shown in SEQ ID No. 6. In another implementation, the antibody contains a variable amino acid sequence of the heavy chain as shown in SEQ ID No. 9. In an additional implementation, the antibody may contain variable amino acid sequence of the heavy chain as shown in SEQ ID No. 12.

In another implementation humanitariannet antibody contains a variable AMI is kislotno sequence of light chain, as indicated in SEQ ID No. 6, and variable amino acid sequence of the heavy chain as shown in SEQ ID No. 9. Alternatively, the antibody may contain variable amino acid sequence of the light chain as shown in SEQ ID No. 6, and variable amino acid sequence of the heavy chain as shown in SEQ ID No. 12.

Variable regions gumanitarnogo antibodies can be linked, at least part of the immunoglobulin constant region of human immunoglobulin. In one implementation humanitariannet the antibody contains a constant region of the light chain and heavy chain. The constant region of the heavy chain typically includes CH1, hinge section, CH2, CH3 and, sometimes, the scope of CH4. In one implementation of the constant region gumanitarnogo antibodies represent the areas of human isotype IgG4.

The present invention relates to isolated nucleic acid molecules coding for data monoclonal antibodies against CCR5 or humanized versions. The molecule of nucleic acid can be an RNA, DNA or cDNA. In one implementation, the nucleic acid molecule encodes the light chain. In one implementation, the nucleic acid molecule encodes a heavy chain. In one implementation, the nucleic acid molecule encodes heavy and light chain. In one of the westline one or more nucleic acid molecules encode Fab-fragment. In one implementation, one or more nucleic acid molecules encode CDR fragments. In one implementation, the nucleic acid molecule encodes the variable domain. In another implementation, the nucleic acid molecule encodes the variable domain and one or more constant domains.

Preferably, the analogs are shown, for example, humanized antibodies against CCR5 differ from those shown, for example, humanized antibodies against CCR5 conservative substitutions of amino acids. With the purposes of classification of substitutions of amino acids as conservative or not conservative amino acids can be grouped as follows: group I (hydrophobic side chains): met, ala, val, leu, ile; group II (neutral hydrophilic side chains): cys, ser, thr; group III (acidic side chains): asp, glu; group IV (basic side chains): asn, gln, his, lys, arg; group V (residues influencing chain orientation): gly, pro; and group VI (aromatic side chains): trp, tyr, phe. Conservative substitutions include substitution of amino acids of the same class. Non-conservative substitutions are substitution of the representative of one of these classes to the representative of another.

Analogues of humanized antibodies against CCR5 demonstrate substantial identity of amino acid sequences here, for example, humanized PRO140 No. 1 or international narcotics the new PRO140 No. 2. Variable regions of the heavy and light chain analogues are encoded by sequences of nucleic acids, hybridization with encoding the variable regions of the heavy and light chain gumanitarnogo PRO140 No. 1 or gumanitarnogo PRO140 No. 2 nucleic acids, or their degenerate forms in hard conditions.

Due to the degeneracy of the genetic code humanitariannet antibody against CCR5 according to the present invention is encoded by the set of all sequences of nucleic acids. In certain implementations, the antibody molecule encodes a nucleic acid vysokoekonomichny the above nucleic acid molecules. Preferably homologous to the nucleic acid molecule includes a nucleotide sequence at least about 90% identical to the provided nucleotide sequence. More preferably, the nucleotide sequence was, at least about 95% identical, at least about 97% identical, at least about 98% identical, or at least about 99% identical to the provided nucleotide sequence. The homology can be calculated using various, publicly available software tools, a well-known work, the Ista in this area. Examples of tools include the BLAST system available from the web page of the National Center for Biotechnology Information (NCBI) at the National Institutes of Health.

One way of identifying vysokomolochnyh nucleotide sequences represents the hybridization of nucleic acids. Thus, the invention also includes humanized antibodies against CCR5 properties contact CCR5 described here and other functional properties, which are encoded by nucleic acid molecules, hybridization with the above nucleic acid molecules in very hard conditions. Identification of related sequences can also be achieved using polymerase chain reaction (PCR) and other methods of amplification, suitable for cloning of related sequences of nucleic acids. Preferably, the primers for PCR opt for amplification of parts of a specific sequence of nucleic acid, such as CDR.

As used in the present description, the term "very strict conditions" refers to well-known in this field parameters. The parameters of the hybridization of nucleic acids can be found in the references, which collected information about these methods, for example, Molecular Cloning: A Laboratory Manual, J. Sambrook, et al., eds., Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989, or Current Protocols in Molecular Biology, F. M. Ausubel, et al., ed., John Wiley & Sons, Inc., New York. One example of stringent conditions is a hybridization at 65 degrees centigrade in hybridization buffer (3,5× SSC, 0.02% ficoll, of 0.02% polyvinylpyrrolidone, 0.02% of bovine serum albumin, 2.5 mm NaH2PO4(pII7), 0,5% SDS, 2 mm EDTA). SSC is a 0.15 M sodium chloride/0.015 g M sodium citrate, pH 7; SDS is sodium dodecyl sulphate and EDTA is an ethylenediaminetetraacetic acid. After hybridization, the membrane to which the transferred nucleic acid, washed, for example, a 2× SSC at room temperature and then in 0.1 to 0.5× SSC/0,1× SDS at temperatures up to 68 degrees Celsius.

The nucleic acid sequences are expressed in hosts after the sequences are functionally connected (i.e. have to ensure the functioning of) controlling the expression sequence. Data expressing vectors, typically replicated organisms owners or episome, or as an integral part of the chromosomal DNA of the host. As a rule, expressing vectors contain breeding markers, e.g., tetracycline or neomycin, to provide a definition of what data cells transformed with the desired DNA sequences (see, for example, U.S. patent No. 4704362 included here in the quality of the ve links).

E. coliis a prokaryotic host, especially suitable for cloning the DNA sequences of the present invention. Other usable microbial hosts include bacilli, such asBacillus subtilusand other Enterobacteriaceae, such asSalmonella,Serratiaand various species ofPseudomonas. In data prioritiesin hosts can also be obtained expressing the vectors typically contain controls the expression of a sequence that is compatible with your cell host (for example, start replication). In addition, you may attend any number of a variety of well-known promoters, such as the lactose promoter system, a tryptophan (trp) promoter system, β-lactamania promoter system, or a promoter system of the phage. Promoters typically control expression, optionally with the help of the operator sequence, and are sequence binding site with the ribosome, etc. for initiating and completing transcription and translation.

Also suitable for the expression may be other microorganisms, such as yeast. The preferred host isSaccharomyceswith suitable vectors having controls the expression sequences, such as promoters, including 3-phosphoglycerate or other the glycolytic enzymes and start replication termination sequences and the like, as necessary.

Other than micro-organisms for the expression and obtain polypeptides of the present invention can also be applied tissue cell culture mammal (see, Winnacker, "From Genes to Clones", VCH Publishers, New York, New York (1987)). Essentially eukaryotic cells are preferred, because in this area has developed a number of suitable lines of host cells capable of secreting intact immunoglobulins, and they include lines of CHO cells, different cell line COS, HeLa cells, preferably cell line myeloma, etc. and transformed B-cells or hybridoma. Expressing the vectors for these cells can include controlling the expression of a sequence, such as the beginning of replication, a promoter, an enhancer (Queen et al., Immunol. Rev., 89, 49-68 (1986), included in the present description by reference) and the required areas of information processing, such as binding sites of ribosomes, the sites of RNA splicing, polyadenylation sites and the sequence termination of transcription. Preferred controls the expression promoters are promoters derived from immunoglobulin genes, SV40, adenovirus, cytomegalovirus, human papilloma virus of cattle, etc.

Containing the desired DNA vectors (e.g., encoding heavy and became the eighth circuit sequence and the sequence controlling the expression) can be transferred into the cell host using well-known methods, which vary according to the type of cellular host. For example, prokaryotic cells, typically used transfection with calcium chloride, whereas for other cellular hosts, you can apply the treatment with calcium phosphate or electroporation (in General, see Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press (1982), included here as a reference).

After expression, the whole antibodies, their dimers, individual light and heavy chains, or other forms of immunoglobulins of the present invention can be cleaned by standard means this area, including the deposition of ammonium sulfate, affinity column chromatography on column, gel electrophoresis and the like (in General, see R. Scopes, "Protein Purification", Springer-Verlag, New York (1982)). For pharmaceutical applications, the preferred substantially purified immunoglobulins of at least about 90 to 95% homogeneity, and most preferably from 98 to 99% homogeneity. After partial purification or purification to homogeneity, as desired, the polypeptides can then be used therapeutically (including in vitro use) or for the development and procedures of analysis, immunofluorescence stains, etc. (in General, see Immunological Methods, Vos. I and II, Lefkovits and Pernis, eds., Academic Press, New York, New York (1979 and 1981)).

For the purposes of diagnosis or detection antibodies can be either labeled or unlabeled. Unlabeled antibodies can be used in combination with other labeled antibodies (second antibodies)that interacts with a humanized antibody, such as antibodies, specific for the constant regions of human immunoglobulin. Alternatively, antibodies can be marked directly. You can apply a wide variety of labels, such as radioisotopes, fluorescent means, enzymes, substrates, enzymes, cofactors of enzymes, enzyme inhibitors, ligands (particularly haptens) and the like To determine expressing CCR5 cells or determine changes CCR5 on cells that can Express the CCR5 available and well known to specialists in this field are numerous types of immunological tests.

The present invention also relates to conjugates of the antibody fragment-polymer, with a visible size or molecular weight, increasing the half-life in serum, increasing the average retention time in the circulation system (MRT) and/or reduce the clearance of serum compared to rederivation fragments of antibodies.

Conjugates of the antibody fragment-polymer according to the invention can be obtained by derivatization of the desired fragment antic the La inert polymer. It is clear that for use in the design of conjugates of the antibody fragment-polymer according to the invention any suitable inert polymer, providing a conjugate desirable mid-size or have selected the actual molecular weight.

For use in medicinal preparations suitable many of inert polymers. See, for example, Davis et al., Biomedical Polymers: Polymeric Materials and Pharmaceuticals for Biomedical Use, pp. 441-451 (1980). All implementations of the invention use non-protein polymers. Non-protein polymer, as a rule, is a hydrophilic synthetic polymer, i.e. the polymer, or in the nature is not occurring. However, it is also suitable polymers that exist in nature and are obtained by recombinant methods or waysin vitroand the polymer isolated from natural sources. In the scope of the present invention are hydrophilic polyvinyl polymers, e.g. polyvinylalcohol and polyvinylpyrrolidone. Particularly suitable are polyalkylene ethers such as polyethylene glycol (PEG); polyoxyalkylene, such as polyoxyethylene, polyoxypropylene and block copolymers of polyoxyethylene and polyoxypropylene (Pluronics); polymethacrylates; carbomer; branched or unbranched polysaccharides containing sacharine monomers D-mannose, D - and L-galactose, fucose, fructose, D-xylose, L-arabinose,D-glucuronic acid, sialic acid, D-galacturonic acid, D-mannurone acid (for example, polymannuronic acid or alginic acid), D-glucosamine, D-galactosamine, D-glucose and neuraminic acid; including homopolysaccharides and heteropolysaccharides such as lactose, amylopectin, starch, gidroxiatilkrahmal, amylose, extrasolar, dextran, dextrins, glycogen, or the polysaccharide subgroup of acid mucopolysaccharides, e.g. hyaluronic acid; polymers of sugar alcohols such as Polysorbate and pelimannit, heparin or heparin. The polymer to cross-linking does not necessarily have to be, but preferably is water-soluble but the resulting conjugate must be water-soluble. Preferably, the conjugate showed a solubility in water of at least about 0.01 mg/ml and more preferably at least about 0.1 mg/ml, and even more preferably at least about 1 mg/ml in Addition, the polymer should not be highly immunogenic in the form of a conjugate or to have a viscosity, as it is not compatible with intravenous infusion or intravenous injection, if the conjugate is intended for the introduction of such paths.

In one implementation, the polymer contains only one interacting group. This helps to avoid the decussation of the CSOs binding protein molecules. However, the scope of the present invention includes the maximum improvement of the reaction conditions for the reduction of cross-linking or purification of the reaction products by gel-filtration or ion exchange chromatography to obtain essentially homogeneous derivatives. In other implementations, the polymer contains two or more interacting groups for the purposes of linking several fragments of antibodies with a polymer basis. You can also apply the gel filtration or ion exchange chromatography to obtain the desired derived essentially in homogeneous form.

The molecular weight of the polymer can be in the range of about to 500,000 and preferably constitutes at least about 20000 Yes, or at least approximately 30,000 Yes, or at least about 40,000 Da. Selected molecular weight may depend on the effective amount of the conjugate that is to be obtained, the nature (for example, patterns such as linear or branched) polymer and the degree of derivatization, i.e. the number of polymer molecules on the fragment of the antibody, and the site or sites of attachment of polymer to the antibody fragment.

The polymer may be covalently associated with the fragment of the antibody through a multifunctional tool for the formation of cross-links, interaction of the plans with the polymer and one or more amino acid residues of the fragment of the antibody, want to link. However, the scope of the invention also include direct formation of cross-links the polymer through interaction derivatizing polymer with a fragment of the antibody or Vice versa.

The sites of formation of covalent cross-links on the fragment of the antibody include N-terminal amino group and e-amino groups found on lysine residues, as well as other amino, imino-, carboxyl, sulfhydryl, hydroxyl or other hydrophilic groups. The polymer can covalently bind directly with the fragment of the antibody without the use of a multifunctional (usually bifunctional) means for the formation of cross-links, as described in U.S. patent No. 6458355.

The degree of substitution in such a polymer, as a rule, depends on the number of interacting sites on the antibody fragment, the molecular weight, hydrophilicity and other characteristics of the polymer and the specific sites of derivatization on the fragment of the antibody. Typically, the conjugates contain from 1 to 10 polymer molecules, but also consider the large number of polymer molecules attached to the fragments of the antibodies according to the invention. The desired level of derivatization easily be achieved by applying the experimental matrix, in which changing the degree of substitution change the time, temperature is round and the other reaction conditions, then the degree of substitution of the polymer in the conjugate are determined by gel filtration or other known in the field of ways.

Functionalityand PEG polymers for modification of fragments of the antibodies according to the invention are available in Shearwater Polymers, Inc. (Huntsville, Ala.). Such commercially available PEG derivatives include as non-limiting examples of amino-PEG esters of PEG with amino acids, PEG-hydrazide, PEG-thiol, PEG-succinate, karboksimetilirovaniya PEG, PEG-propionic acid, PEG-amino acids, PEG-succinimidylester, PEG-succinimidylester, Succinimidyl ester carboxymethylamino PEG, PEG-succinimidylester, Succinimidyl esters PEG-amino acids, PEG-oxycarbonate, PE nitrophenylarsonic, PEG-TResult, PEG-glycidyl ether, PEG-aldehyde, PEG-vinylsulfonic, PEG-maleinimide, PEG-orthopaedically, heterofunctional PEG, vinyl derivatives of PEG, the PEG silanes and pospolity PEG. The reaction conditions for binding data derived PEG vary depending on the protein, the desired level of PEG and used a derivative of PEG. Some factors relating to the choice of PEG derivatives include desirable area connection (for example, lysine or cysteine R-group), hydrolytic stability and reactivity derivatives, stability, toxicity and antigenicity of communication, the camera is lnost for analysis, etc. Exact instructions for applying any particular derivative available from the manufacturer. The conjugates of this invention are separated from not entered into the interaction of initial substances a gel filtration or ion-exchange high-performance chromatography.

Antibodies against CCR5 or their fragments can be used in combination with one or more additional antiviral agents selected from the group consisting of non-nucleoside reverse transcriptase inhibitors (NNRTIS), nucleoside reverse transcriptase inhibitors, protease inhibitors HIV-1, inhibitor of viral penetration or combinations thereof.

Known connections NNRTIS, which can be used in compositions of the present invention, include as non-limiting examples of efavirenz, UC-781, HBY 097, nevirapine (11-cyclopropyl-5,11-dihydro-4-methyl-6H-dipyrido[3,2-b:2'3'-][1,4]diazepin-6-one), delavirdine (Rescriptor™; decision Upjohn Pharmacia) (piperazine,1-[3-[(1-methylethyl)amino]-2-pyridyl]-4-[[5-[(methylsulphonyl)amino]-1H-indol-2-yl]carbonyl]-(nanomeasurement), SJ-3366 (1-(3-cyclopenten-1-yl)methyl-6-(3,5-dimethylbenzoyl)-5-ethyl-2,4-pyrimidinedione), MKC-442 (6-benzyl-1-(ethoxymethyl)-5-isopropylbenzyl), GW420867x (S-3-ethyl-6-fluoro-4-isopropoxycarbonyl-3,4-dihydroquinoxaline-2(1H)-he; Glaxo), HI-443 (N'-[2-(2-thiophene)ethyl]-N'-[2-(5-bromopyridin)]thiourea), etc.

Nucleoside inhibitors of reverse transcr PTZ, which can be used in the composition in combination with at least one antibody against CCR5 or its fragment according to the present invention include as non-limiting examples of abacavir (Ziagen™, GlaxoSmithKline) ((1S,cis)-4-[2 - amino-6-(cyclopropylamino)-9H-purine-9-yl]-2-cyclopenten-1-methanosulfonate (Sol)), lamivudine (Epivir™, GlaxoSmthKline) ((2R,cis)-4-amino-1-(2-hydroxymethyl-1,3-oxathiolan-5-yl)-(1H)-pyrimidine-2-one), zidovudine (Retrovir™; GlaxoSmithKline) (3'-azido-3'-deoxythymidine), stavudine (stavudine, zerit; Bristol-Myers Squibb) (2',3'-didehydro-3'-deoxythymidine), decitabine (Hivid™; Roche Laboratories) (4-amier-1-β-D2',3'-dimethoxybiphenyl-2-(1H)-pyrimidone), didanosine, etc.

The protease inhibitors of HIV-1, which can be used in compositions in combination with the antibody against CCR5 or fragments of the present invention, include as non-limiting examples of lopinavir (1S-[1R*,(R*),3R*,4R*]]-N-4-[[(2,6-dimethylphenoxy)acetyl]amino]-3-hydroxy-5-phenyl-1-(phenylmethyl)pentyl]tetrahydro-α-(1-methylethyl)-2-oxo-1-(2H)-pyrimidinylidene), saquinavir (N-tert-buildimage-2-[2(R)-hydroxy-4-phenyl-3(S)-[[N-(2-hinolincarbonova)-L-asparaginyl]amino]butyl]-(4aS,8aS)-isoquinoline-(3S)-carboxamide), nelfinavir mesilate ([3S-[2 (2S*,3S*),3A,4β,8aβ]]-N-(1,1-dimethylethyl)decahydro-2-[2-hydroxy-3-[(3-hydroxy-2-methylbenzoyl)amino]-4-(phenylthio)butyl]-3-ethanolinduced nanomeasurement), indin the Vira sulfate (([1(1S,2R),5(S)] -2,3,5-trideoxy-N-(2,3-dihydro-2-hydroxy-1H-inden-1-yl)-5-[2-[[(1,1-dimethylethyl)amino]carbonyl]-4-(3-pyridinylmethyl)-1-piperazinil]-2-(phenylmethyl)-D-eletropitanija sulfate (1:1) salt), APV ((3S)-tetrahydro-3-furyl-N-[(1S,2R)-3-(4-amino-N-isobutylacetophenone)-1-benzyl-2-hydroxypropyl]carbamate), ritonavir ((10-hydroxy-2-methyl-5-(1-methylethyl)-1-[2-(1-methylethyl)-4-thiazolyl]-3,6-dioxo-8,11-bis(phenylmethyl)-2,4,7,12-tetraazatricyclo-13-OIC acid, 5-triazolylmethyl ester, [5S-(5R*,8R*,10R*,11R*)]), etc.

Fusion inhibitors with HIV-1 or penetration of the virus, which can be used in combination with the antibody against CCR5 or fragments of the present invention, include PRO 542 (Progenics Pharmaceuticals, Inc., Tarrytown, NY), T-20 (Trimeris, Inc., Durham, NC) (U.S. patent No. 5464933; 6133418; 6020459), T-1249 (U.S. patent No. 6345568; 6258782), etc.

To ensure the combination of antibody against CCR5 or its fragment according to the present invention it is possible to introduce the subject before, after or simultaneously with one or more conventional anti-viral drugs.

The invention will be more clear from the following details of the experiments. However, the person skilled in the art will easily understand that the particular piece of the methods and the results are shown only to illustrate the invention, described more fully in the claims following hereafter.

The details of the experiments:

Example 1

A. Materials and methods

1)Reagents

MAb 2D7 acquired in Pharmingen (San Diego, CA), and CC - and CXC-chemokines were obtained from R&D Systems (Minneapolis, MN). CD4-IgG2 (1), dissolve IMY(e) CD4 (2) and recombinant gp120 of HIV-1 JR-FLreceived from Progenics Pharmaceuticals, Inc. (59).

2)Isolation and purification of mAb against CCR5

CCR5+-L1.2 cells (63) incubated for 16 hours in the presence of 5 mm sodium butyrate, activating transcription from a promoter of cytomegalovirus (CMV), which controls the expression of CCR5, which resulted in a 10-fold increase in the density of receptor on the cell surface. Female Balb/c mice were immunized intraperitoneally 107CCR5+-L1.2 cells with 3-week intervals and re were immunized by intravenous injection of 107L1.2 CCR5+cells three days before removal of the spleen. Splenocytes were merged with the cell line Sp2/0. When you first skanirovaniya tested supernatant ten thousand cultures hybrid; a hundred and twenty of them in the analysis of resonant energy transfer (RET), as described previously (19, 38), inhibited mediated membrane of HIV-1 cell fusion PM1 (10), by nature expressing CCR5 and CD4, cells and HeLa-EnvJR-FL+. Hybridoma, producing most effectively inhibiting supernatant and also coloring CCR5+cells were subclinically by limiting dilutions. By Harlan Bioproducts for Science, Inc. (Indianapolis, IN) in Balb/c mice, which were injected with hybridoma producing mAb against CCR5 PA8, PA9, PA10, PA11, PA12, and PA14, got ascitic fluid. mAb separately purified to > 95% homogeneity precipitat what she ammonium sulfate followed by chromatography with protein A. All mAb resuspendable in phosphate-buffered saline (PBS) at a final concentration of 5 mg/ml

3)Analysis of fluorescence-activated sorting of cells (FACS) and mapping of epitopes of mAb against CCR5

To determine the reactivity of mAb PA8-PA12 and PA14 to cell surface CCR5 was used flow cytometry. Treated with sodium butyrate CCR5+-L1.2 cells (106) for 20 min, incubated with 25 µg of antibody at 4°C in 50 µl PBS, Dulbecco (DPBS) with 0.1% of sodium azide (NaN3). mAb 2D7 against CCR5 was used as a positive control, nonspecific mouse IgG1 was used as a negative control. Cells were centrifuged, washed, and incubated with labeled with phycoerythrin goat antibodies against IgG of mice (Caltag, Burlingame, CA)diluted 1:100 in the same conditions as the first incubation with the antibody. In conclusion, cells were analyzed by flow cytometry. PBMC were isolated and stimulated as described previously (60), and stained using similar methods.

A similar procedure was used for epitope mapping mAb against CCR5. Described panel of seventy point mutants in CCR5 (20, 24, 52). Coding sequences of these proteins were subcloned into the vector pcDNA3.1 (Stratagene), with which it is possible to carry out transcription under the control of the 5' promoter, the T7 polymerase. The mutant CCR5 C-end are gamig outinnovate (HA) tag from 9 residues for the determination of protein in cell lysates or by flow cytometry. HeLa cells (2×106) 5 hours incubated in OPTI-MEM (Life Technologies, Gaithersburg, MD) with 20 μg/ml lipofectin and an equal number expressing the CCR5 wild-type or mutant CCR5 plasmid. The cells are then 12 hours infected 2×107p.f.u. vTF7 (23) to enhance the expression of CCR5 was separated by a 2 mm ethylenediaminetetraacetic acid (EDTA) in PBS and once washed with a buffer for binding (1% BSA, 0.05% of NaN3in DPBS). The surface of the cells (2×106) were labeled mAb, as described in the previous paragraph, once washed with incubation buffer and 30 minutes resuspendable in 1 ml of 1× FACSlyse in water (Becton Dickinson) at room temperature for violations of the permeability of the cell membrane. The cells are then centrifuged, washed with incubation buffer and 1 hour at 37°C incubated with 4 μg/ml fluorescein labeled-isothiocyanato (FITC) mouse mAb against HA (BabCo, Richmond, CA) for intracellular labeling. In conclusion, cells once washed with the buffer for binding and once DPBS, resuspendable in 1% formaldehyde in PBS and analyzed by flow cytometry. The amount of bound peroxidase mutant CCR5 mAb was determined by equation (m.f.i. mutant CCR5 with PE / m.f.i. wt CCR5 with PE) / (m.f.i. mutant CCR5 with FITC /m.f.i. wt CCR5 with FITC) × l00%. It normalizes the binding of mAb for levels of expression of the mutant receptor.

4)analysis of the binding of gp120/sCD4

the gp120 biotinyl zerouali using NHS-Biotin (Pierce, Rockford, IL) according to manufacturer's instructions, and unbound Biotin was removed by diafiltration. Treated with sodium butyrate CCR5+-L1.2 cells for 1 hour at room temperature and incubated with different dilutions of equimolar mixture of sCD4 and biotinylated gp120 or 1.25 μg/ml sCD4 and 2.5 μg/ml biotinylated gp120 in the presence of various concentrations of mAb PA8-PA12, PA14, 2D7 against CCR5 or nonspecific mouse IgG1 in DPBS with 0.1% NaN3. Cells were washed with incubation buffer and 1 hour at room temperature, incubated with streptavidin-PE (Becton Dickinson)diluted 1:50. In conclusion, the cells were washed with buffer for binding and analyzed using reader fluorescence tablets (Perspective Biosystems, Framingham, MA).

5)Inhibition mediated membrane fusion cell-cell and entry of HIV-1 by mAb against CCR5

Mediated membrane of HIV-1 fusion cells HeLa-EnvJR-FL+and PM1 were determined using analysis of RET. Equal amounts (2×104) labeled fluorescein-octadecenoyl complex ether (F18), expressing the membrane of the cells and labeled with octadecylamine (R18) PM1 cells were placed in 96-well plates in 15% fetal calf serum in DPBS and 4 hours at 37°C incubated in the presence of various concentrations of mAb PA8-PA12, PA14, 2D7 against CCR5 or nonspecific mouse is x IgG1. Fluorescence in RET was measured using a Cytofluor plate reader (PerSeptive Biosystems), and the % RET was determined as described previously (38).

Viruses NLluc+env-supplemented intransthe glycoprotein shell of JR-FL or Gun-1, was obtained as described previously (20). Cells U87MG-CD4+CCR5+(14) in the presence of different concentrations of selected mAb infected chimeric reporter viruses containing 50-100 ng/ml p24. After 2 hours at 37°C containing the virus, the medium was replaced with fresh, containing mAb environment. After 12 hours was again added to fresh medium without antibodies. After 72 hours in total to the cells was added to 100 ál lyse buffer (Promega) and measured the luciferase activity (r.l.u.), as described (20). Percentage (%) inhibition of transmission of HIV-1 was defined as [1-(r.l.u in the presence of the antibody/r.l.u in the absence of antibody)] × 100%.

6)Analysis of signal transmission using calcium

To treated with sodium butyrate CCR5+-L1.2 cells were added fluorochrome Indo-1AM (Molecular Probes, Eugene, OR) at a final concentration of 5 μm. After 30 min incubation at 37°C cells once washed and resuspendable in the buffer saline Hanks. Cells (106sequentially stimulated with mAb against CCR5 or PBS and 60 C. RANTES. MAb PA8-PA12 and PA14 were used at a concentration of 100 µg/ml, 2D7 - at 20 µg/ml) and RANTES - at 250 ng/ml Inhibition of release of calcium by PA14 and 2D7 were also tested for wide is the first range of mAb concentrations, in the range from 0 to 100 μg/ml Monitoring intracellular levels of calcium was carried out using the fluorescence spectrophotometer Perkin-Elmer LS-50S by measuring the ratio of spuskanii fluorescence at 402 nm (associated dye) and 486 nm (free dye) after excitation at 358 nm.

B. Results and discussion

1)Selection of monoclonal antibodies against CCR5 PA8, PA9, PA10, PA11, PA12 and PA14

Found that the respective extracellular domains of CCR5 peptides ineffective to stimulate specific responses in the form of antibodies with high titers against native receptor on the cell surface (50). Therefore, Balb/C mice were immunized CCR5+-L1.2 cells, and supernatant hybrid cultures in the analysis of RET tested for their ability to inhibit mediated membrane JR-FL fusion of membranes with CD4+CCR5+-PM1 cells (19, 38). Although supernatant, in quantities significantly greater than a hundred, inhibited the fusion of cells by more than 50%, only six indicated PA8, PA9, PA10, PA11, PA12, and PA14, specifically and intensively stained CCR5+-L1.2, but not the parent L1.2 cells, as demonstrated by flow cytometry (data not shown). Based on previous experience suggested that the other is able to inhibit the fusion of cell-cell mAb probably directed about the Yves adhesion molecules on the cell surface, such as LFA-1 (37). By ittipiboon using ELISA (Cappell, Durham, NC) has determined that hybridoma PA8-PA12 and PA14 secrete IgG1 mAb. In Balb/C mice, which were injected with these six hybridomas were obtained ascitic fluid was purified fractions IgG1. PA8, PA9, PA11, PA12 and PA14 showed different profiles in isoelectric focusing, while PA10 had a very similar profile to the profile PA9 and, therefore, may represent a second isolate of the same mAb (data not shown).

2)Binding of MAb to CCR5+cells

None of purified mAb against CCR5 were not stained with the parent cell line L1.2 (data not shown). However, mAb PA9-PA12 and PA14 were stained with >90%, and PA8 stained ≈70%, CCR5+-L1.2 cells as determined by flow cytometry, demonstrating that they recognize CCR5 (figure 1). mAb against CCR5 2D7, who served in the experiments of the authors of the present invention control, also stained >90% CCR5+-L1.2 cells. PA8-PA12 and PA14 all are IgG1 and equally well interact with goat antibodies against mouse IgG, whereas 2D7 represents IgG2a and can interact with reporter antibody differently. Therefore, directly comparable are the average fluorescence intensity (m.f.i.) only mAb PA8-PA12 and PA14. The rank order of average intensities of fluorescence intensity (m.f.i.) not only is l a PA12 ≈ PA11 > (2D7=) PA14 ≈ PA10 ≈ PA9 > PA8. The difference between the m.f.i. PA12 and m.f.i. PA8 was threefold. Differences in the intensity of staining between PA8 and other mAb remained constant in a wide range of concentrations (data not shown) and probably do not correspond to differences in offendeth mAb to CCR5. This means that PA8 interacts only with a subset of CCR5 molecules present on the surface CCR5+-L1.2 cells.

Compared with CCR5+-L1.2 cells mitogen-stimulated PBMC showed different profiles staining by mAb against CCR5. 2D7 and PA14 were stained with >20%, PA11 and PA12 stained ≈10%, PA8, PA9 and PA10 stained <5% of PBMC (figure 1). The average intensity of fluorescence stained PBMC were about ten times lower than the average fluorescence intensity obtained with CCR5+-L1.2 cells for each mAb; their rank order were (2D7 >) PA14 > PA12 ≈ PA11 ≈ PA10 ≈ PA9 ≈ PA8. This again was a little bit different from the order of reactivity observed for CCR5 transfectants. The difference between the m.f.i. PA9 and m.f.i. PA14 was seven. Other groups have observed similar differences in the ability of mAb against CCR5 to paint stable CCR5+cells compared to PBMC (28). This can occur due to specific cells of the differences in conformation, posttranslational modifications or CCR5 oligomerization. lternative, for different cells may be different Association with other molecules on the cell surface. As the obvious choice for such a molecule could be a cell surface antigen CD4, absent on CCR5+-L1.2 cells present in PBMC, the authors present invention also tested the ability PA8-PA12, PA14 and 2D7 to stain HeLa cells, transtorno expressing CCR5 separately or together with CD4. The ability of any of the mAb to paint CCR5 on the cell surface in the presence of CD4 differences were not observed (data not shown). If there is an Association between these two properties, it is not involved epitopes, identified available the authors present invention mAb against CCR5. Alternatively, the Association between CCR5 and CD4 can only exist in primary lymphocytes.

3)Mapping of mAb epitopes using alanine mutant CCR5

None of the antibodies were not able to detect reduced and denatured protein CCR5 in Western-blotting, indicating that they recognize conformation-dependent epitopes (data not shown). Using a panel of seventy-alanine point mutants in residues in Nt and ECL CCR5 conducted research on the mapping of mAb epitopes. HeLa cells were subjected to lipofectin coding sequences of the mutant CCR5 or CCR5 wild type with the relax is nymi C-the end of the HA-tagged and infected vTF7 (23) to increase expression of the coreceptor. Then cells were incubated with mAb against CCR5, and their binding was detected by PE labeled goat antibody against mouse IgG. Secondly, performed intracellular staining with FITC labeled mAb against HA (BabCo). This internal control has allowed immediate normalization of staining by mAb against CCR5 levels of expression of mutant receptor on the cell surface. Therefore, the binding of mAb with each mutant is expressed as a percentage of binding to CCR5 wild-type (figure 4).

Certain point mutations reduced the binding of all antibodies to CCR5 by more than 50%. Typically, this class of mutants, including mutants pair cysteine C101A and C178A, mutants Y10A, D11A, K25A in Nt, mutants D95A in ECL1, mutants K171A/E172A, Q188A, K191A/N192A in ECL2, and mutants F263A and F264A in ECL3, had the biggest influence on PA8-PA12 and less influenced PA14 and 2D7 (figure 1). One interpretation is that these residues are not part of the mAb epitopes, but changing them to alanine residues causes conformational perturbations, providing the overall effect on the binding of all mAb. The authors of this invention have assumed that if the mutation reduced the binding of individual mAb more than 75%, and did not reduce the binding of most of the other antibodies, the rest probably was a direct participant epitopes are detected the data by mAb. Using these simple principles, concluded that seven mAb against CCR5 recognize overlapping, but different epitopes (figure 4). Binding of MAb PA8 with CCR5 depended on N13 and Y15 in the Nt. For MAb PA9 and PA10 needed was D2, Y3, Q4, P8 and N13 in the Nt, and Y176 and T177 in ECL2. For MAb PA9 was necessary S7 in the Nt. Binding of MAb PA11 and PA12 depended on Q4 in the Nt. For PA14 was necessary to D2 in the Nt and R168 and Y176 in ECL2. Finally, mAb 2D7 to contact CCR5 needed was Q170 and K171/E172 in ECL2.

4)Signaling by chemokines in the presence of mAb against CCR5

Binding receptors chemokines funds may be antagonists or, more rarely, agonist-mediated receptor signaling inside the cell. Alternatively, they may not have any effect on signal transmission. CCR5 is capable of binding with three CC-chemokines, RANTES, MIP-1α and MIP-1βand to transmit a signal regulating cytosolic calcium levels. Therefore, the authors of this invention have tested the activity of different concentrations of mAb PA8-PA12, PA14 and 2D7 as agonists/antagonists. Changes in intracellular calcium concentrations were measured in CCR5+-L1.2 cells to put them in Indo-1. The absence of stimulation by mAb changes (Ca2+i meant that they are not CCR5 agonists. PA8-PA12 was also unable to inhibit the release of Ca2+, inducere the config RANTES (figure 5 and data not shown), even at such high concentrations as 100 μg/ml, demonstrating that they are not antagonists. These concentrations provide a saturating binding of mAb to CCR5+-L1.2 cells as shown by flow cytometry and analysis of binding gp120/CCR5 (figure 6D and data not shown). However, mAb PA14 and 2D7 blocked induced RANTES mobilization of calcium, although with different efficiencies (figures 5A, 5B). IC50for inhibiting release of calcium by PA14 was 50 µg/ml, which is approximately 8 times higher than the IC50for 2D7 (figure 5B). Each of the induced RANTES, MIP-1α and MIP-1β outputs calcium inhibited by similar concentrations of PA14 (data not shown). None of the mAb had no effect on induced SDF-1 mobilization of calcium in CCR5+-L1.2 cells, expressing endogenous CXCR4 (data not shown). Finally, neither mAb or CC-chemokines did not affect cytosolic levels of calcium in the parent L1.2 cells (data not shown).

5)Inhibition by mAb functions CCR5 as coreceptor

MAb PA8-PA12 and PA14 initially selected based on their ability to inhibit mediated membrane of HIV-1 fusion cells. This activity confirmed and measured for purified mAb. As expected, all six mAb and mAb 2D7 blocked the merger of PM1 cells CD4+CCR5+and cells HeLa-EnvJR-FL+in the analysis of RET. The rank order of effectiveness was 2D7 ≈ PA14 > PA12 > PA11 > PA10 ≈ PA9 ≈ PA8 (figure 6A). The values of the IC50for PA14 and 2D7 was 1.7 μg/ml and 1.6 µg/ml, respectively, for PA11 and PA12 they accounted for 25.5 μg/ml and 10.0 μg/ml, respectively (figure 3). PA8, PA9 and PA10 at 300 µg/ml inhibited the fusion of only 10-15%. None of the mAb had no effect on cell fusion PM1 cells and HeLa-EnvLAI+expressing full-sized envelope protein of the virus X4 (data not shown).

Also tested the ability of various mAb against CCR5 inhibit the penetration of prototypical R5 virus, JR-FL and R5X4 virus, Gun-1 in one round, based on replication luciferase analysis of penetration. The rank order of effectiveness in the analysis of penetration was similar to the procedure defined in the analysis of merge cells (figure 6B). To get more than 50% inhibition of penetration of JR-FL or Gun-1 using PA8-PA11 was impossible. The value of the IC50for PA12 was 2.5 µg/ml, However, to obtain the inhibition of penetration when using this mAb more than 60% failed. Has determined that the value of the IC50for inhibiting the penetration of JR-FL for PA14 and 2D7 were 0,024 and was 0.026 µg/ml, respectively (figure 3) and was 60 times smaller than the values of the IC50obtained in the analysis of mergers. Penetrated owenie Gun-1, having double trapnest was 2-3 times more sensitive to inhibition by mAb against CCR5 than the penetration of JR-FL (data not shown).

MAb against the receptor can inhibit mediated membrane fusion, or directly affecting the interaction of gp120/CCR5 or preventing stages after binding, involved in the formation of an active complex to merge. To determine the mechanism of inhibition of adhesion with the virus and penetration of the virus through PA8-PA12 and PA14 tested the ability of various mAb to block the interaction of gp120/CCR5. Was used for this analysis, which defines the binding to CCR5+-L1.2 cells biotinylated gp120 of HIV-1JR-FLin complex with sCD4. Binding of biotinylated gp120 in the absence of sCD4 or CCR5, or when used gp120 of HIV-1LAInot observed (figure 6C).

Except PA8 all mAb completely broke the binding of gp120/sCD4 with CCR5+-L1.2 cells (Fig. 6D). Inhibition by PA8 came to ≈40%, which coincides with the data of flow cytometry (figure 1) under the assumption that this mAb binds to only a subset of CCR5 molecules on CCR5+-L1.2 cells. MAb PA9, PA10, PA11 and PA12 inhibited binding with values IC50the components of 0.24, 0,13, 0,33, of 0.24 µg/ml, respectively (figure 3). Suddenly mAb PA14 and 2D7 were the two least effective inhibitors of the binding of gp120/sCD4 with values IC 50constituting 1,58 and 1.38 µg/ml, respectively (figure 3). Therefore, the correlation between the ability of mAb mediated inhibition of gp120/CD4/CCR5 fusion of membranes and penetration and its ability to block the binding of gp120/sCD4 with coreceptor did not exist.

6)Synergistic inhibition of fusion with HIV-1 by the combination of mAb against CCR5 and other inhibitors of viral penetration

Specific to the receptor means can operate at many stages of the process of penetration of the virus and to show non-additive effects when used in combination. Based on the perspectives for clinical practice, it is important to determine the interaction of specific forms of drug candidates with the endogenous chemokines that can provide some level of protection against development of the disease. Therefore, mAb against CCR5 tested in combination with each other mAb or with RANTES or CD4-IgG2 binding to gp120 of HIV-1, for inhibiting the adherence to target cells. These tools, used individually or in combination, in the analysis of merger or penetration got curves dose-response. Data were analyzed using the principle of the median effect (9). The concentration of individual or combinations thereof, required to obtain this effect quantitatively compared using conditions known the spas as the combination index (CI). The value of CI greater than 1 indicates antagonism, CI≈1 indicates an additive effect, and CI<1 indicates a synergistic effect, where the presence of one means increases the effect of another.

The combination of PA12 and 2D7 were the most synergistic, with CI values ranging from 0.02 to 0.29, depending on the ratio of antibody (figure 7 and figure 2). It is known that the degree of synergy varies depending on the stoichiometric ratios of the funds. The results of the merge with the virus and penetration of the virus in identifying combinations of mAb, which vysokoenergeticheskie, PA12 and 2D7; moderately synergistic, PA12 and PA14; additive, PA11 and PA12, and weakly antagonistic, PA14 and 2D7, as a rule, coincide. The lack of synergy between PA14 and 2D7 is not unexpected, as these mAb mutually compete for binding to CCR5+cells as determined by flow cytometry (data not shown). Observation additive effect between PA11 and PA12 may be an indication that the data mAb bind to CCR5 with slightly different epitopes, thus separating the dependence of the balance Q4 Nt.

Also tested the ability of mAb PA12, PA14 and 2D7 synergy with RANTES in blocking the merger of cells. The combination of PA12 and RANTES showed moderate synergy (figure 2). PA14 and 2D7 did not show synergy with RANTES, which is consistent with the fact that these mAb ablauts the inhibitor for binding and signal transmission RANTES (figure 5A, 5B). Finally, the authors present invention tested the synergy between mAb PA12, PA14, 2D7 and CD4-IgG2, which interact with gp120. The authors of this invention have observed moderate synergy between PA12 and CD4-IgG2, but the lack of synergy between PA14 or 2D7 and CD4-IgG2 (figure 2).

Discussion experiment

Isolated and characterized six murine IgG1 mAb against CCR5. While PA8, PA9, PA11, PA12 and PA14 are separate molecular species, PA9 and PA10 was indistinguishable through analyses and, therefore, probably are one and the same mAb. All selected mAb recognize complex conformational epitopes, which is often the case with mAb raised against native proteins on the cell surface. Mapping of epitopes was performed for all mAb using panel alanine point mutant CCR5. Suggested that the remains, similarly affecting the binding of all mAb, cause conformational perturbations in coreceptor and are not part of the epitope mAb data. It is shown that only two such balance, Y10 and D11, affect the penetration of HIV-1 (20, 52). Epitopes PA8, PA11 and PA12 are located exclusively in the Nt domain. In agreement with this result in the analysis of ELISA PA8 was able to communicate with biotinylated peptide Nt containing residues from D2 to R31 (data not shown). However, PA11 and PA12, linking which strictly depends only on 4, not contacted with the peptide Nt in solution (data not shown). One possible explanation is that the peptide Nt does not accept the correct conformation for recognition by PA11 and PA12, while linking PA8 may be less dependent on conformation. Alternatively, PA11 and PA12 may interact with those residues that have not been subjected to mutagenesis by the authors of the present invention, or form weak links with amino acids that are located in other domains of CCR5, or contact the atoms in the peptide chain, the presentation of which was not changed during the mutagenesis. Antibodies PA9, PA10 and PA14 recognize epitomy comprising residues in Nt and ECL2 domain CCR5, whereas the epitope 2D7 was located exclusively in the ECL2 domain.

Epitope PA14 contains and D2 in the Nt, and R168 in ECL2, which means that these two residue located close to one another according to the results of Footprinting mAb. They even can directly interact with one another by means of their opposite charges.

MAb PA8-PA12 and PA14 were stained with CCR5+cells with different intensities, depending on the cell type way. All mAb, except PA8, dyed in more than 90% of CCR5+-L1.2 cells, with the highest average intensity of fluorescence observed for PA11 and PA12. However, PA14 and 2D7 were stained with the highest percentage of PBMC, and created the greatest average interest is cunosti fluorescence of these cells. Hill et al. (28) have recently characterized a panel of mAb against CCR5, which similarly stained transfetsirovannyh cells, but only two of the eight were stained PBMC and none were stained with primary monocytes. Low affinity to CCR5, probably due to the lack of interaction between the two of mAb with primary cells, but it is unlikely that this is the explanation of the failure to response for the other four. In panel mAb authors of the present invention, the authors observed the most intense staining of PBMC by mAb 2D7 and PA14 with epitopes located entirely or partially in the first ten ECL2 residues. However, Hill et al. reported that are specific to Nt and ECL1 mAb stained PBMC, whereas specific to ECL2 and ECL3 mAb is not stained PBMC, so that a consistent profile of reactivity is not identified. One explanation for the type-dependent cell staining by mAb can be activated PBMC (and monocytes) secrete CC-chemokines that bind to CCR5 on the cell surface, masking some of the mAb epitopes. However, it can be expected that this will be especially true for PA14 and 2D7, which are antagonists induced cytokine mobilization of calcium and possibly, compete with the CC-chemokines for binding to CCR5. However, these mAb stained PBMC with the greatest intensity. Alternatively, different the exposure of the epitope CCR5 may reflect specific to cell type oligomerization of the receptor, Association with other molecules on the cell surface or different post-translational modifications such as glycosylation. The authors of this invention have shown that differences in the binding of mAb, probably do not reflect the specific types of cells, differences in the interactions of CD4/CCR5.

MAb PA8-PA12 not inhibited induced CC-chemokines mobilization of calcium in CCR5+cells and did not oposredovany signaling through CCR5. MAb 2D7 and PA14 were inhibitors induced CC-chemokines mobilization of calcium, but 2D7 was almost an order of magnitude more efficient than PA14. This can occur because the epitope PA14 less overlaps with the binding of CC-chemokines domain on CCR5 than the epitope 2D7. All data mAb also blocked the penetration of HIV-1-mediated membrane of HIV-1 fusion of membranes, but the inhibition of fusion of cells in some cases demanded by almost two orders of magnitude more antibodies than was necessary to block the penetration of the virus. It is likely that during the merging of cells is established and operates in conjunction greater number of interactions of gp120/CD4/CCR5, as well as interactions between adhesion molecules, compared with the fusion of the virus-cell, making it more difficult for inhibition. Generally, it is observed for antibodies to LFA-1 or to the glycoprotein shell of the HIV-1 (45, 51). PA8, PA9 and PA10 AC is ALIS unable to block the merger of cells by more than 15% and penetration of the virus by more than 40% even at the highest concentrations of antibodies. However, more than 90% inhibition of fusion was achieved with the use of PA11, PA12, and PA14, and more than 90% inhibition of penetration was achieved with the use of PA14. The most effective of the six mAb in blocking mergers and penetration was PA14, which was as effective as 2D7. Suddenly, PA14 and 2D7 were one of the least effective inhibitors of binding of gp120/sCD4 with CCR5+-L1.2 cells, whereas PA9-PA12 blocked with similar efficiencies, and PA8 was unable to block more than 40% of the binding of gp120/sCD4. These observations raise questions about the nature of CCR5 molecules presented on different cells, and the mechanisms of inhibition of adhesion with the virus and virus attack. It may be that CCR5 on the L1.2 cells used in the assays of the binding of mAb and gp120, is not in a conformation identical to the conformation of CCR5 in PBMC used for analysis of binding of mAb, or conformation of CCR5 on the PM1 cells and U87MG used for analyses of fusion and penetration.

Weak staining of PBMC and partial inhibition of fusion and penetration of some of the mAb authors of the present invention indicates that they are able to communicate with only a subset of CCR5 molecules expressed on cell lines and primary lymphocytes, PM1 and U87MG-CD4+CCR5+. However, all mAb, except PA8 are able okra is tested more than 90% of CCR5 +-L1.2 cells and completely block the binding of a complex of gp120/sCD4 with these cells. At least one difference between CCR5+-L1.2 cells and other cells, which were used by the authors of the present invention, is the density protein receptor on the cell surface. Indeed, the authors of this invention have determined that CCR5+-L1.2 cells Express a number of coreceptor on the cell surface from 10 to 100 times greater than the PM1 cells and U87MG-CD4+CCR5+. But when HeLa cells were designed so that transtorno to Express the same number of receptor as CCR5+cell line L1.2, the authors present invention still could not detect binding of gp120/sCD4 with them (data not shown). Therefore, overexpression of CCR5 on L1.2 together with other specific cell factors may favor the conformation of the receptor which exhibits markedly Nt, making it more accessible to mAb, and for gp120. Such a conformation can induce oligomerization of the receptor, weakened or altered in Association with proteins on the cell surface or the interaction of the receptor with G-proteins (25, 62). Can multiple conformations CCR5 co-exist at the cell surface, and whether they contribute to the penetration of the virus? Profiles of reactivity mAb allow predpolojite such since in the presence of mAb concentrations, saturating the epitopes required for the binding of gp120 to CCR5+-L1.2 cells, can occur penetration of HIV-1 and fusion of HIV-1, although at lower levels. The authors of the present invention support the hypothesis that the CCR5+-L1.2 cells coreceptor molecules have the same conformation, suitable for entry of HIV-1, whereas the CCR5 molecule on PBMC, PM1 and CCR5+U87MG are just suitable for penetration of the States, which demonstrates the different reactivity of the mAb. While PA14 and 2D7 can recognize all conformations, other mAb can't. Why L1.2 cells contribute to specific and appropriate to merge conformation, is still needed to find out.

It was recently shown that the domain of binding to gp120 lies in the first twenty residues Nt-domain of CCR5. MAb to the domain binding to gp120 to CCR5 effectively block this interaction, but they are not equally effective in the inhibition of fusion with HIV-1 and entry of HIV-1 into target cells, as PA14 and 2D7, epitopes which lie outside this area. PA14 recognizes the end of the Nt and residues in ECL2, whereas the epitope 2D7 is located exclusively in ECL2. On the mechanism of action of these mAb can only speculate. It may be that linking them with the first few ECL2 residues induces conformational changes in the receptors, that prevents the fusion of membranes. Alternatively, blockade of epitopes in ECL2 can prevent oligomerization of the receptor and the formation of a suitable fusion protein complex. Another possibility is that the balances on ECL2 facing the inside of the pores of the merger, and the binding of mAb inhibits gp41 to implement fusion protein in the plasma membrane. Opposite mAb PA8-PA12 probably inhibit fusion and penetration only through the creation of direct obstacles to associate with complexes of gp120/CD4. The authors of this invention have no information about whether other than the exposure of the epitope and affinity to CCR5, the parameters of the efficiency of inhibition of viral penetration data mAb. It remains unclear why the inhibition subsequent to the interaction of gp120/coreceptor stages may be more effective than direct blocking of this interaction. One possible explanation for this may be the assumption that the reduction of binding of gp120 to CCR5 is substantially less than the increase in the binding of mAb to CCR5. Thus, every time when mAb is separated from the molecules of the receptor associated with the virion gp120 molecule replaces it casinoplatinum way, because each interaction leads to the fusion of membranes.

Synergy between combinations of mAb against CCR5, probably, PR is dstanley a result of their interaction with different epitopes, involved in mutually independent successive stages of entry of HIV-1. The degree of the observed synergy between PA12 and 2D7 (CI<0,1 for many conditions) is unusual, because the values CI<0.2 for combinations of antibodies against HIV-1 (33, 35, 61), reverse transcriptase inhibitors (29) or protease inhibitors (44) observed rarely. Because of its efficiency combination PA12:2D7 investigated in many types of analyses and in many ratios of concentrations that were consistently observed high levels of synergy. Moderate synergy was observed for PA12, combined with PA14. The authors of this invention have observed moderate synergy between PA12 and CD4-IgG2. Complex CD4/gp120 metastable and, if he is not able to interact with the receptor, falls to the state, not able to merge (45-48). As PA12 directly blocking the binding site of gp120 to CCR5, his presence can shift the equilibrium towards the inactivation of the complex gp120/CD4. This may explain why the authors of this invention have observed synergy between CD4-IgG2 and mAb PA12 with respect to inhibition of fusion and penetration. The lack of synergy between mAb PA14 and CD4-IgG2 suggests that they act on two non-consecutive and independent stages of an infection. To determine the exact mechanisms of synergy between the various compounds in relation to inhibition with whom iania with the virus and penetration of virus required complex further research.

The above results are consistent with a model in which the penetration of HIV-1 occurs in three different stages, including receptor binding, the binding of receptor and receptor-mediated fusion of membranes. To assume a separate stage of the coreceptor binding and merge the lack of correlation between the ability of monoclonal antibodies to block the binding of gp120 and fusion/penetration of HIV-1. The chronology of events during the merge additionally suggest the profiles of the observed synergy. Tools such as PA12, effectively inhibiting the mid stage of the process, i.e. the binding of gp120, act synergistically with inhibitors of the previous or subsequent stages.

Example 2

Prerequisites:The increased proportion of drug polyresistance HIV-1 makes the search for new classes of anti-retrovirus. CCR5 is a need to merge primary isolates of HIV-1 co-receptor is a promising target for antiviral therapy. PRO140 is a monoclonal antibody against CCR5, effectively inhibiting penetration and replication of HIV-1 at concentrations that do not affect the activity of the receptor for chemokines CCR5in vitro. In this study the authors of this invention have evaluated therapeutic potential of PRO140in vivousing t rapeutically models of infection of HIV-1 on animals.

Methods:Mice CD-17 SCID restored normal human PBMC and infected with the R5 isolate HIV-1JR-CSF. When reaching the stationary state of the virus in animals administered intraperitoneally was subjected to PRO140, or control antibodies and watched viral load using Roche Amplicor Assay. In initial studies tested a single dose of 1 mg PRO140. In the study of multiple doses of PRO140 was administered once every three days for three weeks at doses comprising from 0.1 to 1.0 mg. In a separate experiment to test the possibility of reducing the number of lymphocytes after injection PRO140 was used flow cytometry.

The results:And single and multiple doses of PRO140 reduced viral load to undetectable levels in all affected animals, and a reduction in viral load was in the range of up to 1.8 log 10. After a single injection PRO140 watched temporary control of virus replication, whereas repeated injections resulted in long-lasting control without signs of recovery of the virus during treatment. Observed dose-dependent differences in the kinetics mediated PRO140 reductions in viral load. Analysis of flow cytometry showed that treatment by PRO140 did not lead to a reduction in the number of lymphocytes, confirming that the effect on the viral bulk packing : spindle is the situation in vivohappened only because of the blockade of CCR5.

Conclusions:PRO140 is highly effective for control obtained in models of infection of HIV-1 mice (hu-PBL-SCID infection of HIV-1. These facts provide crucial experimentsin vivofor treatment through PRO140, in particular, for treatment with a CCR5 inhibitor, in General.

Example 3

Methods:

Humanitariannet antibody against CCR5 (huPRO140) tested for the ability to block induced RANTES mobilization of calcium in CCR5+-L1.2 cells and block the replication of HIV-1 CASE C 1/85 in human PBMC using methods described here.

The results:The results, shown in figure 19, show that humanitariannet antibody against CCR5 effectively blocks HIV-1, but not RANTES.

LINKS

1. Allaway, G.P., K.L. Davis-Bruno, B.A. Beaudry, E.B. Garcia, E.L. Wong, A.M. Ryder, K.W. Hasel, M.C. Gauduin, R.A. Koup, J.S. McDougal and P.J. Maddon. 1995. Expression and characterization of CD4-IgG2, a novel heterotetramer that neutralizes primary HIV type 1 isolates. AIDS Res Hum Retroviruses11:533-539.

2. Allaway, G.P., A.M. Ryder, G.A. Beaudry and P.J. Maddon. 1993. Synergistic inhibition of HIV-1 envelope-mediated cell fusion by CD4-based molecules in combination with antibodies to gp120 or gp41. AIDS Res Hum Retroviruses9:581-587.

3. Amara, A., S.L. Gall, O. Schwartz, J. Salamero, M. Montes, P. Loetscher, M. Baggiolini, J.L. Virelizier and F. Arenzana-Seisdedos. 1997. HIV coreceptor downregulation as antiviral principle: SDF-1a-dependent internalization of the chemokine receptor CXCR4 contributes to inhibition of HIV replication. J. Exp. Med.186:139-146.

4. Berger, E.A. 1997. HIV entry and tropism: the chemokine receptor connection. ADS 11 (suppl A):S3-S16.

5. Bieniasz, P.D. and B.R. Cullen. 1998. Chemokine receptors and human immunodeficiency virus infection. Frontiers in Bioscience3:d44-58.

6. Bieniasz, P.D., R.A. Fridell, I. Aramori, S.S.G. Ferguson, M.C. Caron and B.R. Cullen. 1997. HIV-1-induced cell fusion is mediated by multiple regions within both the viral envelope and the CCR5 co-receptor. EMBO16:2599-2609.

7. Brelot, A., N. Heveker, O. Pleskoff, N. Sol and M. Alizon. 1997. Role of the first and third of increasing interest among domains of CXCR4 in human immunodeficiency virus coreceptor activity. J. Virol.71:4744-4751.

8. Chan, V.S. and P.S. Kim. 1998. HIV entry and its inhibition. Cell93:681-684.

9. Chou, T.C. and B.C. Rideout. Synergism and antagonism in chemotherapy. New York: Academic Press, 1991

10. Cocchi, F., A.L. DeVico, A. Garzino-Berno, S.K. Arya, R.C. Gallo and P. Lusso. 1995. Identification of RANTES, MIP-1α and MIP-1β as the major HIV-suppressive factors produced by CD8 T - cells. Science270:1811-1815.

11. Connor, R.I., K.E. Sheridan, D. Ceradini, S. Choe and N.R. Landau. 1997. Change in co-receptor use correlates with disease progression in HIV-1 infected individuals. J. Exp. Med.185:621-628.

12. Crump, M.P., J.H. Gong, P. Loetscher, K. Rajarathnam, A. Amara, F. Arenzana-Seisdedos, J.L. Virelizier, M. Baggiolini, B.D. Sykes and I. Clark-Lewis. 1997. Solution structure and basis for functional activity of stromal-cell derived factor-1; disassociation of CXCR4 activation from binding and inhibition of HIV-1. EMBO16:6996-7007.

13. Dalgleish, A.G., P.C.L. Beverly, P.R. Clapham, D.H. Crawford, M.F. Greaves and R. A. Weiss. 1984. The CD4 (T4) antigen is an essential component of the receptor for the AIDS retrovirus. Nature312:763-766.

14. Deng, H.K., R. Liu, W. Ellmeier, S. Choe, D. Unutmaz, M. Burkhart, P. DiMarizio, S. Marmon, R.E. Sutton, C.M. Hill, S.C. Peiper, T.J. Schall, D.R. Littman and N.R. Landau. 1996. Identification of a major co-receptor for primary isolates of HIV-1. Nature381:661-666.

15. Dimitrov, D.S. 1997. How do viruses enter cells? The HIV Co-receptors teach us a lesson of complexity. Cell91:721-730.

16. Donzella, G.A., D. Schols, S.W. Lin, K.A. Nagashim, P.J. Maddon, G.P. Allaway, T.P. Sakmar, E.D. Clercq and J.P. Moore. 1998. JM3100, a small molecule that interacts with the CXCR4 co-receptor to prevent HIV-1 entry. Nat. Med.4:72-77.

17. Doranz, B.J., K. Grovit-Ferbas, M.P. Sharron, S.H. Mao, M. Goetz, E.S. Daar, R.W. Doms and W.A. O'brien. 1997. A small molecule inhibitor directed against the chemokine receptor CXCR4 prevents its use as an HIV-1 co-receptor. J. Ex. Med.186:1395-1400.

18. Doranz, B.J., Z.-H. Lu, J. Rucker, T.-Y. Zhang, M. Sharron, Y.-H. Cen, Z.-X. Wang, H.-H. Guo, J.-G. Du, M.A. Accavitti, R.W. Doms and S.C. Peiper. 1997. Two distinct CCR5 domains can mediate co-receptor usage by human immunodeficiency virus type 1. J. Virol.71:6305-6314.

19. Dragic, T., Litwin V., G.P. Allaway, S.R. Martin, Y. Huanh, K.A. Nagashima, C. Cayanan, P.J. Maddon, R. A. Koup, J.P. Moore and W.A. Paxton. 1996. HIV-1 entry into CD4+ cells is mediated by the chemokine receptor CC-CKR-5. Nature381:667-673.

20. Dragic, T., A. Trkola, X.W. Lin, K.A. Nagashima, F. Kajumo, L. Zhao, W.C. Olson, L. Wu, C.R. Mackay, G.P. Allaway, T.P. Sakmar, J.P. Moore and P.J. Maddon. 1998. Amino terminal substitutions in the CCR5 co-receptor impair gp120 binding and human immunodeficiency virus type 1 entry. J. Virol.72:279-285.

21. Dragic, T., A. Trkola and J.P. Moore. 1997. HIV co-receptors: Gateways to the cell. Advances in Research and Therapy7:2-13.

22. Farzan, M., H. Choe, L. Vaca, K. Martin, Y. Sun, E. Desjardins, N. Ruffing, L. Wu, R. Wyatt, N. Gerard, C. Gerard, and J. Sodroski. 1998. A tyrosine-rich region in the N-terminus of CCR5 is important for human immunodeficiency virus type l entry and mediates an association between gp120 and CCR5. J. Virol.72:1160-1164.

23. Fuerst, T.R., E.G. Niles, F.W. Studier and B. Moss. 1986. Eukaryotic transient-expression system based on recombinant vaccinia virus that synthesizes bacteriophage T7 RNA polymerase. Proc. Natl. Acad. Sci. USA.83:8122-8126.

24. Genoud, S., F. Kajumo, Y. Guo, D.A.D. Thompson and T. Dragic. CCR5-mediated human immunodeficiency virus entry depends on an amino-terminal domain of gp120-binding site and on the conformational integrity of all four of increasing interest among domains. J. Virol. submitted.

25. Gether, U. and B.K. Kobilka. 198. G protein - coupled receptors. J. Biol. Chem273:17979-17982.

26. Gordon, C., M. Muesing, A.E.I. Proudfoot, C.A. Power, J.p. Moore and A. Trkola. 1998. Enhancement of human immunodeficiency virus type 1 infection by the CC-chemokine RANTES is independent of the mechanism of virus-cell fusion. J. Virol. in press.

27. Heveker, N., M. Montes, L. Germeroth, A. Amara, A. Trautmann, M. Alizon and J. Schneider-Mergener. 1998. Dissociation of the signaling and antiviral properties of SDF-1-derived small peptides. Current Biology8:369-376.

28. Hill, C.M., D. Kwon, M. Jones, C.B. Davis, S. Marmon, B.L. Daugherty, J.A. DeMartino, M.S. Springer, D. Unutmaz and D.R. Littman. 1998. The amino terminus of human CCR5 is required for its function as a receptor for diverse human and simian immunodeficiency virus envelope glycoproteins. Virology248:357-371.

29. Johnson, V.A., D.P. Merrill, J.A. Videler, T.C. Chou, R. E. Byington, J.J. Eron, R.T. D'aquila and M. S. Hirsch. 1991. Two-drug combinations of zidovudine, didanosine, and recombinant interferon-alpha A inhibit replication of zidovudine-resistant human immunodeficiency virus type 1 synergistically in vitro. J Infect Dis164:646-655.

30. Klatzmann, D., E. Champagne, S. Chamaret, J.M. Gruest, D. Guetard, T. Hercend, J.C. Gluckman and L. Montagnier. 1984. T-lymphocyte T4 molecule behaves as the receptor for human retrovirus LAV. Nature312:382-385.

31. Kuhmann, K.E., E.J. Platt, S.L. and D. Kozak Rabat. 1997. Polymorphism in the CCR5 genes of African green monkeys and mice implicate specific amino acids in infections by simian and human immunodeficiency viruses. J. Virol.71:8642-8656.

32. Kwong, P.D., R. Wyatt, J. Robinson, R.W. Sweet, J. Sodroski and W.A. Hendrickson. 1998. Structure of an HIV gp120 envelope glycoprotein in complex with the CD4 receptor and a neutralizing human antibody. Nature393:648-659.

33. Laal, S., S. Burda, M.K. Corny, S. Karwowska, A. Buchbinder and S. Zolla-Pazner. 1994. Synergistic neutralization of human immunodeficiency virus type l by combinations of human monoclonal antibodies. J. Virol.68:4001-4008.

34. Labrosse, B.,A.Brelot, N. Heveker, N. jSol, D. Schols, E.D. Clercq and M. Alizon. 1998. Determinants for sensitivity of human immunodeficiecy virus co-receptor CXCR4 to the bicyclam AMD3100. J. Virol.72:6381-6388.

35. Li, A., H. Katinger, M.R. Posner, L. Cavacini, S. Zolla-Pazner, M.K. Gorny, J. Sodroski, T.C. Chou, T.W. Baba and R. Ruprecht. 1998. Synergistic neutralization of simian-human immunodeficiency virus SHIV-vpu+ by triple and quadruple combinations of human monoclonal antibodies and high-titer anti-human immunodeficiency virus type 1 immunoglobulins. J. Virol.72:3235-3240.

36. Littman, D.R. 1998. Chemokine receptors: keys to AIDS pathogenesis. Cell93:677-680.

37. Litwin, V., unpublished results.

38. Litwin, V., K. Nagashima, M. Ryder, C.H. Chang, J.M. Carver, W.C. Olson, M. Alizon, K.W. Hasel, P.J. Maddon and G.P. Allaway. 1996. Human immunodeficiency virus type 1 membrane fusion mediated by a laboratory-adapted strain and a primary isolate analyzed by resonance energy transfer. J. Virol.70:6437-6441.

39. Loetscher, P., J.H. Gong, B. Dewald, M. Baggioloni and I. Clark-Lewis. 1998. N-terminal peptides of stromal cell derived factor-l with CXC chemokine receptor 4 agonist and antagonist activities. J. Biol. Chem.273:22279-22283.

40. Mack, M., B. Luckow, P.J. Nelson, J. Cihak, G. Simmons, P.R. Clapham, N. Signoret, M. Marsh, M. Stangassinger, F. Borlat, T.N.C. Wells, D. Schlondorff and A.E.I. Proudfoot. 1998. Aminooxypentane-RANTES dosage CCR5 internalization but inhibits recycling: a novel inhibitory mechanisms of HIV infectivity. J. Ex. Med.187:1215-1224.

41. Maddon, P.J., A.G. Dalgleish, J.S. McDougal, P.R. Clapham, R. A. Weiss and R. Axel. 1986. The T4 gene encodes the AIDS virus receptor and is expressed in the immune system and the brain. Cell47:333-348.

42. McDougal, J.S., M.S. Kennedy, J.M. Sligh, S.P. Cort, A. Mawle and J.K.A. Nicholson. 1986. Binding of HTLVIII / LAV to T4+T cells by a complex of the 110K viral protein and the T4 molecule. Science231:382-385.

43. McKnight, A., D. Wilkinson, G. Simmons, S. Talbot, L., Picard, M. Ahuja, M. Marsh, J.A. Hoxie and P.R. Clapham. 1997. Inhibition of human immunodeficiency virus fusion by a monoclonal antibody to a co-receptor (CXCR4) is both cell type and virus strain dependent. J. Virol.71:1692-1696.

44. Merrill, D.P., D.J. Manion, T.C. Chou and M. S. Hirsch. 1997. Antagonism between human immunodeficincy virus type 1 protease inhibitors indinavir and saquinavir in vitro. J Infect Dis176:265-268.

45. Moore, J.P., Y. Cao, L. Qing, Q.J. Sattentau, J. Pyati, R. Koduri, J. Robinson, C.F. Barbas, D.R. Burton and D.D. Ho. 1995. Primary isolates of human immunodeficiency virus type 1 are relatively resistant to neutralization by monoclonal antibodies to gp120 and their neutralization is not predicted by studies with monomeric gp120. J. Virol.69:101-109.

46. Moore, J.P., B.A. Jameson, R.A. Weiss and Q.J. Sattentau. The HIV-cell fusion reaction. Boca Raton: CRC Press Inc., 1993 (J. Bentz, ed. Viral Fusion Mechanisms).

47. Moore, J.P., J.A. McKeating, Y. Huang, A. Ashkenazi and D.D. Ho. 1992. The Virions of primary human immunodeficiency virus type 1 isolates resistant to soluble CD4 (sCD4) neutralization differ in sCD4 binding and glycoprotein gpl20 retention from sCD4-sensitive isolates. J. Virol.66:235-243.

48. Moore, J.P. and R.W. Sweet. 1993. The HIV gpl20 - CD4 interaction: a target for pharmacological and immunological intervention. Prospect in Drug Discovery and Design1:235-250.

49. Murakami, T., T. Nakajima, Y. Koyanagi, K. Tachibana, N. Fujii, H. Tamamura, N. Yoshida, M. Waki, A. Matsumoto, O. Yoshie, T. Kishimoto, N. Yamamoto and T. Nagasawa. 1997. A small molecule CXCR4 inhibitor that blocks T cell line-tropic HIV-1 infection. J. Ex. Med.186:1389-1393.

50. Olson, W.C. unpublished results.

51. Pantaleo, G., G. Poli, L. Butini, C. Fox, Dayton, A.I. and A.S, Fauci. 1991. Dissociation between syncytia formation and spreading HIV. Suppression of syncytia does not necessarily reflect inhibition of HIV infection. Eur. J. Immunol.21:1771-1774.

52. Rabut, G.E.E., J.A. Konner, F. Kajumo, J.P. Moore and T. Dragic. 1998. Alanine substitutions of polar and non-polar residues in the amino-terminal domain of CCR5 differently impair entry of macrophage - and dual-tropic isolates of the human immunodeficiency virus type 1. J. Virol.72:3464-3468.

53. Rizzuto, C., R. Wyatt, N. Hernandez-Ramos, Y. Sun, P. Kwong, W. Hendrickson and J. Sodroski. 1998. Identification of a conserved human immunodeficiency virus gp120 glycoprotein structure important for chemokine receptor binding. Science280:1949-1953.

54. Rucker, J., M. Samson, B.J. Doranz, F. Libert, J.F. Berson, Y. Yi, R.J. Smyth, R.G. Collman, CC. Broder, G. Vassart, R.W. Doms and M. Parmentier. 1996. Regions in the β-chemokine receptors CCR-5 and CCR-2b that determine HIV-1 cofactor specificity. Cell87:437-446.

55. Schols, D., S. Struyf, J.V. Damme, J.A.'este, G. Henson and E.D. Clercq. 1997. Inhibition of T-tropic HIV strains by selective antagonization of the chemokine receptor CXCR4. J. Ex. Med.186:1383-1388.

56. Simmons, G., P.R. Clapham, L. Picard, R. E. Offord, M.M. Rosenkilde, T.W. Schwartz, R. Buser, T.N.C. Wells and A.E.I. Proudfoot. 1997. Potent inhibition of HIV-l infectivity in macrophages and lymphocytes by a novel CCR5 antagonist. Science276:276-279.

57. Simmons, G., D. Wilkinson, J.D. Reeves, M.T. Dittmar, S. Beddows, J. Weber, G. Carnegie, U. Desselberger, P.M. Gray, R.A. Weiss and p. R. Clapham. 1996. Primary, syncytiutn-inducing human immunodeficiency virus type-1 isolates are dual-tropic and most can use either LESTR or CCR5 as co-receptor for virus entry. J. Virol.70:8355-8360.

58. Strizki, J.M., J. Davis-Turner, R.G. Collman, J. Hoxie and F. Gonzalez-Scarano. 1997. A monoclonal antibody (12G5) directed against CXCR4 inhibits infection with the dual-tropic human immunodeficiency virus type 1 isolate of HIV-1 89.6 but not the T-tropic isolate of HIV-1 HxB. J. Virol.71:5678-5683.

59. Trkola, A., T. Dragic, J. Arthos, J. Binley, W.C. Olson, G.P. Allaway, C. Cheng-Mayer, J. Robinson, P.J. Maddon and J.P. Moore. 1996. CD4-dependent, antibody-sensitive interactions between HIV-1 and its co-receptor CCR-5. Nature384:184-187.

60. Trkola, A., W.A. Paxton, S.P. Monard, J.A. Hoxie, M.A. Siani, D.A. Thompson, L. Wu, C.R. Mackay, R. Horuk and J.P. Moore. 1997. Genetic subtype - independent inhibition of human immunodeficiency virus type-l replication by CC - and CXC chemokines. J. Virol.72:396-404.

61. Vijh-Warrier, S., A. Pinter, Honnen WJ and S.A. Tilley. 1996. Synergistic neutralization of human immunodeficiency virus type 1 by a chimpanzee monoclonal antibody against the V2 domain of gp120 in combination with monoclonal antibodies against the V3 loop and the CD4-binding site. J. Virol.70:4466-4473.

62. Ward, S.G., K. bacon andJ.Westwick.. 1998. Chemokines and lymphocytes: more than an attraction. Immunity9:1-11.

63. Wu, L, N.P. Gerard, R. Wyatt, H. Choe, C. Parolin, N. Ruffing, A. Borsetti, A.A. Cardoso, E. Desjardin, W. Newman, C. Gerard, and J. Sodroski. 1996. CD4-induced interaction of primary HIV-1 gp120 glycoproteins wi-th the chemokine receptor CCR-5. Nature384:179-183.

64. Wu, L., G. LaRosa, N. Kassam, C.J. Gordon, H. Heath, N. Ruffing, H. Chen, J. Humblias, M. Samson, M. Parmentier, J.P. Moore and C.R. Mackay. 1997. Interaction of chemokine receptor CCR5 with its ligands: multiple domains for HIV-1 gpl20 binding and a single domain for chemokine binding. J. Exp. Med.186:1373-1381.

65. Wyatt, R., P.D. Kwong, E. Desjardins, R. Sweet, J. Robinson, W. Hendrickson and J. Sodroski. 1998. The antigenic structure of the human immunodeficiency virus gp120 envelope glycoprotein. Nature393:705-711.

66. Wyatt, R. and J. Sodroski. 1998. The HIV-1 envelope glycoproteins: fusogens, antigens and immunogens. Science280:1884-1888.

67. Ylisastigui, L., J.J. Vizzavona, E. Drakopoulou, Paindavoine, P., C.F. Calvo, M. Parmentier, J.C. Gluckman, C. Vita andA.Benjouad. 1998. Synthetic full length and truncated RANTES inhibit HIV-l infection of primary macrophages. AIDS12:977-984.

68. Zhang, J.L., H. Choe, B.J. Dezube, M. Farzan, P.L. Sharma, X.C. Zhou, L.B. Chen, M. Ono, S. Gillies, Y. Wu, J.G. Sodroski and C.S. Crumpacker. 1998. The bis-azo compound FP-21399 inhibits HIV-l replication by preventing viral entry. Virology244:530-541.

69. Cairns, J.S., D'souza. M.P., 1998. Chemokines and HIV-1 second receptors: therapeutic connection. Nature Medicine. 1998. Vol 4, No. 5: 563.

70. Kilby, J.Michael, et al. 1998. Potent suppression of HIV-l replication in humans by T-20, a peptide inhibitor of gp41-medicated virus entry. Nature Medicine. Vol. 3, No. 11: 1302.

71. U.S. patent No. 4816567, issued March 28, 1989 Cabilly et al.

72. U.S. patent No. 5225539, issued July 6, 1993 Gregory Winter.

73. U.S. patent No. 5585089, issued December 17, 1996 Queen et al.

74. U.S. patent No. 5693761, issued December 2, 1997 Queen et al.

75. PCT International Application no PCT/US89/05857, filed December 28, 1989 the ode, published on July 26, 1990, WO 90/07861.

1. Antibody against CCR5, containing

(i) two light chains, each light chain contains the expression product of the plasmid designated pVK:HuPRO140-VK deposited in ATSC number of MOUTH-4097,

(ii) two heavy chains, each heavy chain contains the expression product of either a plasmid designated pVg4:HuPRO140 HG2-VH deposited in ATSC number of MOUTH-4098, or by the plasmid designated pVg4:HuPRO140 (mut B+D+I)-VH deposited in ATSC number of MOUTH-4099,

or its fragment, which binds to CCR5 on the surface of human cells.

2. Antibody against CCR5, containing

two light chains, where each chain contains consecutive amino acids, amino acid sequence listed in SEQ ID NO:6, and

two heavy chains, each heavy chain contains the sequence of amino acids, amino acid sequence listed in SEQ ID NO:9 or SEQ ID NO:12,

or its fragment, which binds to CCR5 on the surface of human cells.

3. Nucleic acid selected from the group consisting of

(a) a nucleic acid that encodes a polypeptide containing consecutive amino acids, amino acid sequence listed in SEQ ID NO:6, 9 or 12;

(b) a nucleic acid that contains consecutive nucleotides, POS is egovernance listed in SEQ ID NO:5, 8 or 11; and

(c) a nucleic acid that encodes a polypeptide containing consecutive amino acids, where successive amino acids are amino acids, expressed by the plasmid designated pVK:HuPRO140-VK deposited in ATSC number of MOUTH-4097, by the plasmid designated pVg4:HuPRO140 HG2-VH deposited in ATSC number of MOUTH-4098, or by the plasmid designated pVg4:HuPRO140 (mut B+D+I)-VH deposited in ATSC number of MOUTH-4099.

4. Nucleic acid according to claim 2, where the nucleic acid is an RNA, DNA or cDNA.

5. Composition for inhibiting infection of CD4+ HIV-1 containing at least one antibody against CCR5 or its fragment according to any one of claims 1 and 2 and at least one antiviral agent.

6. The composition according to claim 5, where the antiviral agent is selected from the group consisting of dinucleotides reverse transcriptase inhibitors (NNRTIS), nucleotide reverse transcriptase inhibitors, protease inhibitors HIV-1 and inhibitor occurrence of the virus.

7. Composition for inhibiting infection of HIV-1 containing at least one antibody against CCR5 or its fragment according to any one of claims 1 and 2 and the media.

8. Composition for inhibiting infection of HIV-1 containing at least one antibody against CCR5 or its fragment according to any one of claims 1 and 2 with the attached substance selected from the GRU is dust, consisting of a radioisotope, toxin, polyethylene glycol, cytotoxic tools and detectable labels.

9. The use of antibodies against CCR5 according to claim 1 or its fragment, which binds to CCR5 on the surface of human cells, to obtain a pharmaceutical composition for inhibiting infection by HIV-1 human cell.

10. The use of antibodies against CCR5 according to claim 2 or its fragment, which binds to CCR5 on the surface of human cells, to obtain a pharmaceutical composition for the treatment of patients with infection of HIV-1.

11. The use of claim 10, where the antibody against CCR5 or its fragment is present in the composition at a dose in the range from 0.1 to 100,000 μg/kg of body weight of the patient.

12. Conjugate antibodies against CCR5, containing a fragment of the antibody according to claim 1, conjugated to at least one polymer, where the polymer gives an increased half-life in serum, increased average retention time in the circulation system or a reduced rate of clearance from the serum compared to the unconjugated form of fragments of antibodies.

13. Conjugate antibodies against CCR5 indicated in paragraph 12, where the polymer is selected from the group consisting of hydrophilic polyvinyl polymers, polyalkylene ethers, polyoxyalkylene, polymethacrylates, carbonero, branched polysaccharides, unbranched polysaccharides, polymers, sugar alcohols, and C is Rina and gepirone.

14. Conjugate antibodies against CCR5 indicated in paragraph 13, where the polyalkylene ether is a polyethylene glycol or its derivative.

15. Conjugate antibodies against CCR5 on 14, where the average molecular weight of the polyethylene glycol is at least 20 kDa.

16. Conjugate antibodies against CCR5 on any of PP-15, where the conjugate has at least one of the properties: increased half-life in serum, increased average retention time in the circulation system or a reduced rate of clearance from the serum compared to the unconjugated form of the antibody against CCR5 or its fragment.

17. The conjugate antibodies against CCR5-containing fragment antibodies against CCR5 according to claim 2, conjugated at least one polymer for the treatment of HIV-1 infection in a patient, where the polymer gives an increased half-life in serum, increased average retention time in the circulation system or a reduced rate of clearance from the serum compared to the unconjugated form of fragments of antibodies.

18. Transformed a host cell containing at least two vectors, where at least one vector contains a nucleic acid sequence that encodes a heavy chain antibodies against CCR5, and at least one vector contains a nucleic acid sequence that encodes a light chain of the antibody is Rotel CCR5, where the antibody against CCR5 contains two heavy chain with the amino acid sequence shown in SEQ ID NO:9, or 12, and two light chain with the amino acid sequence shown in SEQ ID NO:6.

19. Transformed a host cell according p, where cell is the cell of a mammal.

20. Transformed a host cell according to claim 19, where the cell is a COS cell, the cell is Cho or myeloma cell.

21. Transformed a host cell according to any one of p-20, where the cell secretes the antibody against CCR5.

22. Transformed a host cell according to any one of p-20, where the vector represents a vector designated pVg4:HuPRO140 HG2-VH deposited in ATSC number of MOUTH-4098 or pVg4:HuPRO140 (mut B+D+I)-VH deposited in ATSC number of MOUTH-4099.

23. Transformed a host cell according to any one of p-20, where the vector is a vector designated pVK:HuPRO140-VK deposited in ATSC number of MOUTH-4097.

24. Transformed a host cell according to any one of p-20, where the nucleic acid sequence that encodes the heavy chain has the nucleotide sequence indicated in SEQ ID NO:8 or 11.

25. Transformed a host cell according to any one of p-20, where the nucleic acid sequence that encodes a light chain, has the nucleotide sequence indicated in SEQ ID NO:5.

26. The expression vector containing the nucleic acid sequence according to claim 3 or 4.

27. A method of obtaining antibodies against CCR5, which includes culturing the host cell containing (i) a plasmid designated pVK:HuPRO140-VK deposited in ATSC number of MOUTH-4097, and (ii) either a plasmid designated pVg4:HuPRO140 HG2-VH deposited in ATSC number of MOUTH-4098, or a plasmid designated pVg4:HuPRO140 (mut B+D+I)-VH deposited in ATSC number of MOUTH-4099, in conditions where the production of antibodies containing two light chains, encoded by nucleic acid plasmid designated pVK:HuPRO140-VK deposited in ATSC number of MOUTH-4097, and two heavy chains encoded by the nucleic acid or plasmid designated pVg4:HuPRO140 HG2-VH deposited in ATSC number of MOUTH-4098, or by the plasmid designated pVg4:HuPRO140 (mut B+D+I)-VH deposited in ATSC number of MOUTH-4099, so that the formed antibody against CCR5.

28. A method of obtaining antibodies against CCR5, which provides

a) transforming the host cell (i) a plasmid designated pVK:HuPRO140-VK deposited in ATSC number of MOUTH-4097, and (ii) or by the plasmid designated pVg4:HuPRO140 HG2-VH deposited in ATSC number of MOUTH-4098, either by the plasmid designated pVg4:HuPRO140 (mut B+D+I)-VH deposited in ATSC number of MOUTH-4099; and

b) culturing the transformed host cell under conditions that allow production of the antibody comprising two light chains, to the dummy nucleic acid plasmids, designated pVK:HuPRO140-VK deposited in ATSC number of MOUTH-4097, and two heavy chains encoded by the nucleic acid or plasmid designated pVg4:HuPRO140 HG2-VH deposited in ATSC number of MOUTH-4098, or by the plasmid designated pVg4:HuPRO140 (mut B+D+I)-VH deposited in ATSC number of MOUTH-4099, so that the formed antibody against CCR5.

29. The method according to any of item 27 or 28, further providing for removing the produced antibodies against CCR5.

30. The method according to any of item 27 or 28, where the host-cell is a cell of a mammal.

31. The method according to item 30, where a host cell of a mammal is a COS cell, the cell is Cho or myeloma cell.



 

Same patents:

FIELD: medicine, immunology, chemistry of peptides.

SUBSTANCE: invention discloses peptides GNA33 representing mimetics of epitopes of microorganisms Neisseria meningitides of the serogroup B showing definite amino acid sequences given in the description and able to induce production of antibodies eliciting complement-mediated bactericidal activity and/or opsonic activity against indicated microorganisms in a mammalian subject. Indicated peptides are used as components of a composition used in a method for production of the immune response reaction against indicated microorganisms in a mammalian subject. Also invention concerns a polynucleotide encoding indicated peptide and recombinant expression vector comprising indicated polynucleotide. Also invention discloses a method for preparing peptide GNA33 by culturing the cell-host comprising recombinant expression vector and assay for the presence of antibodies raised against meningococci B in biological sample using GNA33 peptide. Using the invention provides effectiveness of vaccine raised against MenB and safety for its using for a patient.

EFFECT: valuable biological and medicinal properties of peptides.

11 cl, 6 tbl, 8 dwg, 6 ex

FIELD: biotechnology, biochemistry, molecular biology.

SUBSTANCE: invention represents eucaryotic cell-host that expresses the first polynucleotide encoding the first propeptide and FVII or its functional variants and relates to the first expression unit, and expresses the second polynucleotide encoding the second free propeptide. Each of indicated the first and second propeptides comprises amino acid sequence chosen independently from group consisting of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 and 18. Also, invention relates to a method for preparing vitamin K-dependent proteins, among them and FVII, using abovementioned cell-host, and to a method for preparing this cell-host. Invention provides preparing vitamin K-dependent proteins with the high degree of effectiveness.

EFFECT: improved preparing method of proteins.

38 cl, 3 tbl, 3 dwg, 2 ex

FIELD: biotechnology, molecular biology, proteins.

SUBSTANCE: invention relates to a method for preparing cytokines of class II and can be used in medicine. Prepared proteins zcyto20, zcyto22, zcyto24 and zcyto25 are the most relative with interferon-α at amino acid sequence level. Receptor of cytokines of class II represents a receptor for this family of proteins. Proteins can be prepared by recombinant way using a cell-host transformed with expression vector that comprises nucleic acids corresponding to proteins. Base on proteins xcyto20, xcyto21, zcyto22, zcyto24 and zcyto25 antiviral pharmaceutical composition and specific antibodies are prepared. Invention provides preparing the novel cytokine that stimulates cells of differentiation hemopoietic line and possesses the expressed antiviral activity.

EFFECT: valuable biological and medicinal properties of polypeptide, improved preparing method.

24 cl, 21 tbl, 32 ex

FIELD: microbiology, genetic engineering, medicine, oncology.

SUBSTANCE: invention relates to genetically modified microorganism comprising the following components in its genomes that are expressed: (I) nucleotide sequence encoding at least one the tumor cell antigen epitope, and/or nucleotide sequence of at least one antigen epitope showing specificity to tissue cell generating tumor; (II) nucleotide sequence encoding protein that stimulates cell of the immune system; (IIIA) nucleotide sequence of the transport system providing expression of product of components (I) and (II) expression on external surface of microorganism, and/or secretion of product of component (I) and component (II) expression, and/or (IIIB) nucleotide sequence of protein for lysis of microorganism in mammal cells cytosol and for intracellular releasing plasmas containing in microorganisms to be lysed, and (IV) activating sequence activated in microorganism showing specificity to tissue cell but not specific to cell for expression of one or some components from (I) to (IIIB), and wherein each of components from (I) to (IV) can be represented once or many times. Proposed microorganism is useful for using as a drug as component of anti-tumor vaccine. Also, invention describes plasmid or the expression vector for preparing the genetically modified microorganism and a method for its preparing. Vaccine based on genetically modified microorganism represents the improved and overcome immune tolerance with respect to tumors in case for its using for prophylaxis and therapy of tumors.

EFFECT: valuable properties of microorganisms.

21 cl, 1 dwg, 2 ex

FIELD: biotechnology, medicine, proteins.

SUBSTANCE: invention proposes chimeric polypeptide that comprises cell-specific targeting moiety and granzyme. Polypeptide prepared by recombinant way is used for inducing apoptosis in target-cell. Invention provides carrying out the effective treatment of malignant neoplasms by directed lysis of tumor cells. Invention can be used in therapy of hyperproliferative disorders.

EFFECT: valuable medicinal properties of agents.

24 cl, 3 tbl, 35 dwg, 41 ex

FIELD: biotechnology, immunology.

SUBSTANCE: disclosed are variants of chimerical anti-IL-6 antibodies based on mice CLB-8 antibody. Each antibody contains constant region from one or more human antibodies. Described are variants of nuclear acids encoding anti-IL-6 antibody, vectors and host cells. Developed is method for production of anti-IL-6 antibody by using nuclear acid or vector. Described are variants of composition for application in method for modulation of malignant disease or immune disorder mediated with IL-6. Developed is method for treatment or modulation of malignant disease or immune disorder mediated with IL-6.

EFFECT: variant of chimerical anti-IL-6 antibody with high affinity of mice anti-IL-6 antibody and reduced immonogenicity.

26 cl, 16 dwg, 1 tbl, 8 ex

FIELD: medicine.

SUBSTANCE: claimed vaccine includes DNA construct operatively encoding receptor protein of vessel endothelial growth factor (VEGF) selected from group containing VEGFR-2 (KDR; SEQ ID NO:2), VEGFR-1 (Flt-1; SEQ ID NO:4) or Flk-1 (mice KDR homologue, SEQ ID NO:6) and functional equivalent thereof having of about 80 % homology. Encoding receptor proteins of DNA-sequence are represented in SEQ ID NO: 1, 2 and 5, and in description. Described are methods for uses of DNA-vaccines as inhibitors of proliferating endothelial cells in mammalian by administering of DNA-vaccine to stimulate of immune response in relation to proliferating endothelial cells, in particular to inhibit angiogenesis and tumor growth. Also disclosed is dosage form of DNA-vaccine.

EFFECT: DNA-vaccine against angiogenesis and tumor growth.

14 cl, 13 dwg, 5 tbl, 4 ex

FIELD: biotechnology.

SUBSTANCE: disclosed is isolated polypeptide being acid-proof metalloprotease isolated from Thermoascus aurantiacus. Described are strain Thermoascus aurantiacus CGMCC No 0670 for polypeptide production and method for polypeptide production using said strain. Disclosed is method for plant protein treatment to increase digestion value thereof by using said polypeptide.

EFFECT: protease of good acid resistance useful in feed production.

6 cl, 7 ex, 4 tbl

FIELD: biotechnology, molecular biology.

SUBSTANCE: invention proposes a polynucleotide VEGI-192a encoding polypeptide that inhibits growth of human vascular endothelial cells. Invention describes expressing vector comprising polynucleotide and E. coli cell-host comprising vector. Invention discloses polypeptide encoded by polynucleotide and fused protein based on indicated polypeptide. Invention describes polynucleotide encoding fused protein and expressing vector based on indicated polynucleotide. Invention discloses a pharmaceutical composition used for inhibition of angiogenesis based on polypeptide-inhibitor of growth of human vascular endothelial cells and polynucleotide encoding its. Invention describes therapeutic methods for inhibition of angiogenesis and suppression of tumor growth based on this composition. Invention describes an antibody raised to polypeptide that inhibits growth of human vascular endothelial cells. Using this invention provides novel forms of inhibitor of human growth of vascular endothelial cells and can be used in medicine.

EFFECT: valuable biological and medicinal properties of inhibitor.

27 cl, 27 dwg, 13 tbl, 34 ex

FIELD: biotechnology, genetic engineering.

SUBSTANCE: invention relates to a plasmid vector able to autonomic replication in the strain Escherichia coli K-12 at any temperature. Plasmid comprises region or with mutation, gene rep from pMW119 and DNA fragment. DNA fragment can be integrated into chromosome of microorganism belonging to Escherichia coli W or Escherichia coli B as result of homologous recombination. Plasmid is fused into a microorganism belonging to Escherichia coli W or Escherichia coli B followed by preparing amino acid. Also, invention relates to strains Escherichia coli DH5α/pMTS11910 (FERM BP-6904) and Escherichia coli DH5α/pMTS11914 BP-6905) designated for storage the plasmid vector. Strains Escherichia coli WLA-131 (FERM BP-6902) and Escherichia coli WL-1133 (FERM BP-6903) are producers of amino acid leucine. Invention provides carrying out superexpression of gene and to obtain stable plasmids.

EFFECT: valuable biological properties of plasmids and strains.

13 cl, 2 dwg, 1 tbl, 5 ex

FIELD: biotechnology, immunology.

SUBSTANCE: disclosed are variants of chimerical anti-IL-6 antibodies based on mice CLB-8 antibody. Each antibody contains constant region from one or more human antibodies. Described are variants of nuclear acids encoding anti-IL-6 antibody, vectors and host cells. Developed is method for production of anti-IL-6 antibody by using nuclear acid or vector. Described are variants of composition for application in method for modulation of malignant disease or immune disorder mediated with IL-6. Developed is method for treatment or modulation of malignant disease or immune disorder mediated with IL-6.

EFFECT: variant of chimerical anti-IL-6 antibody with high affinity of mice anti-IL-6 antibody and reduced immonogenicity.

26 cl, 16 dwg, 1 tbl, 8 ex

FIELD: biotechnology, proteins, immunology.

SUBSTANCE: invention proposes fragment of antibody MR1-1 that retains capacity for recognition and binding EGFRvIII and nucleic acid encoding its. Polypeptide is prepared from the known antibody MR1 by mutation of CDR3 VH- and VL-chains. Also, invention describes using a polypeptide for preparing immunotoxin possessing the cytotoxicity property to cells carrying antigen of epidermal growth factor EGFRvIII receptors. Also, invention disclosed immunotoxin based on this polypeptide. Also, invention proposes a method for preparing polypeptide comprising amino acid substitution of at least one amino acid in hypervariable region CDR with amino acid encoded by a codon comprising nucleotide belonging to a hot point motif. The hot point motif is chosen from AGY or RGYW wherein R represents A or G; Y represents C or T, and W represents A or T. Prepared polypeptide is characterized by value Kd for EGFRvIII 7 nM or less. Also, invention describes a method for cell killing that expresses antigen EGFRvIII using a polypeptide. Using this invention provides preparing antibodies showing enhanced cytotoxicity and improved binding EGFRvIII as compared with the parent antibody MR1 that can be used for target-directed delivery of immunotoxins in treatment of malignant neoplasms.

EFFECT: valuable medicinal properties of polypeptide.

34 cl, 7 tbl, 6 dwg, 8 ex

FIELD: biotechnology, genetic engineering.

SUBSTANCE: invention describes recombinant plasmid DNAs constructed in vitro that comprise artificial genes for light and heavy chains of full-scale human antibody prepared by genetic engineering methods. These genes are created on basis of variable fragments of light and heavy chains of recombinant antibody 1F4 and constant human genes IgG1, cytomegalovirus promoter and polyadenylation site BGH. Plasmids provide biosynthesis of recombinant full-scale human antibodies of class IgG1 in mammalian cells. These antibodies interact specifically with smallpox vaccine virus. The affinity constant for prepared recombinant antibodies is 3.54 x 109 ± 0.38 x 109 M-1. Plasmids are used by combined transfection of human cells HEK 293T. Prepared full-scale recombinant antibody against protein of size 27 kDa of smallpox virus vaccine can be used as a base for creature of pharmaceutical preparations used for diagnosis of some post-vaccine complications caused by smallpox virus vaccine. Also, preparations will comprise decreased therapeutic doses of immunoglobulins that will provide minimal undesirable immune response in patients after administration of the preparation.

EFFECT: valuable medicinal properties of plasmid DNA.

4 cl, 7 dwg, 6 ex

FIELD: biotechnology, immunology.

SUBSTANCE: invention describes a monoclonal anti-IFNα antibody that binds with the following subtypes of IFNα: IFNα1, IFNα2, IFNα4, IFNα5, IFNα8, IFNα10 and IFNα21 and comprises three CDR-sites of heavy chain. Amino acid is given in the invention description. Invention discloses heavy chain of anti-IFNα antibody or its fragment that comprise indicated CDR-sites also. Invention describes anti-IFNα antibody that comprises at least one light chain and one heavy chain. Invention discloses variants of nucleic acids encoding indicated antibodies and variants of vectors used for expression of nucleic acids, and variants of transformed host-cells. Among expression vectors invention describes also vectors deposited at № 2881 and № 2882 carrying heavy and light chain of antibody, respectively. Invention describes a method for preparing antibody from indicated cells. Invention discloses the murine hybridoma cell line deposited in ATCC at number № РТА-2917, and antibody produced by indicated cell line. Also, invention describes variants of the antibody-base pharmaceutical composition and a method used for diagnosis of autoimmune disease. Also, invention discloses using antibodies in treatment of disease or state associated with enhanced level of IFNα in a patient. Using the invention provides inhibiting biological activity of at least seven human IFNα subtypes simultaneously, namely: IFNα1, IFNα2, IFNα4, IFNα5, IFNα8, IFNα10 and IFNα12 that can be used in diagnosis and therapy of different human diseases mediated by IFNα, such as insulin-dependent diabetes mellitus or erythematosus lupus.

EFFECT: valuable biological and medicinal properties of antibodies.

53 cl, 4 tbl, 10 dwg, 2 ex

FIELD: biotechnology, immunology, molecular biology, pharmacy.

SUBSTANCE: invention describes variants of MCP-1-binding molecules. One of MCP-1-binding molecule comprises at least one variable region of immunoglobulin (VH) heavy chain comprising of hypervariable sites CDR1, CDR2 and CDR3 while other molecules comprises both light and heavy chains. Invention proposes DNA constructs encoding indicated MCP-1-binding molecules and expressing vector carrying at least one of these DNA constructs. Invention describes a method for preparing MCP-1-binding molecule. Invention discloses a method for treatment of disease or disorder mediated by MCP-1 or eotaxine-1 based on antibody raised to MCP-1 that binds eotaxine-1 by cross mode. Invention describes a pharmaceutical composition based on antibody raised to MCP-1 that binds eotaxine-1 by cross mode and used in treatment of disease or disorder mediated by MCP-1 or eotaxine-1 in a patient. MCP-1-binding molecules inhibit binding MCP-1 with its receptor. The full immobilized antibody is highly specific as far as it binds human recombinant MCP-1 with value KD = (43 ± 2.9) x 1012 and can be used in medicine.

EFFECT: valuable medicinal properties of antibodies, improved method of treatment.

13 cl, 5 dwg, 4 tbl, 2 ex

FIELD: biotechnology, immunology, medicine, oncology.

SUBSTANCE: invention describes variants of monoclonal antibodies showing specificity to TRAIL-receptor DR4. By one of variant antibodies are produced by hybridoma 2E12 recorded in ATCC at number PTA-3798. Each of antibody variants possesses apoptosis-inducing activity both in vivo - in the concentrations less 10 mg/kg in target cells expressing DR4 and in vitro - in the presence of a cross-linking agent in the concentrations less 1 mcg/ml in target cells. Invention discloses variants of methods for selective induction of apoptosis in cells expressing DR4, and variants of methods for inhibition of DR4-expressing cells based on using antibodies. Invention describes variants of compositions, methods for treatment of a patient suffering from inflammatory or autoimmune disease and methods in treatment of a patient suffering from malignant tumor wherein these compositions are based on antibody to DR4 for inducing apoptosis in cells expressing DR4. Also, invention discloses variants of nucleic acids, purified polypeptides, expression vectors and host-cells used in preparing antibody. Using the invention provides delaying tumor growth and decreasing case of its regression that can be used in tumor therapy.

EFFECT: improved and valuable properties of antibody.

103 cl, 169 dwg, 6 tbl, 30 ex

FIELD: biotechnology, microbiology, immunology.

SUBSTANCE: invention proposes peptide from N. meningitides eliciting a sequence SEQ ID NO:10 that is used in treatment or diagnosis. Also, invention proposes polynucleotide encoding this peptide, a host-cell expressing peptide, microorganism, vaccine and antibody showing specificity to peptide proposed. Invention can be used in medicine.

EFFECT: valuable medicinal properties of peptide.

16 cl, 2 tbl

FIELD: gene engineering.

SUBSTANCE: recombinant fragmid DNA pHEN-TAB, containing unique human single-strand antibody gene is selected from constructed in vitro combinatorial phage library udder controlling of lactose operon promoter. Then Escherichia coli HB2151 cells are transformed with obtained fragmid DNA to produce recombinant bacterium strain Escherichia coli HB2151/pHEN-TAB as producer of human single-strand antibody capable of binding of human tumor necrosis factor alpha. Said antibody gas affinity constant of Kaf = 3.96±0.52x108 M-1.

EFFECT: new soluble human single-strand antibody scTAB against human tumor necrosis factor alpha with high affinity.

3 cl, 6 dwg, 6 ex

FIELD: immunology, medicine.

SUBSTANCE: claimed recombinant antibody (Ab) has at least constant regions in heavy and light chains representing human Ab regions. Said At inhibits bonding of integrine recognizing RGD and SVVYGLR sequences to integrine or fragment thereof. Also disclosed are nucleotide sequences (NS) encoding heavy and light chains of recombinant Ab as well as expression vectors containing respective NS. Described are host cell for Ab production, transformed with two vectors for expression of Ab heavy and light chains and method for abovementioned host cell application to produce recombinant Ab. Ab of present invention is useful in diagnosis and treatment of autoimmune diseases, rheumatism and rheumatoid arthritis.

EFFECT: therapeutic methods of increased efficiency.

45 cl, 14 tbl, 28 ex

FIELD: immunology, biotechnology.

SUBSTANCE: invention describes murine antibody and its humanized variant (CDP870) showing specificity to human tumor necrosis factor-alpha. Amino acid sequence is given in the description. Also, invention describes compounds showing affinity with respect to human tumor necrosis factor-alpha based on humanized antibody wherein lysylmaleimide group bound covalently with one or some methoxypoly(ethylene glycol) molecules by lysyl residue is joined to one of cysteine residues by C-end of heavy chain of the humanized antibody. Invention discloses DNA sequences encoding antibodies showing specificity to human tumor necrosis factor-alpha and variants if expression vectors involving indicated DNAs. Also, invention describes variants of a method for preparing a host-cell using expression vectors and variants of a method for preparing antibodies based on prepared host-cells. Invention discloses therapeutic compositions used in treatment of pathology mediated by tumor necrosis factor-alpha based on antibodies. Invention provides providing antibodies showing high affinity: 0.85 x 10-10 M for murine antibodies and 0.5 x 10-10 M for its humanized variant and low immunogenicity for human for humanized antibodies. Part of patients with improved ACR20 in administration of 5 and 20 mg/kg of CDP870 is 75% and 75% in 8 weeks, respectively. Half-life value of CDP870 in plasma is 14 days.

EFFECT: valuable biological and medicinal properties of antibodies.

58 cl, 24 dwg, 6 tbl, 1 ex

FIELD: medicine.

SUBSTANCE: invention relates to application of recombinant antibody having hypervariable sites of CDR region with specific amino acid sequence for treatment and preventing of non-specific ulcer colitis and Crone disease in steroid-resistant patients.

EFFECT: improved preparation for treatment of steroid-resistant patients.

15 cl, 2 ex, 2 tbl

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