RU2731516C1 - Anaphylatoxin c5a antibodies to human - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: invention relates to biochemistry, particularly to an antibody capable of specifically binding to human anaphylatoxin C5a. Also disclosed is an immunosorbent for immunosorption of human anaphylatoxin C5a containing said antibody.

EFFECT: invention provides effective treatment of diseases associated with human C5a.

3 cl, 2 dwg, 2 tbl, 7 ex

Description

The group of inventions relates to the field of biotechnology, in particular to monoclonal antibodies against the complement component, namely, murine and chimeric monoclonal antibodies against human complement anaphylatoxin C5a, as well as to a hemosorbent for sorption of human complement anaphylatoxin C5a from blood and its derivatives.

Analysis of the mechanisms of pathogenesis of a number of inflammatory diseases, including systemic inflammatory response syndrome (SIRS) and sepsis, made it possible to select the main targets that play a key role in the development of these severe conditions [Chousterman B.G., Swirski F.K., Weber G.F. Cytokine storm and sepsis disease pathogenesis // Semin Immunopathol, 2017; 39 (5): 517-528]. The activation of cells of the immune system caused by bacteria and their decay products: neutrophils, macrophages and mononuclear leukocytes, makes them the main source of endogenous pro-inflammatory agents - cytokines. Proinflammatory cytokines such as tumor necrosis factor alpha (TNFα), interleukin-1 beta (IL-1β), interleukin-6 (IL-6), interleukin-8 (IL-8) appear in the blood relatively quickly after a bacterial stimulus. In turn, TNFα and IL-1β stimulate the production of other pro-inflammatory cytokines, and IL-6 stimulates the synthesis of proteins in the acute phase of inflammation. Ultimately, this leads to overproduction of cytokines and a life-threatening patient escalation of generalized inflammation [McILwain R.B., Timpa J.G., Kurundkar A.R., et al. Plasma concentrations of inflammatory cytokines rise rapidly during ECMO-related SIRS due to the release of preformed stores in the intestine // Lab Invest., 2010; 90 (1): 128-139].

Complement activation products are important pathogenetic factors in sepsis. Complement activation can occur through the interaction of bacteria and their decay products with the lectin-binding protein MBL (lectin pathway), C3 component (alternative path), and with the appearance of antibodies and the formation of immune complexes, with the C1q subcomponent (classical path) [Charchaflieh J., Rushbrook J., Worah S., Zhang M. Activated Complement Factors as Disease Markers for Sepsis // Dis Markers., 2015; 2015: 382463]. Whichever way the complement activation takes place, its result is the appearance of small molecules - anaphylatoxins C3a, C4a, C5a - products of limited proteolysis of the corresponding complement components. They interact with specific receptors and mediate a variety of inflammatory reactions [Hawksworth O.A., Li X.X., Coulthard L.G., et al. New concepts on the therapeutic control of complement anaphylatoxin receptors // Mol Immunol., 2017; 89: 36-43]. It has been established that in sepsis C5a is the most dangerous, since it stimulates many functional disorders in various organs and tissues, for example, contributes to dysfunction of the heart and blood coagulation. Acting synergistically with a number of cytokines, C5a causes an oxidative burst in neutrophils, enhances phagocytosis and the release of granular enzymes, it is a strong chemoattractant for neutrophils, as well as macrophages and monocytes, and also increases the permeability of the vascular walls [Ward P.A. Role of C5 activation products in sepsis // Scientific World Journal., 2010; 10: 2395-2402; Ward P.A. The harmful role of c5a on innate immunity in sepsis // J Innate Immun., 2010; 2 (5): 439-445]. Excessive accumulation of C5a in the blood is dangerous to the patient's life [Fattahi F., Ward P.A. Complement and sepsis-induced heart dysfunction // Mol Immunol., 2017; 84: 57-64]. It has been shown that neutralization or removal of C5a molecules has a positive effect on the outcome of diseases leading to SIRS and septic shock, as well as a number of infectious diseases [Guo R.F., Ward P.A. C5a, a therapeutic target in sepsis // Recent Pat Antiinfect Drug Discov., 2006; 1 (1): 57-65; Niederbichler A.D., Hoesel L.M., Westfall M.V., et al. An essential role for complement C5a in the pathogenesis of septic cardiac dysfunction // J Exp Med., 2006; 203 (1): 53-61].

To neutralize or remove C5a from the bloodstream, it is advisable to use antibodies that specifically bind this protein. In particular, antibodies that block the binding of C5a to receptors are expediently administered to the patient parenterally; immobilized antibodies that bind C5a can also be used for hemosorption.

Various antibodies against human C5a are known (EA 028899, EA 201691764, EA 201691481, EA 201200795, WO 0115731, WO 0305819, WO 2018175833, WO 2018184739, WO 20160181133, WO 212088247, EP 3372243).

The technical problem to be solved by the present invention is to expand the spectrum of antibodies that bind human anaphylatoxin C5a. Another technical problem is the creation of a hemosorbent capable of removing anaphylatoxin C5a from human blood and its derivatives (serum and blood plasma).

The technical result was achieved by creating a monoclonal mouse antibody to human C5a protein, specifically binding human C5a protein, as well as by creating a chimeric antibody to human C5a protein, which specifically and with high affinity (K _D = 5.976⋅10 ^-11 M) binds to human C5a. In addition, on the basis of the claimed antibodies, an immunosorbent was created, which allows efficient removal of the C5a protein from human blood and its derivatives, in particular, the sorbent with 66 μg of the claimed mouse or chimeric monoclonal antibodies binds about 90% of C5a from 70 ml of activated blood plasma.

The mouse monoclonal antibody contains the hypervariable regions of the variable region of the heavy chain according to SEQ ID NO: 3-5 and the hypervariable regions of the variable region of the light chain according to SEQ ID NO: 6-8. Chimeric monoclonal antibody contains a heavy chain according to SEQ ID NO: 9 and a light chain according to SEQ ID NO: 10. Comparative analysis of the CDR sequences of the heavy and light chains showed the absence of homology between the known antibodies to C5a and the claimed antibodies.

The technology for obtaining a murine antibody included the immunization of animals with an antigen, which is the C5a protein from the activated blood serum of donors. The same protein was used as antigens for the selection of positive producers of monoclonal antibodies when performing an enzyme-linked immunosorbent assay (ELISA) that was used for immunization. Subsequently, in the sequences of the light and heavy chains of the obtained mouse monoclonal antibody, the constant regions were replaced with the constant regions of human immunoglobulin G to obtain a chimeric (mouse-human) antibody. Chimeric antibody to human C5a was produced in transfected cultured cells of the Chinese hamster line CHO-K1, purified and immobilized on an inert carrier.

The claimed group of inventions are illustrated by the following graphic materials:

FIG. 1 shows the results of electrophoresis of purified human C5a protein in 15-25% polyacrylamide gel with sodium dodecyl sulfate. 1 - markers of molecular weight, 2 - C5a.

FIG. 2 shows the dynamics of removal of C5a from the model blood plasma using immunosorbents containing monoclonal antibodies to the human C5a protein.

The essence and industrial applicability of the invention are illustrated by the following examples:

Example 1. Obtaining monoclonal murine antibodies to human anaphylatoxin C5a.

Example 1.1. Obtaining human C5a protein. Anaphylatoxin C5a was obtained from activated blood serum of donors. To activate the serum, baker's yeast was used, which was preliminarily prepared by incubating the yeast suspension in a boiling bath for 20 min, and then washed three times, followed by centrifugation. One liter of serum was heated to 37 ° C, magnesium chloride was added to it to a final concentration of 10 mM, a carboxypeptidase-B inhibitor (MERGEPTA) to a final concentration of 0.1 nM, and prepared yeast at a rate of 20 g / L. The serum treated in this way was left for 1 hour at a temperature of 37 ° C and stirring. The activation reaction was stopped by the addition of ethylenediaminetetraacetic acid (EDTA) to a final concentration of 20 mM and the serine protease inhibitor phenylmethylsulfonyl fluoride (PMSF) to a final concentration of 0.5 mM. The activated serum was loaded onto a column with a commercial antiC5a-Sepharose immunosorbent (Boehringer, Germany). After removal of nonspecifically sorbed material by elution with 1 M NaCl, adsorbed C5a was eluted with 0.1 M glycine-HCl buffer solution, pH 2.2. The eluate was neutralized, concentrated, and freed from excess antibodies by gel filtration on a TSK G-2000SW 21.5 × 600 mm column (Toyo Soda, Japan) in HPLC mode. The purity of the preparations was checked by electrophoresis according to Laemmli in a 15-25% polyacrylamide gel gradient (Fig. 1). The resulting protein had a molecular weight of about 10 kDa, characteristic of C5a, and its purity was at least 98% according to the results of stained gel densitometry. Using a test system (LLC "Cytokin", Russia), it was shown that the obtained protein binds to antibodies to human anaphylatoxin C5a. The yield of protein C5a from 1 L of serum was 0.52 mg.

G. and Milstein C. Continuous cultures of fused cells secreting antibody of predefined specificity // Nature, 1975, 256 (5517): 495-497]. Селекцию гибридов проводили в среде RPMI-1640 с добавлением 10% телячьей эмбриональной сыворотки, содержащей 10^-4 М гипоксантина, 4⋅10^-7 М аминоптерина и 1,6⋅10^-5 М тимидина, после чего рассевали на отдельные клоны, которые культивировали в лунках 96-луночных культуральных планшетов в среде RPMI-1640 с 10% телячьей эмбриональной сыворотки.Example 1.2. Obtaining hybridomas producing mouse monoclonal antibodies to human C5a protein. To obtain monoclonal antibodies (MAT), Balb / c mice were immunized with the obtained C5a protein. For the first immunization, the antigen was emulsified in Freund's complete adjuvant (PAF), after which it was injected into the plantar aponeurosis of the hind limbs at a dose of 5-10 μg / mouse. After 4 weeks, the immunized mice were boosted by intravenous administration of 5 μg of antigen. On the fourth day after injection, lymphocytes of inguinal and abdominal lymph nodes were collected from anesthetized animals for hybridization with myeloma. The resulting lymphocytes were mixed with SP 2/0 myeloma cells in a 2: 1 ratio. Hybridization in 50% polyethylene glycol solution was carried out according to the method of Milstein and Keller [

G. and Milstein C. Continuous cultures of fused cells secreting antibody of predefined specificity // Nature, 1975, 256 (5517): 495-497]. The selection of hybrids was carried out in RPMI-1640 medium supplemented with 10% fetal calf serum containing 10 ^-4 M hypoxanthine, 4⋅10 ^-7 M aminopterin and 1.6⋅10 ^-5 M thymidine, after which they were disseminated into separate clones, which were cultured in the wells of 96-well culture plates in RPMI-1640 medium with 10% fetal calf serum.

To select clones that stably produce antibodies to the C5a protein, the concentration of antibodies to the C5a protein was determined in the culture fluid samples conditioned with the obtained clones by the method of indirect enzyme-linked immunosorbent assay, registering the binding of antibodies to the C5a protein. For this, the C5a protein obtained in example 1.1. Was immobilized in the wells of a plate with a high sorption capacity in an amount of 5 μg of protein per well. Samples of conditioned culture fluid were added to the wells with immobilized C5a and kept for 1 hour at 37 ° C, then washed, antibodies against mouse immunoglobulins labeled with horseradish peroxidase (Sigma, USA) were added to the wells of the plates. The plate was incubated at 37 ° C for 40 min, washed, after which the chromogenic substrate tetramethylbenzidine (TMB) was added. After stopping the reaction by adding sulfuric acid, the plates were scanned on a plate analyzer, measuring the optical density at 450 nm.

Selected positive clones were repeatedly cloned by the final dilution method on peritoneal macrophages with the selection of the most productive and stable clones. As a result, three clones were selected, designated as AC5a-2, AC5a-7 and AC5a-11.

In order to test the stability of the hybridomas, they were cultured in RPMI-1640 medium containing 20% fetal bovine serum, 2 mM L-glutamine, and 50 μg / ml gentamicin. Subculture of hybridoma cells was performed 2 times a week with a dilution ratio of 1: 2-1: 3. The inoculum concentration of cells was 10 ⁵ in 1 ml of medium.

The maximum concentration of hybrid cells during cultivation was 10 ⁵ in 1 ml of medium. The studied hybrid clones AC5a-2, AC5a-7, and AC5a-11 did not lose their ability to synthesize antibodies during the entire observation period (15 passages), which indicated their stability.

Example 1.3. Purification of monoclonal antibodies. The production of monoclonal antibodies produced by clones AC5a-2, AC5a-7, AC5a-11 was carried out in the peritoneal cavity of inbred BALB / c mice, which were injected intraperitoneally with 0.5 ml of 14-tetramethylenepentadecane (pristane) before hybridoma cells were injected. After 14 days, the animals were also injected intraperitoneally with 10 ⁶ hybrid cells per mouse. When the abdominal cavity reached a noticeable volume, ascitic fluid (3-5 ml / mouse) containing monoclonal antibodies was taken, in which the concentration of antibodies was determined by ELISA. The concentration of monoclonal antibodies in the ascitic fluid samples from mice varied from 2 to 7 mg / ml.

Monoclonal antibodies were isolated from ascitic fluid by precipitation with ammonium sulfate followed by chromatography on a column with a MabSelect affinity sorbent (GE-healthcare Life Science, USA). After isolation and purification, each antibody was concentrated by ultrafiltration in a cell with a polyethersulfone membrane and a nominal retention mass of 30 kDa (Sartorius, Germany) to a protein concentration of approximately 10 mg / ml. As a result, monoclonal antibodies (MAB) AC5a-2 in the amount of 117.7 mg, AC5a-7 in the amount of 97.8 mg and 107.1 mg of MAB AC5a-11 were obtained.

Example 2. Study of the properties of monoclonal antibodies AC5a-2, AC5a-7, AC5a-11 to human C5a protein.

Antibodies were characterized by polyacrylamide gel electrophoresis under reducing conditions, which showed the presence of two polypeptide chains with molecular weights of 53 and 23 kDa, which corresponds to the heavy and light chains of immunoglobulins. Isotyping of the obtained MABs using a commercial ISO-2 kit (Sigma, USA) showed that all three types of MABs belong to IgG1.

The parameters of the interaction of MAT with C5a were measured by surface plasmon resonance (SPR) at 25 ° C using chips with carboxymethylated dextran (CM5), on a Biacore X100 device with a double flow cell (GE-Healthcare Life Science, 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) antibodies against mouse IgG was injected at a rate of 5 μl / min for 18 min (5 μg / ml in 10 mM sodium acetate buffer, pH 5.0). The free groups of the chip were then 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.

Increasing concentrations of C5a protein in the 12.5-800 nM range were injected above the chip after MAb injection and washing with HBS-P + buffer solution (150 mM NaCl, 10 mM Hepes-NaOH, pH 7.5, 0.05% polyoxyethylene sorbitan) at a rate of 10 μl / min. Each experiment consisted of 6 stages:

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

2) injection of MAT to C5a for 3 min;

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

4) injection of C5a within 1 min;

5) stage of dissociation (washing with HBS-P) for 5 min;

6) regeneration with 3M magnesium chloride solution for 2 min.

A part of the flow cell without immobilized secondary (anti-species) antibodies against mouse IgG was used as a control to determine the nonspecific binding of anti-C5a and C5a mAbs to the chip (<2% of Rmax). Finally, for each concentration of C5a, the control binding curve was subtracted from the corresponding binding curve with immobilized secondary (anti-species) antibodies. The equilibrium dissociation constant K _{D of} the “MAT to C5a - C5a” complex was determined from the dependence of the steady-state response value (RU) on the C5a concentration, while the data corresponded to the Langmuir equation for the single-center binding model:

where R _eq is the response (RU) when equilibrium is reached, R _max is the response (RU) when the surface is saturated with analyte. The kinetic parameters: k _a is the rate constant of association, k _d is the rate constant of dissociation, K _D is the equilibrium constant of dissociation, and R _max is the maximum binding capacity of the chip surface, were calculated using the 1: 1 binding model.

The results are shown in Table 1.

Analysis of the data in Table 1 shows:

- MAT AC5a-2, in comparison with the other two MAT, are characterized by the largest association constant K _a = 83.95 1 / Ms, the lowest equilibrium dissociation constant K _D = 5.69⋅10 ^-9 M and the average kinetic dissociation constant K _d = 0.477 1 / s. Consequently, MAB AC5a-2 has the highest affinity for the C5a protein.

- MAT AC5a-11 shows the highest Rmax value and, therefore, it has the ability to bind (adsorb) the greatest amount of antigen. In addition, MAB AC5a-11 is characterized by the lowest value of _Kd = 0.0027 1 / s, which indicates its property of irreversibly binding the antigen.

- MAT AC5a-7 have the worst parameters of interaction with the antigen.

Example 3. Obtaining and study of the effectiveness of immunosorbents when removing anaphylatoxin C5a from the model solution.

In order to select the optimal antibodies for removing C5a from the solution, three samples of immunosorbents were synthesized using MAT AC5a-2, AC5a-7, and AC5a-11 as ligands, and CNBr-activated Sepharose-4 FF as a matrix (GE Healthcare Life Science ", USA). Immunosorbents were designated as IS- (AC5a-2), IS- (AC5a-7), IS- (AC5a-11). The synthesis was carried out according to the manufacturer's instructions, to obtain a ligand concentration of 10 mg MAT / ml sorbent.

The resulting immunosorbents with a volume of 0.2 ml each were placed in chromatographic columns 5 × 10 mm in size. The capacity of the columns was assessed by passing blood plasma through them, into which the C5a protein was added to a final concentration of 1.5 μg / ml. 150 ml of this plasma was passed through each column in a continuous cycle for 16 hours. After the end of the cycle, the column was washed first with physiological buffer solution and then with a buffer solution containing 1 M sodium chloride. After that, adsorbed C5a was eluted from the column with glycine buffer solution, pH 2.5, and the amount of adsorbed protein was determined using ELISA. The capacity of all sorbents was approximately the same and equal to 110 μg.

To study the efficiency of the removal of protein C5a by the obtained immunosorbents, blood plasma was prepared, which was activated using baker's yeast according to the method described in example 1. The concentration of C5a in the activated plasma, determined by ELISA, was 550 ng / ml.

Through columns with immunosorbents IS- (AC5a-2), IS- (AC5a-7), IS- (AC5a-11), 120 ml of activated plasma with a concentration of C5a 0.55 μg / ml was passed. Chromatography was performed at a rate of 0.5 ml / min, taking samples every 10 ml. The last sample was taken after the entire volume of serum was applied. The concentration of C5a in the samples was determined by ELISA. The results are shown in FIG. 2, it follows from these results that all three samples of immunosorbents had the ability to remove C5a from the model solution. At the same time, the immunosorbent IS- (AC5a-2) with immobilized antibodies AC5a-2 possessed the highest efficiency, with which, under the conditions of a model experiment, it was possible to remove about 90% of C5a when passing 70 ml of model blood plasma, while immunosorbents IS- (AC5a-7) and IS- (AC5a-11) removed 90% of protein C5a when passing 10 and 30 ml of blood plasma, respectively, after which the efficiency of binding of protein C5a by these immunosorbents decreased. Based on the results obtained in examples 2 and 3, monoclonal antibody AC5a-2 was chosen for further work.

Example 4. Determination of nucleotide sequences and analysis of genes of the variable regions of the light and heavy chains of the murine monoclonal antibody AC5a-2.

Example 4.1. Obtaining and analysis of DNA sequences encoding the variable regions of the heavy and light chains of the murine monoclonal antibody AC5a-2. Ribonucleic acid (RNA) was isolated from cells of clone AC5a-2 using the GeneJET RNA Purification kit (Thermo scientific, Lithuania), and then cDNA was obtained on the template of this RNA by reverse transcription using primer oligo (dT) 18 and reverse transcriptase MMLV (JSC Evrogen, Russia) according to the manufacturer's instructions. Then, using the method of polymerase chain reaction (PCR) using known sets of primers and a high-precision mixture of DNA polymerases High Fidelity PCR Enzyme Mix (Thermo scientific, Lithuania), DNA fragments encoding the variable regions of the heavy and light chains of the antibody were obtained. The reaction was carried out in a C1000 thermal cycler (Bio-Rad, USA) under the following conditions:

- primary denaturation at a temperature of 94 ° C - 5 minutes;

- next 37 cycles:

- denaturation at a temperature of 95 ° C - 15 sec .;

- annealing at a temperature of 55 ° С - 20 sec .;

- amplification at 72 ° C - 20 sec .;

- final amplification at 72 ° C - 15 min.

PCR products of the variable fragments of the light and heavy chains of immunoglobulins were analyzed by electrophoresis in 1.5% agarose gel. Product bands of the expected size in the region of 450 base pairs were excised from the gel, eluted using a kit of reagents for DNA isolation from agarose gel (ZAO Evrogen, Russia). Thereafter, the resulting PCR products were analyzed by sequencing. Sequencing was performed on an AB3500 capillary sequencer (Applied Biosystems, USA) using a commercial BigDye® Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, USA), samples were purified before analysis using a BigDye® XTerminator ™ Purification Kit (Applied Biosystems, USA).

The found sequences of the obtained DNA fragments encoding the variable regions of the heavy and light chains of the monoclonal antibody AC5a-2 are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively.

Functional and structural analysis of the variable fragments of the light and heavy chains of the antibody under study, carried out using the IMGT / V-QUEST tool and the IMGT database [www.imgt.org], showed the location of the frame and complementarity-forming regions (CDRs). The found CDR sequences are shown in the sequence listing according to Table 2, in which the CDRs of the heavy chain are designated CDRH and the CDRs of the light chain are designated as CDRL.

Example 5. Obtaining genes of a chimeric (mouse-human) antibody to human C5a protein and plasmid vectors expressing the light and heavy chains of this chimeric antibody.

Amplification of the sequences of genes for the constant regions of the light (IGKC) and heavy (IGHG1) chains of immunoglobulin G1 / kappa human was performed on a cDNA template from human blood mononuclear cells, while the reverse primers contained a recognition site for the restriction nuclease XhoI. The annotated mRNA sequence BC090940.1 (Homo sapiens immunoglobulin heavy constant gamma 1 (G1m marker), mRNA) was used as the main mRNA sequence for the selection of primers for cloning the gene for the constant part of the human IgG1 heavy chain.

The annotated sequence of the J00241.1 kappa locus gene (Homo sapiens immunoglobulin kappa chain constant region (IGKC) gene) was used as the main mRNA sequence for the selection of primers for cloning the gene for the constant part of the human kappa light chain.

A signal peptide sequence containing a recognition site for the EcoRI restriction enzyme was artificially synthesized. The amplification of the variable regions of the light and heavy chains of murine antibodies was performed using specially selected primers, which included regions complementary to the regions of the signal sequence and the constant region flanking the variable region of the chimeric antibody.

The amplified DNA fragments were purified by preparative electrophoresis in 1.5% agarose gel (Gibco, USA). An agarose section containing a DNA band of the required size was excised, and the DNA fragment was purified using a DNA gel purification kit (Evrogen, Russia) according to the manufacturer's instructions.

Splicing by Overlap-Extension PCR (SOE-PCR) was used to assemble sequences containing an open reading frame for the full-length light or heavy chain of the chimeric antibody. The synthesized signal peptide sequence and the PCR products purified at the previous stage containing the sequences of the variable region of the light or heavy chains of a murine antibody and the constant region of the light or heavy chains of human IgG, respectively, were used as starting elements. The sequences of the obtained heavy and light chains of the chimeric (mouse-human) antibodies, verified by sequencing, are shown in SEQ ID NO: 9 and SEQ ID NO: 10. Next, DNA fragments encoding the heavy and light chains of the chimeric anti-C5a antibody were treated with EcoRI and XhoI restriction enzymes (Fermentas, Lithuania) and ligated with EcoRI and XhoI digested vector pOptiVec.

After the end of the ligation reaction, the resulting plasmids were transformed into competent E. coli cells. The transformed cells were scattered into individual clones, from which plasmid DNA was isolated, the correct assembly of which and the absence of possible mutations were checked by sequencing. Thus, the plasmids pOptiVec / C5a2H and pOptiVec / C5a2L were obtained, encoding the heavy and light chains of the chimeric (mouse-human) antibodies to the human C5a protein, respectively.

Example 6. Obtaining a chimeric antibody and studying the parameters of its binding to human C5a protein.

Example 6.1. Temporary expression of recombinant immunoglobulins in HEK293 cells. Plasmids pOptiVec / C5a2H and pOptiVec / C5a2L were isolated from E. coli cells using a commercial QIAGEN Plasmid Mini Kit (Qiagen, United States). Transfection of HEK293 cells, pre-seeded into the wells of a 24-well plate at 1.3⋅10 ⁵ cells per well in DMEM medium (Biolot, Russia), was performed using the liposomal reagent TurboFect ™ (Fermentas, Lithuania) according to the manufacturer's instructions, while in the wells 1 μg of plasmids pOptiVec / C5a2H and pOptiVec / C5a2L was added in a ratio of 1: 1, 1: 2, 2: 1, 3: 2, 2: 3. The transformed cells were cultured for 3 and 4 days, after which the culture medium was taken from the wells, in which the concentration of human IgG was determined using an enzyme-linked immunosorbent assay with mouse anti-human IgG mAb (FGUP GosNII OChB, Russia) according to the manufacturer's procedure. The maximum concentration of human IgG was obtained using plasmid DNA in a ratio of 2: 3 on the 4th day of cultivation and amounted to 1.38 μg / ml. Further operating time of the chimeric monoclonal antibody was performed in 5 T175 culture flasks (Costar, USA), each of which was seeded at ⁷ 2,7⋅10 HEK293 cells following transfection plasmid DNA (50 ug pOptiVec / C5a2H and 50 ug pOptiVec / C5a2L on bottle) according to the method described above. After 5 days of cultivation, the chimeric antibody against the C5a protein was purified from the culture medium as described in example 3.

The determination of the binding of the chimeric antibody to the C5a protein was carried out by surface plasmon resonance, as described in example 2, using antibodies to human IgG immobilized in the flow cell of the device. As a result, the following characteristics of the binding of the antibody to the human C5a protein were obtained:

association constant К _a = 2.245⋅10 ⁸ 1 / Ms;

dissociation constant K _d = 0.01342 1 / s;

equilibrium dissociation constant K _D = 5.976⋅10 ^-11 M.

Example 7. Preparation and study of the effectiveness of an immunosorbent based on a chimeric antibody in removing the C5a protein from a model solution. Immobilization of 2 mg of purified chimeric antibody against C5a and the study of the efficiency of binding of protein C5a from activated human serum to the obtained immunosorbent was carried out as described in example 3. It was shown that at least 90% of protein C5a was removed when 70 ml of activated serum passed through the column, Thus, the efficiency of binding of the C5a protein with the immobilized chimeric antibody does not differ from the efficiency of the binding of the C5a protein by the immobilized mouse monoclonal antibody AC5a-2.

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Sequence listing

<110> Federal State Unitary Enterprise

"State Research Institute of Highly Pure Biological Products" of the Federal Medical and Biological Agency

Federal State Unitary Enterprise “State Research Institute of Highly Pure

Biopreparations "of the Federal Medical and Biological Agency

<120> Antibodies against human anaphylatoxin C5a

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Ser Met Lys Leu Ser Cys Val Ala Ser Gly Phe Thr Phe Ser Asp Ala

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TGG ATG GAC TGG GTC CGC CAG TCT CCA GAG AAG GGG CTT GAG TGG GTT 144

Trp Met Asp Trp Val Arg Gln Ser Pro Glu Lys Gly Leu Glu Trp Val

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GCT GAA GTT AGA ATC AAA ACT AAT AAT CAT GCA ACA TAC TAT GCT GAG 192

Ala Glu Val Arg Ile Lys Thr Asn Asn His Ala Thr Tyr Tyr Ala Glu

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TCT GTG AAA GGG AGG TTC ACC ATC TCA AGA GAT GAT TCC AAA AGT AGT 240

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ser

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GTC TAC CTG CAA ATG AAC AGC TTA AGA CCT GAA GAC ACT GCC ATT TAT 288

Val Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Ile Tyr

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TAC TGT ACT ATG ATT GAT TAT GCT ATG GAC TAC TGG GGT CAA GGA ACC 336

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Gln Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly

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GAG AAG GTC ACC ATG ACC TGC AGT GCC AGC TCA AGT GTA AGT TAC ATG 96

Glu Lys Val Thr Met Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Met

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TAC TGG TAC CAG CAG AAG CCA GGA TCC TCC CCC AGA CTC CTG ATT TAT 144

Tyr Trp Tyr Gln Gln Lys Pro Gly Ser Ser Pro Arg Leu Leu Ile Tyr

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GAC ACA TCC AAC CTG GCT TCT GGA GTC CCT GTT CGC TTC AGT GGC AGT 192

Asp Thr Ser Asn Leu Ala Ser Gly Val Pro Val Arg Phe Ser Gly Ser

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GGG TCT GGG ACC TCT TAC TCT CTC ACA ATC AGC CGA ATG GAG GCT GAA 240

Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Met Glu Ala Glu

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GAT GCT GCC ACT TAT TAC TGC CAG CAG TGG AGT AGT TAC CCG CTC ACG 288

Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Tyr Pro Leu Thr

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TTC GGT GCT GGG ACC AAG CTG GAG CTG AAA C 319

Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys

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<213> Mus musculus

<220>

<222> (1) ... (8)

<223> Amino acid sequence of the CDR-1 region of the heavy chain variable region of the mouse monoclonal antibody C5a-2

<400> 3

Gly Phe Thr Phe Ser Asp Ala Trp

1 5 8

<210> 4

<211> 10

<212> protein

<213> Mus musculus

<220>

<222> (1) ... (10)

<223> Amino acid sequence of the CDR-2 region of the heavy chain variable region of the mouse monoclonal antibody C5a-2

<400> 4

Val Arg Ile Lys Thr Asn Asn His Ala Thr

1 5 10

<210> 5

<211> 9

<212> protein

<213> Mus musculus

<220>

<222> (1) ... (9)

<223> Amino acid sequence of the CDR-3 region of the heavy chain variable region of the mouse monoclonal antibody C5a-2

<400> 5

Thr Met Ile Asp Tyr Ala Met Asp Tyr

1 5 9

<210> 6

<211> 5

<212> protein

<213> Mus musculus

<220>

<222> (1) ... (5)

<223> Amino acid sequence of the CDR-1 region of the light chain variable region of the mouse monoclonal antibody C5a-2

<400> 6

Ser Ser Val Ser Tyr

15

<210> 7

<211> 3

<212> protein

<213> Mus musculus

<220>

<222> (1) ... (3)

<223> Amino acid sequence of the CDR-2 region of the light chain variable region of the mouse monoclonal antibody C5a-2

<400> 7

Asp Thr Ser

13

<210> 8

<211> 9

<212> protein

<213> Mus musculus

<220>

<222> (1) ... (9)

<223> Amino acid sequence of the CDR-3 region of the light chain variable region of the mouse monoclonal antibody C5a-2

<400> 8

Gln Gln Trp Ser Ser Tyr Pro Leu Thr

1 5 9

<210> 9

<211> 470

<212> protein

<213> Artificial sequence

<220>

<222> (1) ... (470)

<223> Amino acid sequence of the heavy chain of the chimeric antibody C5a-2x

<400> 9

Met Asp Phe Gln Val Gln Ile Phe Ser Phe Leu Leu Ile Ser Ala Ser

1 5 10 15

Val Ile Ile Ser Arg Gly Glu Val Lys Leu Glu Glu Ser Gly Gly Gly

20 25 30

Leu Val Gln Pro Gly Gly Ser Met Lys Leu Ser Cys Val Ala Ser Gly

35 40 45

Phe Thr Phe Ser Asp Ala Trp Met Asp Trp Val Arg Gln Ser Pro Glu

50 55 60

Lys Gly Leu Glu Trp Val Ala Glu Val Arg Ile Lys Thr Asn Asn His

65 70 75 80

Ala Thr Tyr Tyr Ala Glu Ser Val Lys Gly Arg Phe Thr Ile Ser Arg

85 90 95

Asp Asp Ser Lys Ser Ser Val Tyr Leu Gln Met Asn Ser Leu Arg Pro

100 105 110

Glu Asp Thr Ala Ile Tyr Tyr Cys Thr Met Ile Asp Tyr Ala Met Asp

115 120 125

Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser Ala Ser Thr Lys

130 135 140

Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly

145 150 155 160

Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro

165 170 175

Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr

180 185 190

Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val

195 200 205

Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn

210 215 220

Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro

225 30 235 240

Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu

245 250 255

Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp

260 265 270

Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp

275 280 285

Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly

290 295 300

Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn

305 310 315 320

Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp

325 330 335

Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro

340 345 350

Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu

355 360 365

Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn

370 375 380

Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile

385 390 395 400

Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr

405 410 415

Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys

420 425 430

Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys

435 440 445

Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu

450 455 460

Ser Leu Ser Pro Gly Lys

465 470

<210> 10

<211> 235

<212> protein

<213> Artificial sequence

<220>

<222> (1) ... (235)

<223> Amino acid sequence of the light chain of the chimeric antibody C5a-2x

<400> 10

Met Asp Phe Gln Val Gln Ile Phe Ser Phe Leu Leu Ile Ser Ala Ser

1 5 10 15

Val Ile Ile Ser Arg Gly Gln Ile Val Leu Thr Gln Ser Pro Ala Ile

20 25 30

Met Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Ser Ala Ser

35 40 45

Ser Ser Val Ser Tyr Met Tyr Trp Tyr Gln Gln Lys Pro Gly Ser Ser

50 55 60

Pro Arg Leu Leu Ile Tyr Asp Thr Ser Asn Leu Ala Ser Gly Val Pro

65 70 75 80

Val Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile

85 90 95

Ser Arg Met Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp

100 105 110

Ser Ser Tyr Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys

115 120 125

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

130 135 140

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

145 150 155 160

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

165 170 175

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

180 185 190

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

195 200 205

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

210 215 220

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

225 230 235

<---

Claims (3)

1. A monoclonal antibody capable of specifically binding to human anaphylatoxin C5a, comprising the hypervariable regions of the heavy chain of SEQ ID NO: 3-5 and the hypervariable regions of the light chain of SEQ ID NO: 6-8. 2. The antibody according to claim 1, having a heavy chain sequence according to SEQ ID NO: 9 and a light chain sequence according to SEQ ID NO: 10. 3. Immunosorbent for immunosorption of human anaphylatoxin C5a, containing an antibody according to claim 1 or 2.

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