RU2630647C1 - Humanized antibody to conformational c3 epitope constituent of human complement, dna sequence (versions), expression vector containing dna sequence (versions), and ovarian cells' strain of chinese hamster cho-humc34-producer of this humanized antibody - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to a humanized antibody, which binds to the C3 epitope of the human complement component. DNA encoding the mentioned antibody, an expression vector, for expressing the mentioned antibody, and also a ovarian cells' strain of chinese hamster which produces the mentioned antibody are also disclosed.

EFFECT: invention has the ability to bind specifically to the C3 epitope of the human complement component, which allows the effective treatment of diseases associated with the C3 component of the human complement.

12 cl, 6 dwg, 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 inhibit the activation of the complement system in an alternative way.

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.

At the same time, when the balance between activation proteins and regulatory proteins is violated, complement overactivation occurs, which leads to anak on their own cells and tissues, which causes the onset and development of serious diseases, including autoimmune diseases, operative interventions, extensive injuries, neurodegenerative processes, organ transplantation and tissues.

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 conditions of 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 C3 to recognizable motifs on the surface of the pathogen.

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 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, attracting 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.

An alternative pathway (AP) of activation refers to one of the factors of innate immunity and is aimed at recognizing and destroying pathogens, as well as at the clearance of cells that died due to apoptosis, necrosis, and their derivatives [Merle NS, Church SE, Fremeaux-Bacchi V, Roumenina LT . Complement System Part I - Molecular Mechanisms of Activation and Regulation. Front Immunol. 2015; V. 6: Article 262]. Activation of complement along an alternative pathway occurs continuously due to the slow spontaneous hydrolysis of the thioether bond in the C3 molecule, resulting in a change in its conformation and transition from an inactive form to a bioactive form of C3 (H ₂ O). The lifetime of the active form of C3 (H ₂ O) is calculated in milliseconds, after which the molecule turns into an inactive form, denoted by C3i, and is rapidly excreted from the body. The amount of modified C3 in the form of C3 (H ₂ 0) and / or C3i is 1-2% of the total circulating C3 and is approximately 1 μg.

Next, a cascade of activation reactions unfolds in a certain sequence. C3 in the form of C3 (H ₂ O) binds to factor B (FB). The resulting complex C3 (H ₂ O) B is attacked by a unique serine protease factor D (FD) with the cleavage of part of the molecule of factor B - Ba. The other part of FB - Bb remains bound to C3 (H ₂ O). The newly formed complex C3 (H ₂ O) Bb is a liquid phase C3 convertase, the substrate of which is native C3. As a result of the action of the newly formed enzyme, C3 circulating in the blood is split into two fragments - C3b and C3a. The latter remains in solution and performs its own functions of anaphylatoxin, while reactive C3b can covalently bind via amide or ether bonds to suitable acceptor sites on the surface of activator (A) located in the microenvironment, forming the A-C3b complex. Next, a transformation cycle occurs, leading to the formation of C3 and C5 convertases and the generation of additional amounts of C3b.

The efficiency of this process is small, but it can be enhanced through a positive feedback mechanism. In this case, A-C3b interacts with the same factors B and D, forming a more stable solid-state C3 convertase A- (C3b) _n Bb, which is further stabilized by another unique protein, properdine or factor P (FP). The resulting complex A- (C3b) _n BbP is already an enzyme with new properties - C3 / C5 convertase, which effectively attacks its substrates C3 and C5.

The C5 component, like C3, is cleaved into a large C5b fragment and small C5a (high potency anaphylatoxin). C5b interacts with a suitable target, becoming the first link in the formation of the membrane-attacking complex (MAC), which causes the death of the target cell by perforating its membrane [Merle NS, Church SE, Fremeaux-Bacchi V, Roumenina LT. Complement System Part I - Molecular Mechanisms of Activation and Regulation. Front Immunol. 2015; V. 6: Article 262]. The activators (A) of the alternative pathway are not only microorganisms that have fallen into the microenvironment, but also their own cells. However, all cells of our body are reliably protected by inhibitors called regulatory proteins, which interrupt the complement activation cascade at the stages of formation of C3 / C5 convertases and at the terminal stage of MAK formation, avoiding damage to their own cells. At the same time, microorganisms that do not have such a protective apparatus undergo lysis and subsequent elimination of complement.

Inhibitory regulatory molecules include, in particular, a soluble cofactor protein called factor H (FH), membrane cofactor protein MCP (CD46), type 1 complement receptor CR1 (CD35) and DAF converting factor convert factor (CD55) [Merle NS, Church SE, Fremeaux-Bacchi V, Roumenina LT. Complement System Part I - Molecular Mechanisms of Activation and Regulation. Front Immunol. 2015; V. 6: Article 262; Merle NS, Noe R, Halbwachs-Mecarelli L, Fremeaux-Bacchi V, Roumenina LT. Complement System Part II: Role in Immunity. Front Immunol. 2015; V. 6: Article 257.]. Mentioned cofactors, which include CRI, interact with C3b bound to the activating surface and change its conformation, making factor I (FI) accessible for attack by the complement serine protease, which cleaves the fragment and transforms it into the inactive form of iC3b. The functions of regulatory proteins are modulated by a number of glycoproteins on the cell surface [Merle NS, Noe R, Halbwachs-Mecarelli L, Fremeaux-Bacchi V, Roumenina LT. Complement System Part II: Role in Immunity. Front Immunol. 2015; V. 6: Article 257].

Under physiological conditions, in normal blood serum there is a balance between proteins involved in activation by an alternative pathway: C3, FB, FD, FP - and regulatory proteins: FH, FI, CD35, CD46, and CD55. However, there are many reasons for upsetting this balance. In particular, in the case of genetic disorders associated with mutations in the genes responsible for the production of at least one of the regulatory proteins, unregulated activation occurs, which leads to a complement attack of their own cells, which causes serious diseases. Among them, paroxysmal nocturnal hemoglobinuria, atypical hemolytic syndrome, age-related retinal degeneration are well studied. Such diseases are referred to as complement-associated diseases.

In addition, as a result of external influences, such as injuries, surgical interventions, organ and tissue transplantation, hemodialysis, hemosorption, as well as internal disturbances: ischemic injuries (heart attacks, strokes, cerebral edema), autoimmune processes, sepsis, complement activation intensifies and increased generation of pro-inflammatory molecules C3a, C5a and macromolecular complex C5b-9, which are chemo-attractants for neutrophils, monocytes, mast cells and basophils migrating to the site of inflammation, by inducers of c itokines and adhesion molecules, and also stimulate apoptosis. Thus, unregulated activation of complement leads to pathological inflammation and serious illness [Holers V.M. Complement and Its Receptors: New Insights Into Human Disease. Annu. Rev. Immunol. 2014.32: 433-59; He S, Atkinson C, Qiao F, Cianflone K, Chen C, Tomlinson S. 2009. A complement-dependent balance between hepatic ischemia / reperfusion injury and liver regeneration in mice. J. Clin. Investig. 119: 2304-16; Chen M, Daha MR, Kallenberg CG. The complement system in systemic autoimmune disease. J Autoimmun. 2010; 34: J276-86; Parkes M, Cortes A, van Heel DA, Brown MA. 2013. Genetic insights into common pathways and complex relationships among immune-mediated diseases. Nat. Rev. Genet. 14: 661-73; Holers V.M. The spectrum of complement alternative pathway-mediated disease. Immunol. Rew. 2008, V. 223, P. 300-316; Mastellos DC, Ricklin D, Hajishengallis E, Hajishengallis G, Lambris JD. Complement therapeutics in inflammatory diseases: promising drug candidates for C3-targeted intervention. Mol Oral Microbiol. 2016; 31 (1): 3-17].

To solve the above problems as therapeutic agents, it is advisable to use artificial complement blockers that can interrupt the cascade of activation reactions at any of its stages. The most effective seems to be blocking the activation of the alternative pathway by binding or blocking the activation of the active site on the C3 molecule, which is involved in interaction with other components of the reaction chain at the activation initiation stage, but is not exposed on the native molecule in the patient’s body, for example, using chimeric or humanized antibodies. Moreover, the direct neutralization of the C3 component is impractical due to its high concentration in blood serum.

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 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 antibodies are still used in medical practice due to the insufficient efficiency of the technology for their preparation.

The closest in technical essence to the claimed invention is the previously developed technology for the production of murine monoclonal antibody CC3-4, which recognizes the conformational epitope (neoantigen), which is usually absent in the C3 molecule and appears when it is activated via an alternative pathway, the CC3-4 antibody, which is specific and with high affinity binds to the C3 molecule of the human complement component and blocks the alternative pathway for complement activation [RU 2584582, 2016] based on the use of a hybridoma strain yshi deposited at RKKK RKKK collection under the number (n) 764D and producing the antibody.

The disadvantage of this technology is the production of non-humanized antibodies, which reduces their practical significance. In particular, the analysis of amino acid sequences in the light and heavy chains of the CC3-4 antibody showed the presence of CDR regions that are different from the CDR regions of other known antibodies to the C3 component of the human complement. It was established that the dissociation constant SS3-4 C3 component of human complement to an exposed tiofirnoy bond determined by ELISA and calculated by Scatchard, is only 2,92 × 10 ^-10 M.

The technical problem solved by the authors was to expand the range of antibodies to the human component C3, capable of inhibiting its biological activity, by creating a producer strain of a new humanized antibody to the conformational epitope C3 of the human complement component, which can be tested as a therapeutic antibody to block unregulated activation complement systems in an alternative way.

The technical result is achieved by creating a group of inventions, including a monoclonal humanized antibody hC34, characterized by the presence of altered (humanized) sequences of light and heavy chains that contain regions that bind the conformational epitope C3 of the human complement component (CDR regions) of the mouse monoclonal antibody CC3-4, surrounded by fragments of human IgG, as well as constant regions of human IgG, which specifically and with high affinity binds to the C3 molecule cient human complement and blocked alternative pathway complement activation, and a strain of Chinese hamster ovary CHO-humC34-producing cells humanized recombinant antibodies and humanized antibodies against a conformational epitope (neoantigenu) an activated form of human complement component C3.

In this case, the amino acid sequences CDRH-1, CDRH-2, CDRH-3 of the mouse CC3-4 antibody are used, which are involved in the formation of the pTVK4 / humC34-H expression vector and the amino acid sequences CDRL-1, CDRL-2, CDRL-3 of the mouse CC3 antibody -4, which are involved in the formation of the expression vector pTVK4d / humC34-L.

The expression vector pTVK4 / humC34-H includes a promoter of the early cytomegalovirus proteins; the heavy chain gene of a humanized anti-human C3 conformational epitope antibody; RNA polyadenylation site from HSV thymidine kinase gene; promoter of early SV40 virus proteins; neomycin resistance gene; polyadenylation site of early SV40 virus proteins; ampicillin resistance gene and regulatory element that enhances the expression of the target gene.

The expression vector pTVK4d / humC34-L includes a promoter of early cytomegalovirus proteins; light chain gene of a humanized antibody to a human C3 conformational epitope; RNA polyadenylation site from HSV thymidine kinase gene; promoter of early SV40 virus proteins; dihydrofolate reductase gene; polyadenylation site of early SV40 virus proteins; ampicillin resistance gene and regulatory element that enhances the expression of the target gene.

The obtained strain of Chinese hamster ovary cells CHO-humC34 was characterized by the following properties.

Pedigree strain: Parent cell line CHO ^dhfr- . Cotransfection with pTVK4 / humC34-H and pTVK4d / humC34-L plasmids, selection of a stable, highly productive clone on a medium without hypoxanthine / thymidine with 200 μg / ml G-418 and a gradual increase in methotrexate concentration. The number of passages at the time of certification and deposit: 20.

Standard growth conditions: CDM4CHO Wednesday (HyClone) without hypoxanthine / thymidine with 6 mM glutamine, 10 nM Methotrexate, 200 g / ml G-418, 37 ° C, 5% CO _2.

Cultural properties: Suspension cultivation in test tubes, flasks or 6-well plates on an orbital shaker with a rotation speed of 130-180 rpm. Sowing dose 0.3-0.5 × 10 ⁶ / ml, reseeding every 3-4 days. The doubling time is 25-27 hours.

Species data: Cricetulus griseus (PCR analysis with species-specific primers).

Marker signs of the strain and methods for their assessment. The culture is resistant to growth in a medium without hypoxanthine and thymidine in the presence of 10 nM methotrexate and 200 μg / ml G-418

Contamination control by bacteria, fungi, mycoplasmas and viruses: no contamination.

Characteristics of the beneficial substance produced by the strain. Recombinant humanized hC34 antibodies (IgG1 isotype, kappa) to the neoantigen of the activated form of component C3 of the human complement, blocking the activation of the complement system in an alternative way (evaluation using a hemolytic test using rabbit erythrocytes, an inhibition test for the formation of anaphylotoxins C3a and C5a upon activation of zymozan).

Characterization of the biosynthesis of useful products (product yield in the environment, level of activity and method for its determination). Recombinant humanized antibodies are secreted into the culture medium in an amount of about 20 pg per cell per day. Cultivation stability - at least 30 passages.

Cryopreservation method: 45% fresh CDM4CHO medium, 45% conditioned medium, 10% DMSO, 3-5 × 10 ⁶ cells / ml, freezing to -70 ° C at a rate of 1 ° C / min, then placing in liquid nitrogen, viability after Defrosting and washing from the cryopreservative 90% (with trypan blue).

Other features of the strain: It is possible to cultivate cells in DMEM / F12 medium with 10% fetal serum (adhesive growth type) without additional adaptation.

The strain was deposited in the collection of the Russian Academy of Sciences under the number RKKK (P) 776D 03/31/2016 The resulting monoclonal antibody recognizes the conformational epitope (neoantigen), which is usually absent in the C3 molecule and appears when it is activated via an alternative pathway. The 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 characteristics of the generated antibodies and sequences are given in the proposed list of sequences.

In FIG. 1 shows a comparison of the sequences of the variable regions of the H and L chains of the CC3-4 antibody and the variable regions of the chains AAC09074 and ABA26038, where CC3-4-H is the amino acid sequence in the variable region of the heavy chain of the CC3-4 antibody; CC3-4-L is the amino acid sequence in the variable region of the light chain of the CC3-4 antibody. CDRs are underlined. Amino acids in the frame sections of the chains of the mouse CC3-4 antibody subjected to replacement are highlighted in bold italics.

List of sequences:

Seq ID No. 1 Amino Acid Sequence of CDRH-1 of a Mouse CC3-4 Antibody

Seq ID No. 2 Amino Acid Sequence of CDRH-2 of a Mouse CC3-4 Antibody

Seq ID No. 3 Amino Acid Sequence of CDRH-3 of a Mouse CC3-4 Antibody

Seq ID No. 4 The amino acid sequence of the CDRL-1 mouse antibody CC3-4

Seq ID No. 5 Amino Acid Sequence of CDRL-2 of a Mouse CC3-4 Antibody

Seq ID No. 6 Amino Acid Sequence of CDRL-3 of a Mouse CC3-4 Antibody

Seq ID No. 7 The amino acid sequence of the variable region of the H chain of the humanized antibody hC34.

Seq ID No. 8 The amino acid sequence of the variable region of the L chain of the humanized antibody hC34, option L-0

Seq ID No. 9 The amino acid sequence of the variable region of the L chain of the humanized antibody hC34, option L-2

Seq ID No. 10 The amino acid sequence of the variable region of the L chain of the humanized antibody hC34, option L-4

Seq ID No. 11 The amino acid sequence of the variable region of the L chain of the humanized antibody hC34, option L-5

Seq ID No. 12 The amino acid sequence of the variable region of the L chain of the humanized antibody hC34, option L-7

Seq ID No. 13 DNA sequence encoding the H chain of the humanized antibody hC34 (with signal peptide).

Seq ID No. 14 DNA sequence encoding a L-0 variant of the L chain of the humanized hC34 antibody (with signal peptide).

Seq ID No. 15 DNA sequence encoding a L-2 variant of the L chain of the humanized hC34 antibody (with signal peptide).

Seq ID No. 16 DNA sequence encoding the L-4 variant of the L chain of the humanized hC34 antibody (with signal peptide).

Seq ID No. 17 DNA sequence encoding the L-5 variant of the L chain of the humanized hC34 antibody (with signal peptide).

Seq ID No. 18 DNA sequence encoding a L-7 variant of the L chain of the humanized hC34 antibody (with signal peptide).

Features of the group of inventions is further illustrated by the following drawings:

In FIG. 2 and 3 show patterns of expression vectors used to create a highly productive producer strain of the humanized antibody hC34 - in FIG. 2 pTVK4 / humC34-H, in FIG. 3 - pTVK4d / humC34-L.

The following notation is used:

- CMV promoter - promoter of early cytomegalovirus proteins;

- hC34-H is the heavy chain gene of a humanized antibody to the conformational epitope of human C3;

- hC34-L - light chain gene of a humanized antibody to the conformational epitope of human C3;

- TK polyA — RNA polyadenylation site from the HSV thymidine kinase gene;

- SV40 promoter - promoter of the early proteins of the SV40 virus;

- DHFR - dihydrofolate reductase gene;

- Neo - a gene of resistance to neomycin;

- SV40 poly A - site polyadenylation of early proteins of the virus SV40;

- pUC ori - replication start point;

- Amp - gene for resistance to ampicillin;

- RE is a regulatory element that enhances the expression of the target gene.

In FIG. 4 shows the results of the analysis of the affinity of humanized

hC34 antibody variants, where A is an analysis of the affinity of antibodies obtained by coexpressing chimeric (chemical) and humanized (hum) antibody chains to the conformational epitope C3 of the complement component (L-0 was used for the light chain); B - analysis of the affinity of antibodies obtained by coexpression of the humanized heavy chain and various light chain variants (Chem - chimeric variant, L-0, L-2, L-4, L-5, L-7 - corresponding variants of the humanized light chain) antibodies to the conformational epitope C3 of the complement component.

In FIG. Figure 5 shows the concentration of human immunoglobulins in the supernatants of single hC34 antibody producing clones ranked by this indicator.

In FIG. Figure 6 shows the blocking of the alternative complement activation pathway (APC) by CC3-4 and hC34 antibodies.

The invention is illustrated by the following examples:

Example 1. Humanization of the variable regions of a monoclonal antibody CC3-4.

1.1. Analysis of the structure of antibodies and the construction of a humanized antibody to the conformational epitope of the C3 component of human complement.

Based on their previously determined amino acid sequences in the variable regions of the heavy (H) and light (L) chains of the mouse CC3-4 antibody, using the Kabath analysis method (2), sequences of regions determining the complementary interaction of the antibody with antigen (CDR regions) were calculated ) and the surrounding frame sections (Fig. 1). Further, using the Ig-BLAST program, light and heavy chains of known human IgG were found, the sequences of which are most similar to the sequences of similar chains of the CC3-4 antibody. The human antibodies were AAC09074 for the heavy chain and ABA26038 for the light chain, in the sequences of which CDR regions and framework regions were also isolated. Comparison of the sequences of the frame sections of AAC09074 and ABA26038 with the sequences of the corresponding sections of the CC3-4 antibody showed that the "mouse" frame sections differ from the "human" substitutions of amino acids, of which only 31 were found in the heavy chain and 37 in the light chain (Fig. 1) .

The humanization of the variable regions of the chains of the mouse CC3-4 antibody was carried out by replacing the frame sections with the corresponding frame sections of the human antibodies AAC09074 and ABA26038 in the heavy and light chains, respectively. Designed in this way, a humanized antibody was designated hC34. The amino acid sequences of the variable fragments of the light (L) and heavy (H) chains of the hC34 antibody are shown in Seq. ID No. 7 and Seq. ID No 8. For the light chain, in addition to the variant designed in the described way and designated as L-0, an additional 4 variants were designed that differ from L-0 by substituting two, four, five or seven amino acids. These options were designated L-2, L-4, L-5, and L-7, respectively. Their amino acid sequences are presented in Seq. ID No. 9-12.

1.2. Obtaining genes encoding the variable regions of the chimeric and variants of the humanized antibody to the conformational epitope of the C3 component of the human complement, and expression plasmids containing them.

DNA sequences encoding the variable fragments of the heavy and light chains of the murine monoclonal antibody CC3-4 were obtained using PNR on a cDNA matrix obtained from hybridoma CC3-4 (described by us earlier in RU 2584582, 2016).

DNA sequences encoding the variable fragments of the heavy and L-0 variants of the light chains of a humanized antibody were obtained synthetically. When designing sequences, nucleotide triplets encoding the necessary amino acids were selected taking into account the frequency of codon usage in the genome of the Chinese hamster (Cricetulus griseus), presented in the codon usage database (kazusa.or.jp).

The DNA sequence encoding the mouse immunoglobulin light chain signal peptide, MDFQVQIFSFLLISASVIISRG and the Kozak sequence, were assembled from synthetic oligonucleotides. The vectors pAL-TA / IGG1const and pAL-TA / IGKconst, which we created earlier (RU 2550262), were used as a source of genes for the constant part of the heavy and light chains. Further gene assembly was performed using overlapping PCR. For the light chain, a fragment containing a signal peptide and a Kozak sequence, a fragment of a variable region and a fragment of a constant region were amplified independently, while the primers used provided the necessary overlapping of the ends of adjacent sequences. Next, the whole construct was amplified from the obtained fragments in two stages of overlapping PCR, after which the final amplification product was cloned into the pIRES-DHFR expression vector [RU 2550262, 2015] at the BamHI and NotI restriction sites and the obtained nucleotide sequence corresponded to the sequence planned by sequencing. The heavy chain was assembled in a similar manner. In the first PCR reaction, the Kozak sequences, the signal peptide and the variable region were combined, and then the constant part of the heavy chain was attached and cloned into an identical expression vector in the same way as the light chain. As a result, expression plasmids pID / CC3-4-H and pID / CC3-4-L were obtained that encode the heavy and light chains of a chimeric antibody containing variable fragments of murine antibodies CC3-4 and constant fragments of human IgG, as well as expression plasmids pID / hC34-H and pID / hC34-L0 encoding the heavy and light chains of a humanized hC34 antibody.

To obtain genes encoding the light chain variants of the humanized antibody L-2, L-4, L-5 and L-7, mutagenesis was performed by PCR. The plasmid pID / hC34-L0 was used as a matrix. Changes in the nucleotide sequence were made using two complementary primers of at least 24 acting in length containing the necessary mutations. After PCR and removal of the original plasmid by restriction enzyme treatment with DpnI, the reaction mixture was used to transform competent cells. Plasmids were isolated from the obtained colonies and the presence of the necessary mutations was determined by sequencing. The resulting plasmids were designated pID / hC34-L2, pID / hC34-L4, pID / hC34-L5 and pID / hC34-L7, respectively.

The nucleotide sequences of the variable regions of the heavy and light chains (variants L-0, L-2, L-4, L-5 and L-7) of the humanized hC34 antibody are presented in Seq ID No. 13-18.

Example 2. Analysis of the binding of the C3 component of the complement to various variants of the humanized hC34 antibody in human serum.

To obtain analytical samples of humanized hC34 antibody variants, the HEK-293 cell culture was cotransfected with a mixture of plasmids encoding the antibody heavy and light chains. At the first stage, pID / hC34-H or pID / CC3-4-H were used as plasmids encoding a heavy chain, and pID / hC34-L0 or pID / CC3-4-L were used as plasmids encoding a light chain. Transfection was performed in 24-well plates using a commercial liposome reagent. Supernatants for analysis were collected 5 days after transfection, then the concentration of human immunoglobulins was determined in them, and the binding of the C3 complement component from human serum was analyzed using enzyme-linked immunosorbent assay (ELISA). For this, monoclonal antibodies I-4 to human IgG were adsorbed into the wells of an ELISA plate at a concentration of 1.5 μg / ml in a volume of 100 μl per cell. The plate was incubated at room temperature for 12-14 hours, after which it was washed with wash buffer. Then, test samples of antibodies were added to the plate, previously diluted to 100 ng / ml in wash buffer with 0.1% bovine serum albumin. Each test sample was added to at least 2 strips (2 × 8 holes). After incubation for 1 hour and washing, serial dilutions of human serum were introduced: from 1/640 to 1/20480 (a series of serial two-fold dilutions) (12 wells of each concentration). The plate was incubated for another 2 hours and, after washing, a conjugate of monoclonal antibodies CC3a-1 to the C3a fragment C3 of the human complement component with horseradish peroxidase at a concentration of 0.5 μg / ml was added. After one-hour incubation and washing, the plate was stained with TMB.

The affinity of the tested antibodies was judged by the angle of inclination of the experimental curves obtained in the wells containing samples of the studied antibodies. The results are shown in FIG. 4A, from which it is seen that when expressing the heavy chain of a humanized hC34 antibody together with the light chain of a chimeric antibody, the affinity of the resulting antibodies does not decrease compared to the chimeric antibody. In this case, antibodies obtained using the L-0 variant of the humanized antibody light chain exhibit less affinity than chimeric immunoglobulins of the corresponding specificity.

In this regard, additional variants of the light chain of a humanized antibody were designed in which several amino acids in positions that, according to the literature are most likely to affect the folding of the antibody light chain, were replaced again with “murine” ones, i.e. antibodies CC3-4 contained in the corresponding positions in the original light chain sequence. Analysis of the binding of recombinant antibodies obtained by cotransfection of the plasmid pID / hC34-H and plasmid encoding one of the variants of the light chain (pID / hC34-L0, pID / hC34-L2, pID / hC34-L4, pID / hC34-L5 or pID / hC34-L7) showed (Fig. 4B) that all four designed variants of the light chain of the humanized antibody in combination with the humanized heavy chain have affinity comparable to the chimeric variant, in particular, variant L-7 shows the best result in this test.

Example 3. Obtaining a strain of cells CHO-humC34 - producer of a humanized antibody to the conformational epitope of human C3.

3.1. Obtaining expression plasmids containing the genes of the heavy and light chains of the hC34 antibody.

To create a producer strain of the hC34 antibody, the vectors pTVK4 and pTVK4d were used, containing, in addition to the expression elements indicated in FIG. 4 and 5, the specialized regulatory element UCOE (3), designated as RE, wherein pTVK4 carries the neomycin resistance gene, and pTVK4d carries the dihydrofo-latreductase gene. The original plasmids pID / hC34-H and pID / hC34-L7 were treated with BamIII and NotI restriction enzymes and a DNA fragment was isolated that contained, in addition to the heavy and light chains of the antibody, a Kozak sequence that provides translation initiation and a sequence encoding a signal peptide from mouse immunoglobulin ( MDFQVQIFSFLLISASVIISRG). Next, DNA fragments encoding the light and heavy chains of the humanized antibody were cloned into pTVK4 and pTVK4d, similarly restricted. As a result, two expression vectors were obtained, pTVK4 / humC34-H and pTVK4d / humC34-L, the schemes of which are shown in FIG. 2 and 3. Restriction analysis and sequencing confirmed the correct assembly of plasmids and the absence of mutations in the chain genes of the humanized antibody.

3.2. Obtaining a producer strain of a humanized antibody hC34

For constant transfection, Chinese hamster ovary line cells deficient in the dihydrofolate reductase (CHO ^dhfr- ) gene, previously adapted to suspension growth under serum-free conditions, were used. Cells were cultured in CDM4CHO medium (HyClone, USA) supplemented with 4 mM alanylglugamine, 0.1 mM hypoxanthine, 0.016 mM thymidine and 0.1% Pluronic F-68 (Sigma, USA) in TubeSpin 50 microbioreactors (" TRP ”, Switzerland) on an Sanyo MIR-S100C orbital shaker (200 rpm), which was placed in a CO ₂ incubator (5% CO ₂ , 37 ° C).

Transfection was performed using the Freestyle liposomal reagent (Life Technologies, USA) according to the manufacturer's instructions, using a mixture of 2.4 μg of plasmid pTVK4 / humC34-H and 3.6 μg of plasmid pTVK4d / humC34-L at 5 × 10 ⁶ cells. 48 hours after transfection, cells were transferred to hypoxanthine / thymidine-free medium supplemented with 200 μg / ml of geniticin G-418 (Life Technologies). In the following days, the medium was changed with an interval of 3 days. After 15 days, the cells were transferred to Erlenmeyer flasks for subsequent growth, after which cloning was carried out using serial dilutions in order to select individual clones with high production. Cloning was performed by plating cells at a density of 1 cell per well of a 96-well plate. In total, 20 96-well plates were seeded in this way. After 10 days, wells containing a single clone were visually selected. 420 clones were identified. After 3 days using the enzyme immunoassay (ELISA), the content of human immunoglobulins in the supernatants of individual clones was evaluated. The result is shown in FIG. 5.

For further work, 40 clones were selected, in the supernatants of which the concentration of immunoglobulins was more than 1000 ng / ml. Selected clones were completely transferred to 48-well plates. After 48 hours, a reanalysis of the concentrations of immunoglobulins in supernatants was performed. According to the results of the second screening, 12 clones were selected with the maximum volumetric production of recombinant antibodies, the concentration of immunoglobulins in the supernatants of which was more than 4800 ng / ml. Selected clones were transferred to 6-well for subsequent cultivation. Next, the cells of each clone were seeded in 12-well plates of 0.5 × 10 ⁶ cells per well in 2 ml of culture medium without G-418. Antibody production was evaluated 3 days after plating. As a result, 8 clones were selected with maximum production on day 3 (more than 5000 ng / ml).

These clones were cultured on a shaker in 5 ml TubeSpin 50 type microbioreactors. The initial density of the culture was 0.5 × 10 ⁶ cells / ml, reseeding was carried out with an interval of 3 days. After the third passage on a shaker, the cells were seeded at a density of 0.5 × 10 ⁶ cells / ml in 5 ml of medium without G-418 and the production of antibodies and cell growth dynamics were analyzed. Based on the data obtained, the specific productivity of clone cells was calculated, which turned out to be the best for clone N3 (4.5 pg per cell per day).

Next, cells of clone N3 were adapted for growth on a medium with 10 nM methotrexate (MTX). After 7 passages in MTX medium, cells were subcloned into 96-well plates in medium with 10 nM MTX and 200 μg / ml G-418, and 80 subclones were obtained. As a result of the sequential selection of subclones with the highest productivity, as described above, it was found that subclone N3 / 8 has the best growth characteristics and productivity: on the 7th day of cultivation, the concentration of viable cells was 4.6 × 10 ⁶ cells / ml with a viability of 90% and the maximum volumetric production of recombinant antibodies was 220 μg / ml.

This subclone was used to create the Main cell bank of the producer strain, and was also deposited in the Russian collection of cell cultures under the number RKKK (P) 776D and the working name "СНО-humC34".

3.3. Analysis of the neutralizing activity of humanized antibodies to the conformational epitope of human C3, secreted by the producer strain CHO-humC34.

The cells of the producer strain CHO-humC34 were cultured on a shaker in Erlenmeyer flasks, resulting in 150 ml of culture fluid, from which antibodies were isolated by affinity chromatography on immobilized protein A. As a result, a laboratory sample of recombinant humanized hC34 antibody was obtained in an amount of 20 mg The ability of hC34 antibodies to block complement activation by an alternative pathway was evaluated in comparison with the CC3-4 murine monoclonal antibody.

Activation of complement along an alternative pathway was carried out in 20 mM HEPES buffer (pH 7.2-7.4) containing 150 mM NaCl, 10 mM EGTA, 5 mM MgCl2, 1 mg / ml BSA (APC buffer) by incubating serum with an activator APC zymosan in a 96-well ELISA plate. 50 μl of a solution of humanized hC34 antibodies or murine CC3-4 antibodies in various concentrations and 100 μl of healthy donor blood serum containing 1 M ε-aminocaproic acid diluted 10 times in the APC buffer were added to the rows of tablet cells. The plate 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 the 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, 10 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 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 anaphylatoxin C5a in the samples after incubation. The neutralizing (blocking) ability was judged by the change in the levels of anaphylactosin C5a in serum samples, depending on the concentration of antibodies.

The results are shown in FIG. 6, which shows that the humanized hC34 antibodies have the ability to block the activation of the alternative complement pathway even more efficiently than the CC3-4 mouse antibodies that were humanized. This makes the humanized hC34 antibodies produced by the producer strain CHO-humC34 promising for therapeutic use in order to block the APC.

Claims (14)

1. A humanized antibody to the conformational epitope C3 of the component of the complement of a person containing a fragment selected from established by Kabatu group, which includes CDRH-1 plot according to Seq ID No: 1; CDRH-2 plot according to Seq ID No: 2; CDRH-3 plot according to Seq ID No: 3; CDRL-1 plot according to Seq ID No: 4; CDRL-2 plot according to Seq ID No: 5; CDRL-3 plot according to Seq ID No: 6 for binding to the conformational epitope C3 component of the human complement. 2. The humanized antibody to the conformational epitope C3 of the human complement component of claim 1, having a sequence of the variable region of the heavy chain according to Seq ID No 7. 3. The humanized antibody to the conformational epitope C3 of the complement component of a person according to claim 1, having the sequence of the variable region of the light chain according to Seq ID No. 8. 4. Humanitariannet antibody to the conformational epitope C3 component of the human complement component according to claim 1, having the sequence of the variable region of the light chain according to Seq ID No 9. 5. The humanized antibody to the conformational epitope C3 of the component of the complement of a person according to claim 1, having the sequence of the variable region of the light chain according to Seq ID No 10. 6. Humanitariannet antibody to the conformational epitope C3 component of the complement of a person according to claim 1, having the sequence of the variable region of the light chain according to Seq ID No 11. 7. Humanitariannet antibody to the conformational epitope C3 component of the complement of a person according to claim 1, having the sequence of the variable region of the light chain according to Seq ID No 12. 8. DNA encoding the heavy chain of a humanized antibody to the conformational epitope C3 of the human complement component of claim 1 or 2, presented on Seq ID No. 13 for binding the conformational epitope C3 of the component of the human complement DNA, encoding the L-0 variant of the light chain of the humanized anti-conformational antibody epitope C3 component of the human complement according to paragraphs. 1 and 3, presented on Seq ID No. 14 for binding of the conformational epitope C3 of the complement component of a person. 9. DNA encoding a variant of the L-2 light chain of a humanized antibody to the conformational epitope C3 of the human complement component of PP. 1 and 4, presented on Seq ID No. 15 for binding of the conformational epitope C3 of the complement component of a person. 10. DNA encoding a variant of the L-4 light chain of a humanized antibody to the conformational epitope C3 of the human complement component of PP. 1 and 5, presented on Seq ID No. 16 for binding a conformational epitope of the C3 component of a human complement. 11. DNA encoding a variant of the L-5 light chain of a humanized antibody to the conformational epitope C3 of the human complement component of PP. 1 and 6, presented in Seq ID No. 17 for binding a conformational epitope of the C3 component of a human complement component DNA encoding a light chain variant L-7 of a humanized anti-conformational epitope of the C3 conformational epitope of the human complement component of pp. 1 and 7, presented on Seq ID No. 18 for binding of the conformational epitope C3 of the complement component of a person. 12. Expression vector pTVK4 / humC34-H, including a promoter of early cytomegalovirus proteins; the heavy chain gene of a humanized anti-human C3 conformational epitope antibody; RNA polyadenylation site from HSV thymidine kinase gene; promoter of early SV40 virus proteins; neomycin resistance gene; polyadenylation site of early SV40 virus proteins; ampicillin resistance gene and regulatory element that enhances the expression of the target gene as well as DNA selected from the group of DNA represented in paragraphs. 8-13, for the expression of a humanized antibody for binding the conformational epitope C3 of the complement component of a person in mammalian cells. 13. The expression vector pTVK4d / humC34-L, including a promoter of the early cytomegalovirus protein; light chain gene of a humanized antibody to a human C3 conformational epitope; RNA polyadenylation site from HSV thymidine kinase gene; promoter of early SV40 virus proteins; dihydrofolate reductase gene; polyadenylation site of early SV40 virus proteins; ampicillin resistance gene and a regulatory element that enhances the expression of the target gene, as well as a DNA sequence selected from the group of DNA sequences presented in paragraphs. 8-13, for the expression of a humanized antibody for binding the conformational epitope C3 of the complement component of a person in mammalian cells. 14. The strain of ovarian cells of the Chinese hamster CHO-humC34, transfected with vectors according to claims. 8 and 13, deposited in RKKK under the number RKKK (P) 776D - producer of a humanized antibody for binding of the conformational epitope C3 of the component of human complement.

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