RU2584582C1 - Monoclonal antibody ss3-4 to conformation human epitope c3, strain of hybrid mouse dna rkkk(p)764d - producer of monoclonal antibody ss3-4 - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention refers to immunology. Disclosed is monoclonal antibody capable of specifically binding to human complement C3 molecule with exposed thioether link, and strain of mouse hybridoma RKKK(P)764D.

EFFECT: disclosed antibody blocks alternative complement activation path and can find further application in creation of antibodies suitable for clinical applications.

4 cl, 5 dwg, 1 tbl, 3 ex

Description

The invention relates to the field of biotechnology, in particular to antibodies that bind the complement component C3 of a person and block the alternative pathway of complement activation.

The complement system plays an important role in the immune defense of the body, in particular, with autoimmune diseases and diseases associated with tissue inflammation. The list of diseases associated with complement activation includes autoimmune diseases such as rheumatoid arthritis, systemic lupus erythematosus, vasculitis, multiple sclerosis, etc. [Ballanti. Immunol Res 56 (2-3): 477-91 (2013)], as well as tissue damage after ischemia and reperfusion, chronic pulmonary distress syndrome, thyroiditis, diabetes mellitus, a number of eye diseases and many other diseases [Walport. N Engl J Med 3441058-1066 (2001)]. The system includes more than 30 proteins that perform regulatory and other functions. Thus, increased concentrations of its components C3a, C5a, C5b-9, circulating in the blood and other body fluids in acute or chronic inflammation, cause and support the activation of neutrophils, monocytes, platelets, vascular endothelial cells.

The complement system is activated in three different ways: classic, alternative, and lectin. [https://ru.wikipedia.org/wiki/ complement system; Pyz et al., Ann Med 38, 242-251 (2006)]. The classical pathway is activated by antigen-antibody complexes, the lectin pathway by recognition of carbohydrate components by lectins, and the alternative pathway by binding of SZ with recognizable motifs on the pathogen surface.

All three activation pathways lead to proteolytic cleavage of C3 with the formation of C3b and C3a fragments, while a thioester is exposed in C3 and C3b molecules, the nucleophilic attack of which leads to covalent binding of C3b to an antigen (usually with the surface structures of a microbial cell). Further cleavage of the bound C3b by specific proteinases leads to the formation of fragments of C3b, C3c and C3dg, which, along with C3b and C3a, are recognized by various phagocyte receptors. C3b located on the surface of microbial cells binds complement factor B, as a result of which factor B becomes a substrate for serine proteinase - factor D. As a result of factorol proteolysis, factor B decomposes into fragments of Ba and Bb. Bb is also an active protease that remains bound to C3b on the surface of the microbial cell. The C3bBb complex is a C3 convertase that attacks C3, the concentration of which in human serum is 1.2 mg / ml. As a result, the amount of C3b increases even more, since one C3 convertase molecule produces about 1000 C3b molecules covalently linked to the microbial cell and opsonizing it for subsequent phagocytosis. In addition, C3 convertase, by adding an additional C3b molecule, becomes a C5 convertase, (C3b) ₂ Bb, cleaving C5 into C5a and C5b. Anaphylotoxins C5a and C3a are neutrophil activators of inflammatory phagocytes and chemoattractants, which attract mast cells to the infection site, which leads to local histamine release, vasodilation, accumulation of phagocytic cells in the inflammatory focus and other inflammatory effects. Further cascade reactions in the complement system also lead to the formation of a membrane-attacking complex that forms pores in the membranes of microbial cells.

Activation of complement along an alternative path includes the so-called amplification loop, which, with the participation of factors B, D and P (pro-perdine) enhances C3 proteolysis by the principle of positive feedback, which in some cases leads to negative consequences for the patient's body. One of the possible mechanisms of inhibiting the formation of active C3-convertase and subsequent blocking of the alternative complement activation pathway is the binding or blocking of C3 activation in the patient’s body, for example, using chimeric or humanized antibodies.

In particular, it is known that all successive transformations of C3 and its daughter components that occur during the activation of the alternative complement pathway are accompanied by conformational rearrangements of the molecules, which leads to the appearance of new antigenic determinants, the so-called neoantigens. Monoclonal antibodies to certain neoantigens were obtained that were able to inhibit the activation of the alternative complement pathway, in particular, murine, chimeric, and humanized antibodies to C3 and C3b and their functional fragments (WO 2004031240, WO 2013152024, RU 2473563, EA 2011101593, WO 2006012621,) selectively suppress an alternative complement activation pathway. However, none of the previously announced chimeric and humanized antibodies are still used in medical practice due to the insufficient efficiency of the technology for their preparation.

Closest to the technical nature of the claimed invention are antibodies against C3b, the Fab fragment of which inhibits an alternative complement cascade with an IC _{50 of} about 100 nM, contains CDR sequences of heavy (SEQ ID NO: 1-4) and / or light (SEQ ID NO: 5-8) chains of the S77 antibody and / or is an S77 antibody or fragment thereof [RU 2473563, 2011].

However, these antibodies have a limited scope, in particular, they do not block the activity of the C3 thioester. In addition, the technology for their preparation is not effective enough.

The technical problem solved by the authors was the expansion of the spectrum of antibodies to the component C3 of a person capable of inhibiting its biological activity, in particular, the creation of antibodies that are able to specifically bind to an antigenic determinant on a C3 molecule with an exposed thioether bond, which appears in the composition of the C3 alternative convertase paths and in the biologically active molecule of the hydrolyzed C3 component of the human complement, and not contact the native molecule C3 of the human component.

The technical result is achieved by creating a group of inventions that include CC3-4 monoclonal antibodies that specifically and with high affinity bind to the C3 molecule of the human complement component with an exposed thioether linkage and block the alternative complement activation pathway, and the mouse sCC3-4 hybridoma producing this monoclonal antibody.

The technology for producing a hybridoma included immunization of animals with an antigen representing human blood serum activated by an alternative route, the use of this modified antigen for immunization of animals, followed by selection of clones producing antibodies that bind the C3 component of the human complement with the exposed thioether linkage and do not bind native C3 , and purification of the obtained antibody.

The resulting murine monoclonal antibody recognizes a conformational epitope (neoantigen), which is usually absent in the C3 molecule and appears when it is activated via an alternative pathway. The CC3-4 monoclonal antibody of the present invention does not bind to native C3, in which the thioether bond is located inside the molecule, isolated from its surface by a loop formed by the α-chain of the molecule fragment and does not block complement activation in the classical way.

The list of drawings and other illustrative material.

In FIG. 1 shows the results of polyacrylamide gel electrophoresis of a mouse monoclonal antibody CC3-4 (4-20% polyacrylamide gel):

a - non-reducing conditions;

b - molecular weight markers (kD);

in - restoring conditions.

In FIG. 2 shows the blocking of MAT CC3-4 activation of an alternative complement activation pathway. The inhibition of APC MAT CC3-4 was shown at various ratios of antibody: antigen.

In FIG. Figure 3 shows the effect of MAT CC3-4 on the activity of C3 convertase. The stop of complement activation along an alternative time path by various agents is demonstrated.

In FIG. 4, the refinement of the epitope specificity of MAT CC3-4 was demonstrated. The results of electrophoresis in SDS page 4-20% C3 of human serum and products of its activation followed by immunoblot with polyclonal rabbit antibodies to human C3d are shown.

In FIG. Figure 5 shows the determination of the binding constant of MAT CC3-4 with C3 treated with hydrazine hydrate using a Scatchard relationship.

List of Consequences

Seq ID NO: 1 The nucleotide sequence encoding the variable region of the heavy chain of a monoclonal antibody CC3-4

Seq ID NO: 2 The nucleotide sequence encoding the variable region of the light chain antibodies CC3-4

Seq ID NO: 3 The amino acid sequence of the variable region of the heavy chain of the monoclonal antibody CC3-4

Seq ID NO: 4 The amino acid sequence of the variable region of the light chain of the monoclonal antibody CC3-4

Seq ID NO: 5 The amino acid sequence of the CDRH-1 site of the monoclonal antibody CC3-4

Seq ID NO: 6 Amino Acid Sequence of CDRH-2 Plot of CC3-4 Monoclonal Antibody

Seq ID NO: 7 The amino acid sequence of the plot CDRH-3 monoclonal antibodies CC3-4

Seq ID NO: 8 The amino acid sequence of the CDRL-1 region of monoclonal antibody CC3-4

Seq ID NO: 9 The amino acid sequence of the CDRL-2 region of the monoclonal antibody CC3-4

Seq ID NO: 10 The amino acid sequence of the CDRL-3 region of the monoclonal antibody CC3-4

The resulting strain of hybrid cultured animal cells (hybridomas), the producer of the CC3-4 monoclonal antibody to the neoantigen of the activated form of the human complement component C3 (the authors name of the strain: SCC3-4), was characterized by the following properties.

1. The pedigree of the strain (hybridoma parent cells - SP2 / 0 mouse myeloma and BALB / C mouse splenocytes; antigen - hydrosinolized C3 component of human complement; fusion was performed using polyethylene glycol followed by cloning of the producer by the method of limiting dilutions).

2. The number of passages at the time of certification and deposit is 20.

3. Standard growing conditions (cultivation at a temperature of 37 ° C, nutrient medium RPMI 1640 with 10% fetal bovine serum).

4. Cultural properties (growth type — suspension, cultivation roller or stationary, sieving ratio 1: 5, subculture time 3-4 days, sowing dose 50 thousand cells / ml).

5. Characterization of culturing a hybridoma in an animal’s body (a hybridoma is cultivated in the peritoneal cavity of syngeneic mice (BALB / C), the dose of cells inoculated 2-3 × 10 ⁶ cells / mouse, the method of sensitization of the animal - preliminary administration of 0.2 ml pristan, time ascites formation or serum selection 12-16 days, transplantability - no more than 10 passages).

6. Species data - BALB / C syngeneic mice.

7. Marker signs and methods for their assessment: the culture produces IgG1 type immunoglobulins that specifically bind the C3 component of human complement.

8. Contamination control by bacteria, fungi, mycoplasmas and viruses (no)

9. Characterization of the beneficial substance produced by the strain (the culture produces IgG1 type immunoglobulins that specifically bind the C3 component of human complement).

10. Characterization of the biosynthesis of useful products, the yield of the product on Wednesday, the level of activity - during cultivation, the hybridoma secretes a monoclonal antibody in the amount of 5-10 · 10 ⁵ μg / ml of the medium during subculture. Productivity control is carried out using enzyme-linked immunosorbent assay. The stability of antibody production is maintained for 15 passages in culture and 10 passages in animals.

11. The method of cryopreservation - cryopreservation is carried out at -70 ° C, long-term storage of the culture is carried out at -70 ° C in the refrigerator or at -196 ° C in liquid nitrogen, heating is carried out by quickly transferring the ampoule with the culture to a water bath with a temperature of 37 ° FROM. Cryopreservation medium - 90% fetal cattle serum and 10% dimethyl sulfoxide.

12. Other features of the strain - hybridoma causes the formation of solid, ascites or mixed tumors when vaccinated in the perionatal cavity of BALB / C mice at a dose of 2-3 · 10 ⁶ cells / mouse.

Hybridoma deposited in the collection under No.RKKK (P) 764D

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

Example 1. Obtaining antigens for immunization and screening of hybridomas, immunization of mice and selection of hybridoma sCC3-4.

1.1. Antigen production

For immunization, human blood serum activated by an alternative route was used. For this, 450 μl of serum was mixed with 50 μl of 20 mM Hepes pH 7.4 buffer containing 150 mM NaCl, 100 mM EGTA, 50 mM MgCl ₂ and 10 mg / ml zymosan. The resulting mixture was incubated with constant stirring for 60 minutes at a temperature of 37 ° C. The concentration of the formed C3a was used to judge the effectiveness of activation, the conversion of C3 was at least 55-60%. The resulting preparation was aliquoted and stored at -80 ° C.

To determine the specificity of monoclonal antibodies, the C3 component of the human complement, treated with hydrazine hydrate, which effectively breaks the thioether bond, was used. The C3 component of complement was obtained from healthy donor serum by immunoaffinity chromatography using monoclonal antibodies CC3a-1 to the human complement component C3 under physiological conditions. To 20 ml of human serum (CNS) was added 1 ml of 1M ε-aminocaproic acid (6-AKK) and 1 ml of 0.5M EDTA. Thus prepared serum was applied to a 2.6 × 30 cm MAT CC3a-1-Sepharose column, equilibrated with 0.02 M Na-phosphate buffer containing 0.3 M NaCl, 0.05 M 6-AKK and 0.02 M EDTA, pH 7, four. Serum was passed at a rate of 15 ml / hour, after which the column was washed at the same speed with 200 ml of equilibration buffer. Next, an additional washing of the column was carried out at the same speed with 180 ml of the same buffer and a fraction of hemolytically active C3 was collected. The solution C3 after dialysis against 0.005 M Tris-HCl buffer pH-7.8 was concentrated by ion exchange chromatography on a 1.6 × 5 cm DEAE-Sefacel column equilibrated with dialysis buffer. C3 elution was performed with a linear NaCL 0-0.5M gradient at a rate of 90 ml / hour. The fraction containing the hemolytic activity of C3 was aliquoted and stored at -80 ° C. According to the results of electrophoresis in PAGE, this fraction contained a protein with a molecular weight of 190 kD, which, under reducing conditions, decomposed into two polypeptide chains of 115 and 75 kD, which corresponds to the human complement component C3.

To obtain a hydrosinalized antigen, a hydrazine hydrate neutralized to pH 7.8 was added to a sample containing 1 mg / ml C3 to a final concentration of 0.1 M and incubated for 1 hour in a 37 ° C thermostat with constant stirring, then dialyzed against 0.02 M phosphate buffer , aliquoted and stored at -80 ° C.

1.2. Mouse immunization and hybridoma screening

Balb / c mice were immunized with the obtained ones as described in pp. 1.1 serum preparation in Freund's complete adjuvant at a concentration of 10 μg / mouse in hind limb aponeurosis. On day 28, mice were re-injected with antigen at a dose of 50 μg / mouse. After another 4 days, spleen cells were isolated and fused with SP2 / 0 myeloma cells according to the standard procedure (Shivanand Pandey. Hybridoma Technology For Production Of Monoclonal Antibodies // International Journal of Pharmaceutical Sciences Review and Research. - Volume 1, Issue 2, March - April 2010; Article 017) After 12-14 days, cloning and determination of the specificity of monoclonal antibodies secreted by clones was performed to suppress the production of C3a and C5a in the complement activation test in an alternative way, selecting neutralizing clones. For this, 50 μl of human serum was added to 50 μl of the selected culture medium, previously diluted 20 times with 20 mM HEPES buffer (pH 7.2-7.4) containing 150 mM NaCl, 10 mM EGTA, 5 mM MgCl ₂ , 1 mg / ml BSA (AIC buffer) and 10 μl of a suspension of zymosan (1 mg / ml) prepared in an AIC buffer. The plate was incubated for 1 hour at 37 ° C, after which 10 μl of a solution containing 0.2 M EDTA, 1 mM PMSF was introduced into the cells. The degree of suppression of complement activation was determined by the content of anaphylatoxins C3a and C5a in the samples after incubation. Concentrations of C3a and C5a were determined by enzyme immunoassay using standardized test systems.

To select clones that bind the C3 component of a human complement with an exposed thioether bond and do not recognize native C3, the culture medium of the clones that suppress alternative complement activation was additionally screened, for which 100 μl of washing buffer was added to the cells of the plate with adsorbed polyclonal antibodies to mouse immunoglobulins, containing 20 mm phosphate buffer and 0.05% Tween-20 and 50 μl of culture medium. After 1 hour of incubation on a shaker at 37 ° C, the cells were washed with washing buffer and 100 μl of washing buffer containing 1 mg / ml BSA and 100 ng / ml or hydrazinalized or native C3 was added. After an hour of incubation, 100 μl of a solution of conjugate of rabbit antibodies to human C3 with peroxidase were added to the cells. Clones that bind only to hydrazinalized C3 were selected for further work.

As a result, sCC3-4 clone was selected that produced the CC3-4 mouse monoclonal antibody that binds the C3 complement component with the exposed thioether linkage and does not bind the native C3 component of human complement.

As a result of subsequent intraperitoneal propagation, cryopreservation and control of cell purity, a hybridoma strain sCC3-4, a producer of monoclonal antibody CC3-4, was obtained.

Example 2. The study of the properties of monoclonal antibodies CC3-4 2.1. Cleaning MAT SS3-4

The CC3-4 antibody was isolated from the ascites fluid of Balb / c mice by the intraperitoneal inoculation of the sCC3-4 hybridoma using affinity chromatography on a Sepharose-C3 sorbent.

2.2. The study of the molecular properties of antibodies CC3-4.

According to the results of electrophoresis in a 4-20% polyacrylamide gel with sodium DDS, the molecular weight of the CC3-4 antibody under non-reducing conditions corresponds to that expected for native mouse IgG (170 kDa), under reducing conditions, the antibody dissociates into heavy (55 kDa) and light (29 kDa) chains (Fig. 1). The isotype of CC3-4 antibody established using the “Mouse monoclonal antibody isotyping reagents” kit manufactured by SIG-MA — IgG1. The preparations of purified CC3-4 antibodies were used to study its biological properties.

2.2. The study of the biological properties of monoclonal antibodies CC3-4.

2.2.1. The study of monoclonal antibody blocking (MAT) CC3-4 alternative complement activation pathway (APC). Activation of complement along the alternative pathway (APC) was carried out in 20 mM HEPES buffer (pH 7.2-7.4) containing 150 mM NaCl, 10 mM EGTA, 5 mM MgCl ₂ , 1 mg / ml BSA (APC buffer) by incubation serum with an activator of the APC zymosan in a 96-well ELISA plate. 80 μl of specific anti-C3 antibodies (Anti-C3 MAT) or nonspecific monoclonal antibodies (MAT) (positive control) and 20 μl of healthy donor blood serum containing 1 M ε-aminocaproic acid were added to the rows of tablet cells. The sample was incubated at 37 ° C with constant stirring for 10 min, after which 10 μl of a suspension of zymosan (with a concentration of 10 mg / ml) prepared in APC buffer was added to the cells. For the negative control, instead of zymosan, an AIC buffer was added. The plate was incubated for 1 hour at 37 ° C with constant stirring on a shaker. Activation of complement was stopped by adding 10 μl of a solution containing 0.2 M EDTA, 1 mM PMSF to the wells. The concentration of antibodies in the incubation samples varied from the equimolar value with respect to the concentration of C3 antigen, which was previously determined in serum samples using ELISA, downward. The established ratios of molar concentrations of Anti-C3 MAT: C3 in experimental samples were 1: 1, 1: 2, 1: 4, 1: 8, 1:16. The degree of complement activation was determined by the content of anaphylatoxins C3a and C5a in the samples after incubation. The degree of spontaneous activation of complement was taken into account by measuring the concentrations of C3a and C5a in samples containing 90 μl of Buffer A and 20 μl of serum after incubation under the same conditions. Concentrations of C3a and C5a were determined by enzyme immunoassay using a standardized test system.

The neutralizing (blocking) ability was judged by the change in the levels of anaphylactosins C3 and C5 in serum samples, depending on the concentration of antibodies.

For quantitative calculations, it was believed that the 100% activation of complement and its components C3 and C5 correspond to the concentrations of C3a and C5a, measured in samples containing non-specific MAT, i.e. in the absence of potential inhibitors. The inhibition of APC was expressed as a percentage of inhibition (PI) and was calculated for each anaphylatoxin separately according to the formula: PI (C5a / C3a) = ((A-C) - (B-C)) / (A-C)) × 100%, where :

A is the concentration of anaphylatoxin, μg / ml, in a sample that does not contain non-specific MAT.

B is the concentration of anaphylatoxin in this sample, μg / ml.

C is the concentration of anaphylatoxins, μg / ml, obtained due to spontaneous activation.

The results presented in FIG. 2 show that 100% inhibition of APC, estimated from production levels of both anaphylatoxins, is achieved with a molar ratio of MAT CC3-4: C3 equal to 1: 8. Since inhibition is achieved when the AT / AG ratio is less than unity, it becomes obvious that in order to achieve effective inhibition, it is sufficient to neutralize only a part of C3 molecules. This fact suggests that antibodies neutralize the proportion of C3 molecules involved in the initiation phase of the APC.

2.2.2. The study of monoclonal antibody (MAT) blocking of CC3-4 of the classic complement activation pathway (CCP).

A hemolytic test was performed to test the ability of MAT CC3-4 to block complement activation along the classical pathway. To activate CPC, sensitized sheep erythrocytes were used. To prepare sensitized sheep erythrocytes, 300 μl of erythrocyte mass containing 3 * 10 ⁹ erythrocytes, erythrocytes were washed three times in veronal saline buffer containing 0.1% gelatin, 2.5 mM MgCl ₂ , 0.75 mM CaCl ₂ (CCP buffer). After washing, the erythrocyte sediment was resuspended in 3 ml of CPC buffer and 3 ml of the same buffer containing the Komplekt reagent (lyophilisate of serum from a guinea pig immunized with ram red blood cells) was diluted 200 times. The suspension was incubated for 30 minutes at 37 degrees with constant stirring. At the end of the incubation, the sessibilized red blood cells were washed three times by centrifugation in a CPC buffer. The erythrocyte sediment was diluted in 14 ml CPC buffer.

50 μl of human serum diluted 10 times in buffer CPC and 50 μl of MAT CC3-4 and anti-C5 MAT solutions as a control with concentrations of 500 μg / ml, 200 μg / were added to each well for immunological work. ml, 100 μg / ml, 50 μg / ml in a CPC buffer containing 1 mg / ml BSA. After a preliminary incubation of 5 minutes, 100 μl of a suspension of red blood cells were introduced into the cells. The plate was incubated for 60 min with constant stirring at 37 ° C. Upon completion of activation, red blood cells were precipitated by centrifugation, supernatants were transferred to flat-bottomed clean plates to measure OD at 415 nm. MAT CC3-4 did not show the ability to block PDA.

2.2.3. Study of the effect of MAT CC3-4 on the activity of alternative pathway C3-convertase.

Since C3-convertase is an alternative pathway: liquid-phase, solid-phase, and the so-called C3-convertase amplification loops are Mg-dependent, the introduction of EDTA into serum stops the alternative complement activation. To study the effect of MAT CC3-4 on all of the above C3 convertases, 80 μl of HEPES buffer (pH 7.2-7.4) containing 150 mM NaCl, 1 mg / ml BSA were added to the wells of a 96-well plate for analysis , 10 mM EGTA, 5 mM MgCl ₂ , 20 μl of human blood serum containing 1M ε-aminocaproic acid and 10 μl of a suspension of zymosan in the APC buffer at a concentration of 10 mg / ml and incubated at 37 ° C and with constant stirring on a shaker in within 1 hour. At different time intervals, 10 μl of a solution containing 0.2 M EDTA, 1 mM PMSF or 10 μl MAT CC3-4 at a concentration of 1 mg / ml was added to the cells. After an hour, the degree of activation was determined by the content of anaphylatoxin C3a in the samples, which was determined by enzyme immunoassay. The inactivation curves of the alternative complement pathway shown in FIG. 3 are identical, which indicates the inactivation of MAT CC3-4 C3 convertase of the alternative pathway.

2.2.3. Clarification of the binding site of MAT CC3-4.

According to preliminary data, the specificity of MAT CC3-4 cannot be detected in a Western blot, since the nondeterminant CC3-4 is conformational and denatures during SDS processing. In order to determine which region C3 the MAT CC3-4 interacts with, C3 and its fragments binding to CC3-4 were isolated from human blood serum zymosan by affinity chromatography on a Sepharose-CC3-4 sorbent. The resulting material was analyzed in a Western blot under non-denaturing conditions. Polyclonal rabbit antibodies to C3d and C3c were used to identify fragments (Behring Diagnostics GmbH, Germany)

From the results presented in Fig. 4, it follows that the smallest fragment of C3 binding to CC3-4 is a fragment with a molecular weight of approximately 39 kDa, which is opened by antibodies to C3c and not opened by antibodies to C3d, which corresponds to a 2 fragment of C3c .

2.2.4. The study of the affinity of MAT SS3-4 to C3 treated with hydrazine hydrate

To determine the affinity of MAT CC3-4 for the antigen, the ELISA was used with the analysis of the binding results according to the Scatchard method (Varfolomeev S.D., Gurevich K.G. // Biokinetics: Practical Course. M., 1999. P. 352), based on the study of the relationship of the ratio of the equilibrium concentration of the antigen-antibody complex to the concentration of free antigen on the concentration of the complex. Human C3 treated with hydrazine hydrate and separated from the native C3 form by reverse phase chromatography was used as antigen.

Sketchard's dependence is presented in fig. 5, from which the value of the dissociation constant KD = 2.92 × 10 ^-10 M was calculated (which corresponds to the binding constant K _b = (3.42 ± 0.10) × 10 ^-9 M).

Example 3. Synthesis and sequencing of cDNA encoding the variable parts of the light and heavy chains of a monoclonal antibody CC3-4.

RNA was isolated from CC3-4 hybridoma cells, on the matrix of which DNA fragments encoding the variable regions of the heavy (VH) and light (VL) chains of the antibody were amplified using synthetic primers (Immunogenetics Information System http://www.imgt.org) . The obtained DNA fragments were cloned into the pALTA vector (Eurogen) and sequenced from external primers (M13). The sequences of DNA fragments encoding VH and VL are shown in SEQ ID No: 1 and SEQ ID No: 2; the calculated amino acid sequences of VH and VL are shown in SEQ ID No 3 and SEQ ID No 4. Analysis of amino acid sequences of the heavy and light chain variable regions CC3-4 monoclonal antibody was produced by Cabot (Kabat E.A., Wu T.T., Perry N., Gottesman K. and Foeller C. (1991) Sequences of Proteins of Immuno-logical Interest, Fifth Edition. NIH Publication No . 91-3242), which allowed to identify sections of the CDR that determine the complementarity of the antibody to the antigen (table. 1).

Comparison of sequences of CDR regions with sequences of known antibodies to components of components C3 and C3b of human complement showed a lack of homology with similar regions of known antibodies.

The CC3-4 monoclonal antibody of the present invention can serve as the basis for the construction of chimeric and humanized antibodies suitable for creating drugs that block the alternative pathway of activation of the complement system. The proposed strain allows to obtain an antibody with a high yield of the target product.

Claims (4)

1. A monoclonal antibody capable of specifically binding to a human complement complement C3 molecule with an exposed thioether linkage and comprising hypervariable regions of the heavy chain with the sequences of SEQ ID NO: 5-7 and hypervariable regions of the light chain with the sequences of SEQ ID NO: 8-10. 2. A monoclonal antibody capable of specifically binding to a human complement complement C3 molecule with an exposed thioether bond according to claim 1, having a heavy chain variable region sequence according to SEQ ID NO: 3 and a light chain variable region sequence according to SEQ ID NO: 4. 3. A monoclonal antibody capable of specifically binding to a human complement complement C3 molecule with an exposed thioether linkage according to claim 2, encoded by a DNA containing the nucleotide sequences of SEQ ID NO: 1 and SEQ ID NO: 2. 4. The mouse hybridoma strain deposited in the RKKK collection under the number RKKK (P) 764D and producing the antibody according to claim 1.

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