RU2722398C1 - Monoclonal antibody to heat shock protein 70 - Google Patents

Abstract

FIELD: chemistry.SUBSTANCE: invention refers to biochemistry, particularly to a monoclonal antibody recognizing a heat shock protein 70 bound to a membrane.EFFECT: invention is capable of effective treatment of tumour diseases.1 cl, 5 dwg, 1 tbl, 6 ex

Description

The invention relates to biotechnology and immunology. A monoclonal antibody is presented against the non-determinants of heat shock protein 70 (HSP70), which is formed by the interaction of HSP70 with the surface of the cytoplasmic membrane of the tumor cell. The antibody can be used to create a new drug for theranostomy of tumors.

When developing a new generation of drugs, priority is given to drugs for targeted therapy. The most interesting and promising objects in this field of pharmacology are therapeutic antibodies that directly interact with the pathogen molecule, neutralizing its effect. A large number of neutralizing antibodies have been developed that are used to treat autoimmune, infectious diseases and certain types of cancer [Ribatti D. From the discovery of monoclonal antibodies to their therapeutic application: An historical reappraisal // Immunology Letters 2014, 161: 96-99 ; Shepard H.M., Phillips G.L., Thanos C., Feldmann M. Developments in therapy with monoclonal antibodies and related proteins // Clin Med (Lond). 2017; 17 (3): 220-232].

The heat shock protein family 70 (HSP70) is a family of ubiquitous proteins that act as chaperones. HSP70, otherwise called HSP1A1, is a stress-induced protein that is overexpressed both in the cytoplasm and on the cytoplasmic membrane of tumor, apoptotic and stressed cells, but is poorly present (almost absent) on the membrane of isotypic and normal cells [Stangl S., Gehrmann M., Riegger J., Kuhs K., Riederer I., Sievert W., Hube K., Mocikat R., Dressel R., Kremmer E., Pockley AG, Friedrich L., Vigh L., Skerra A. , Multhoff G. Targeting membrane heat shock protein 70 (Hsp70) on tumors by cmHsp70.1 antibody // PNAS USA, 2011, 108 (2): 733-738]. It is believed that an increase in the level of HSP70 in the tumor cell, including on its surface, is a sign of tumor aggressiveness, is associated with the resistance of the tumor to chemo and radiotherapy and its ability to metastasize, and also protects the tumor cells from apoptotic death. It was also shown that HSP70 molecules exposed on the plasma membrane of tumor cells are structurally different from both free HSP70 and normal cells associated with membranes [Tamura Y, Tsuboi N, Sato N, Kikuchi K. 70 kDa heat shock cognate protein is a transformation- associated antigen and a possible target for the host's anti-tumor immunity // J Immunol. 1993; 151 (10): 5516-5524]. Therefore, HSP70 on the tumor cell membrane can serve as a promising target for the diagnosis and targeted therapy of cancer using specially prepared neoepitope-specific anti-HSP70 antibodies conjugated with a cytostatic, radionuclide or reporter tag [Shevtsov M, Huile G, Multhoff G. Membrane heat shock protein 70: a theranostic target for cancer therapy // Philos Trans R Soc Lond In Biol Sci. 2017; 373 (1738): 20160526].

In this regard, obtaining monoclonal antibodies that selectively recognize only determinants specific for the HSP70 tumor cell integrated into the membrane of the tumor cell is an urgent task, the solution of which will lead to the creation of a unique tool for tumor theranostics [Fujita Y, Nakanishi T, Miyamoto Y, Hiramatsu M, Mabuchi H, Miyamoto A, Shimizu A, Takubo T, Tanigawa N. Proteomics-based identification of autoantibody against heat shock protein 70 as a diagnostic marker in esophageal squamous cell carcinoma // Cancer Lett. 2008; 263 (2): 280-290; Gehrmann M, Specht HM, Bayer C, Brandstetter M, Chizzali B, Duma M, Breuninger S, Hube K, Lehnerer S, van Phi V, Sage E, Schmid TE, Sedelmayr M, Schilling D, Sievert W, Stangl S, Multhoff G1 Hsp70 - a biomarker for tumor detection and monitoring of outcome of radiation therapy in patients with squamous cell carcinoma of the head and neck // Radiat Oncol. 2014; 9: 131].

Known commercial antibodies such as SPA-820 (Stressgen), H553220-clone7 (BD Pharmingen), clone H5147 BRM-22 (Sigma), 0A500 polyclonal (Dako), MS-482 clone W27 (NeoMarkers), MA3-006 and MA3 -009, which bind to native HSP70, do not recognize the non-determinant of this protein formed on the membrane of the tumor cell, which does not allow us to consider their possible use as drugs or the basis for creating dosage forms.

Monoclonal antibodies are known [RU 2380413, RU 2381271], which bind to HSP70 exposed on the surface of cells. However, these antibodies recognize both HSP70 bound to the membrane of living cells and free soluble HSP70.

A monoclonal antibody cmHsp70.1 (MultiImmune, Germany) is recognized that recognizes the sequence TKDNNLLGRFELSG of the C-terminal domain of HSP70 presented in the membrane-bound tumor cell HSP70 [Stangl S, Gehrmann M, Riegger J, et al. Targeting membrane heat-shock protein 70 (Hsp70) on tumors by cmHsp70.1 antibody. Proc Natl Acad Sci USA. 2010; 108 (2): 733-738; EP1706423]. However, it recognizes both HSP70 bound to the tumor cell membrane and free soluble HSP70 [Stangl S., Gehrmann M., Riegger J., Kuhs K., Riederer I., Sievert W., Hube K., Mocikat R., Dressel R., Kremmer E., Pockley AG, Friedrich L., Vigh L., Skerra A., Multhoff G. Targeting membrane heat shock protein 70 (Hsp70) on tumors by cmHsp70.1 antibody // PNAS USA, 2011, 108, (2): 733-738].

The problem to which the present invention is directed is the expansion of the arsenal of monoclonal antibodies to HSP70 suitable for clinical use in tumor theranostics, as well as the creation of a monoclonal antibody that binds to HSP70 on the surface of the cytoplasmic membrane of a tumor cell and has low affinity for free native HSP70.

The problem is solved by the fact that in the present invention a new monoclonal antibody (MAT) 8D1 is obtained that recognizes HSP70 associated with the membrane of the tumor cell, while it has low affinity for the native (soluble) HSP70. The obtained IgG1 isotype antibody binds to tumor cells, has low affinity for free soluble HSP70, is the dissociation constant of the 8D1-HSP70 complex in solution K _d = 2.25 × 10 ^-5 M, and also has a pronounced antitumor effect in the experiment on the animal model.

Monoclonal antibody 8D1 contains the hypervariable regions (CDR) of the variable region of the light chain according to SEQ ID NO: 1-3 and the hypervariable regions of the variable region of the heavy chain according to SEQ ID NO: 4-6. Comparison of sequences of CDR sites with sequences of known antibodies to HSP70 showed a lack of homology with similar sites of known antibodies.

The technology for producing antibodies included the immunization of animals with an antigen representing a recombinant HSP70 protein sorbed on Octyl Si 100 Polyol 5 nm (Serva, Germany). HPS70 adsorbed on the surface of the plates was used as antigens for the selection of positive producers of monoclonal antibodies during ELISA production (in this case, the antigen underwent conformational changes similar to the immunogen). To exclude producers of antibodies recognizing native (soluble) HSP70, goat immunoglobulins to mouse immunoglobulins adsorbed on the plastic surface of the plates were used. The adsorbed antibody plates were then incubated with the supernatants of cultured clones of hybrid cells, then with native HSP70, which was detected by rabbit polyclonal anti-HSP70 antibodies labeled with horseradish peroxidase. Positive clones were rejected.

The resulting MAT 8D1 can be used to produce chimeric and humanized antibodies and targeted delivery of tags or cytotoxic substances to the tumor cell with their help [Yabbarov N.G., Posypanova G.A., E.A. Vorontsov E.A., O.N. Popova O.N., and E.S. Severin E.S. Targeted Delivery of Doxorubicin: Drag Delivery System Based on RAMAM Dendrimers // Biochemistry (Moscow), 2013, 78 (8): 884-894].

The invention is illustrated by the following graphic materials:

In FIG. 1 shows a histogram of flow cytometry of the binding of antibody 8D1 to cells of the A549 line (human lung carcinoma). The abscissa indicates the fluorescence intensity of 8D1 antibodies labeled with FITC, expressed in arbitrary units of MFI, and the ordinate indicates the number of cells.

In FIG. Figure 2 shows a histogram of flow cytometry of the binding of antibody 8D1 to Sp2 / 0 mouse myeloma cells. The abscissa indicates the fluorescence intensity of 8D1 antibodies labeled with FITC, expressed in arbitrary units of MFI, and the ordinate indicates the number of cells.

In FIG. Figure 3 shows the HSP70 binding sensograms in the concentration range 31.25-500 nM with MAT 8D1 immobilized on the surface of the CM5 chip. The time on the abscissa is in seconds, the response is on the ordinate (RU).

In FIG. 4 shows the survival curves of animals after inoculation of Sp2 / 0 myeloma cells when using MAT 8D1 as a therapeutic agent:

row 1 - mice after administration of Sp2 / 0 myeloma

row 2 - mice after administration of Sp2 / 0 and 8D1 myeloma subcutaneously

row 3 - mice after administration of Sp2 / 0 and 8D1 myeloma intraperitoneally

row 4 - mice after administration of Sp2 / 0 myeloma, opsonized with 8D1

row 5 - mice after the introduction of 8D1

In FIG. 5 shows a histogram of the percentage of surviving animals on the 27th day after inoculation of Sp2 / 0 myeloma cells when using MAT 8D1 as a therapeutic agent:

row 1 - mice after administration of Sp2 / 0 myeloma

row 2 - mice after administration of Sp2 / 0 and 8D1 myeloma subcutaneously

row 3 - mice after administration of Sp2 / 0 and 8D1 myeloma intraperitoneally

row 4 - mice after administration of Sp2 / 0 myeloma, opsonized with 8D1

row 5 - mice after the introduction of 8D1

The invention is illustrated by the following examples.

Example 1. Preparation of HSP70 protein.

HSP70 protein for immunization of mice was sorbed on an Octyl Si100 Polyol 5 nm matrix (Serva, Germany) to give the protein a conformational structure different from that in solution.

Example 2. Immunization of mice and screening of hybridoma.

Balb / c mice were immunized with HSP70, modified as described in Example 1, emulsified in Freund's complete adjuvant (PAF), at a dose of 10 μg per mouse in plantar hind limb aponeurosis. After 4 weeks, animals were injected with 10 μg of the same antigen emulsified in Freund's incomplete adjuvant (NAF), intraperitoneally. On the fourth day after injection, the animals were sacrificed by cervical dislocation and splenocytes were isolated. The resulting cells were mixed with SP 2/0 myeloma cells in a 1: 2 ratio and incubated in a 50% solution of polyethylene glycol (PEG) with a molecular weight of 1,500 Da in a volume of 0.7 ml 1.5 min at room temperature with constant stirring on a Vortex. To the resulting suspension, RPMI-1640 culture medium was added sequentially, in 0.7 ml portions, at intervals of 1 min, until a final volume of 7 ml was reached. After fusion, the cells were precipitated twice by centrifugation to replace the culture medium and seeded in 96-well culture plates of 5 × 10 ⁴ myeloma cells per well, into which daily peritoneal macrophages (5 × 10 ³ cells per well) were previously added. Hybrid breeding was carried out in RPMI-1640 medium with the addition of 20% cow serum containing 10 ^-4 M hypoxanthine, 4ан10 ^-7 M aminopterin and 1.6⋅10 ^-5 M thymidine.

The primary selection of positive clones was performed by binding of monoclonal antibodies secreted by hybridomas to HSP70 using solid-phase ELISA. For this purpose, HSP70 at a concentration of 5 μg / ml was added to the wells of polystyrene ELISA plates (Corning) in 20 mM borate buffer with a pH of 8.0 containing 0.15 M NaCl and kept for 20 hours in a humid chamber so that HSP70 adsorbed on the plastic surface of the tablets. At the end of the adsorption, the plates were washed with wash buffer (20 mM borate buffer, pH 8.0, containing 0.15 M NaCl and 0.05% Tween-20). Then, 100 μl of washing buffer and 50 μl of culture fluid samples containing MAT were added to each well. The plates were incubated at 37 ° C for 1 hour with stirring. At the end of the incubation, the antibodies not bound to the antigen were washed with washing buffer and a solution of horseradish peroxidase conjugated goat immunoglobulins to mouse immunoglobulins (Sigma-Aldrich) was added to the wells according to the manufacturer's instructions. The plates were incubated for 1 hour with stirring at 37 ° C, after which they were thoroughly washed and stained with a solution of a substrate of tetramethylbenzidine (HEMA, Moscow).

To exclude clones producing antibodies that recognize native HSP70, additional screening was performed. For this purpose, the identified positive clones were checked in an experiment on the binding of antigen in solution. For this, goat immunoglobulins were added to the wells of polystyrene plates (Corning) to mouse immunoglobulins at a concentration of 5 μg / ml in 20 mM borate buffer with a pH of 8.0 containing 0.15 M NaCl, which was allowed to sorb for 20 hours in a humid chamber. Then, 100 μl of washing buffer and 50 μl of culture fluid samples from the cells of the selected clones were added to each well. The plates were incubated at 37 ° C for 1 hour with stirring. At the end of the incubation, unbound antibodies were washed with washing buffer and native HSP70 was added to the wells at a concentration of 1 μg / ml. The plates were incubated for 1 hour with stirring at 37 ° C. At the end of the incubation, the unbound antigen was washed with washing buffer and rabbit polyclonal antibodies to HSP70 conjugated with horseradish peroxidase were added to the wells at a concentration of 0.1 μg / ml. The plates were incubated for 1 hour at 37 ° C and stirring, after which they were thoroughly washed and stained with a solution of a substrate of tetramethylbenzidine (HEMA), the ability of antibodies to bind antigen in solution was evaluated by color intensity. Positive clones were rejected. Clones producing antibodies that weakly reacted with the dissolved form of HSP70 were selected for further analysis of the binding / dissociation constant of antibodies to HSP70 by plasmon resonance.

As a result of the screening and determination of the dissociation constant, clone 8D1 was selected that produced specific antibodies to the modified HSP70, which have a weak affinity for HSP70 in solution. Antibodies were accumulated in the ascites of mice, isolated for protein A-Sepharose and subjected to further studies.

Example 3. Analysis of the binding of 8D1 antibodies to tumor cell lines.

An analysis of the binding of 8D1 antibodies to tumor cell lines was performed by flow cytofluorimetry. In FIG. Figures 1 and 2 show histograms of the binding of MAT 8D1 to HSP70 expressed on tumor cell membranes of lines A549 (human lung carcinoma) and Sp2 / 0 (mouse myeloma), which demonstrate the efficiency of antibody binding to tumor membrane-bound HSP70. The results shown in FIG. 1 and 2, indicate that the tested antibodies specifically bind to antigenic determinants on the surface of the studied cells.

Example 4. Determination of the dissociation constant of MAT 8D1 with free HSP70 by surface plasmon resonance.

Measurements were made of the equilibrium dissociation constant and the association of the 8D1 antibody with free recombinant HSP70 in solution. The measurements were carried out using the surface plasmon resonance (SPR) method at a temperature of 25 ° C using carboxymethylated dextran (CM5) chips on a Biacore X100 instrument with a double flow cell (GE-Healthcare, USA). The chip was activated with an equimolar mixture of N-ethyl-N'-dimethylaminopropylcarbodiimide and N-hydroxysuccinimide (0.2 M), then a solution of secondary (anti-species) anti-mouse IgG antibodies (5 μg / ml) was injected for 18 minutes in 10 mm sodium acetate buffer, pH 5.0. Then, the free groups of the chip were blocked with 1 M ethanolamine, pH 8.5. The final level of immobilization of secondary (anti-species) antibodies was 4890 resonance units (RU), which corresponds to 4.9 ng of antibodies per 1 mm ^{2 of} the chip surface.

To assess affinity, increasing analyte concentrations (HSP70) were injected at a rate of 10 μl / min above the chip after injection of MAT 8D1 and washing with 150 mM NaCl buffer solution, 10 mM Hepes-NaOH, pH 7.5, 0.05% polyoxyethylene sorbitan (HBS-P + ) Each experiment consisted of 6 stages:

1) injection of HBS-P + for 1 min;

2) injection of MAT 8D1 for 3 min;

3) washing with HBS-P + for 2 minutes;

4) injection of HSP70 for 1 min;

5) a dissociation step for 5 min (washing with HBS-P + without HSP70);

6) regeneration with 3 M magnesium chloride for 2 minutes

The part of the flow cell without immobilized secondary (anti-species) antibodies against mouse IgG was used as a control to determine the non-specific binding of MAT 8D1 and HSP70 to the chip (<2% of R _max ). As a result, for each HSP70 concentration, the control binding curve was subtracted from the corresponding binding curve with immobilized secondary (anti-species) antibodies. The equilibrium dissociation constant Kd of the antibody complex with HSP70 was determined by the dependence of the steady-state response value (RU) on the concentration of HSP70. The data obtained corresponded to the Langmuir equation for the one-center binding model.

The obtained value of the equilibrium dissociation constant of the complex of the 8D1 antibody with free recombinant HSP70 in the K _d solution was 2.25 × 10 ^-5 M, which indicates a low binding affinity, because K _d antigen-antibody complexes are usually characterized by a magnitude of the order of 10 ^-8 -10 ^-11 M [Varfolomeev SD, Gurevich K.G. Biokinetics: practical course, FAIR-PRESS, M., 1999].

Example 5. Determination of the nucleotide sequence of the genes of light and heavy chains of MAT 8D1.

To determine the amino acid sequence of antibody 8D1, a methodology was used that included the isolation of matrix ribonucleic acid (mRNA) of the corresponding hybridoma, the preparation of a complementary deoxyribonucleic acid (cDNA) based on it, the amplification of cDNA fragments encoding the variable regions of antibodies using the polymerase chain reaction method (PCR ), cloning of fragments encoding the variable parts of the heavy and light chains of the monoclonal antibody into plasmid vectors for banking and production of plasmid DNA in preparative quantities, Sanger sequencing of products and analysis of the structure of the amino acid sequence of the antibody.

Functional and structural analysis of variable fragments of the light and heavy chains of the studied antibodies was performed using the IMGT / V-QUEST tool and the database www.imgt.org.

Analysis of amino acid sequences allowed us to identify structural and functional elements: reference regions (FR) and regions determining complementarity (CDR) of the variable parts of the light and heavy chains of 8D1 antibodies, which are shown in table 1.

Example 6. Antitumor effect of MAT 8D1

In order to evaluate the antitumor effect of the 8D1 antibody, Balb / DBA mice were peritoneally injected with Sp2 / 0 myeloma cells from the culture in an amount of 1 × 10 ⁶ cells per mouse in a volume of 1 ml. After injection, the mice were divided into groups of 20 animals per group. Two different groups of animals immediately after injection of tumor cells and then with a frequency of once every two days were administered MAT 8D1 subcutaneously or intraperitoneally in an amount of 0.5 mg / mouse in a volume of 0.1 ml. Myeloma cells were introduced to a separate group, which were previously incubated with MAT 8D1 (opsonized) at the rate of 10 mg of antibodies per 1 × 10 ⁶ cells for 1 hour at 37 ° C; no antibodies were introduced to this group. Two control groups were also formed, one of which included intact animals, and the other were individuals to which the antibody was not administered.

The efficacy of MAT 8D1 was evaluated by animal survival. The experimental results are shown in FIG. 4 and 5. On days 10-12, the nodules of solid tumors began to be observed and palpated in animals at the injection site, as well as throughout the peritoneal cavity. The first animals died on day 19-20. From 24 to 27 days there was a massive death of animals with introduced myeloma without treatment. On the 27th day of the experiment, with subcutaneous administration of MAT 8D1, survival increased 3 times, with intraperitoneal - 7 times, the total lifespan of mice increased by 5 and 15 days, respectively.

The data obtained indicate that MAT 8D1 has a pronounced antitumor effect, which can be explained by the fact that the formed complex “8D1 - tumor cell” can be attacked by complement of serum, followed by cell death.

The applicant asks to consider the submitted materials of the application "Monoclonal Antibody to HSP70" for the grant of a patent of the Russian Federation for the invention.

Claims (2)

1. A monoclonal antibody that recognizes membrane-bound heat shock protein 70, comprising hypervariable regions of the light chain with sequences of SEQ ID NO: 1-3 and hypervariable regions of the heavy chain with sequences of SEQ ID NO: 4-6. 2. The monoclonal antibody according to claim 1 for the treatment of tumor diseases.

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