RU2551235C2 - Humanised monoclonal antibody specific to syndecan-1 - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: humanised monoclonal antibody (MAb) specific to syndecan-1 (CD138) is proposed, which is characterised by amino acid and nucleotide sequences. According to the present invention, the antibody belongs to isotype IgG4 kappa, at that the constant fragments are fully human and modified by administration of sites IgG3 imparting to antibody the independent antibody-dependent and complement-dependent cytotoxicity, and amino acid substitutions that enable to increase the antibody gun. The variable sites are presented by murine hypervariable and human framework sites, providing an increase in binding affinity with a ligand. Each chain additionally comprises a signal secretory sequence at the N-terminus cleaved upon secretion from the producing cells. The nucleotide sequences are optimised to enhance antibody production in mammalian cells. The present invention may find additional application in the treatment of diseases associated with CD138, including malignant tumours (cancer).

EFFECT: increased efficiency of use of the compounds.

2 cl, 2 dwg, 3 ex

Description

FIELD OF THE INVENTION

This invention relates to immunology, biotechnology and medicine, namely to the field of antitumor drugs based on monoclonal antibodies, and can be used in medicine for the treatment of cancer.

State of the art

Oncological diseases are on the second place in mortality in the world after cardiovascular diseases [1]. Over the past 10 years, the number of patients with cancer, according to various sources, increased by 15%. More than 490 thousand new patients are registered in Russia annually, 2.5 million are under observation and more than 290 thousand patients die every year from various forms of cancer. In view of this, it is advisable to develop and create safe, effective and affordable antitumor drugs.

One of the types of anticancer drugs are biotechnological synthesis products - monoclonal antibodies (Mab). A number of pharmacological solutions are known, including Mab to the epidermal growth factor receptor, vasoendothelial growth factor, which are of proven importance for the vital activity of tumor cells and are of little significance for the functioning of normal cells of various body tissues [2].

Currently, about ten anticancer drugs based on monoclonal antibodies are registered and produced in the world, in particular, Rituximab (Mab to the CD20 receptor), Trastuzumab (the target is the HER2neu receptor), Bevacizumab (VEGF), Cetuximab and Panitumumab (EGFR). There are currently no registered drugs targeting the syndecan-1 surface antigen (CD138).

Protein syndecan-1 (CD138) [4] is a promising target for anticancer therapy with monoclonal antibodies: the important role of this molecule in the vital activity of tumor cells has been proved and its low importance for the functioning of normal cells of various body tissues [5].

This molecule provides intercellular interactions and interactions between cells and the matrix, adhesion and migration. Overexpression of CD138 was demonstrated in most (about 90%) cells of various tumors, including cancer of the breast, colon and stomach, prostate and other epithelial malignant tumors [6, 7]. Also described is the phenomenon of loss of syndecan-1 from the surface of myeloma cells with its accumulation in the stroma, as a rule, in areas of fibrosis [8]. With the loss of syndecan-1, myeloma cells enter TRAIL-induced apoptosis [9]. Functionally, Sindecan-1 is associated with the ability of tumor cells to invasively grow and metastasize. In the aspect of the treatment of malignant tumors, the activity of CD138 is associated with those properties of tumor cells that are practically not amenable to existing therapy.

Localization of CD138 on the surface of tumor cells in a form accessible for recognition by antibodies was shown [10]. Moreover, there is evidence of vaccination efficacy using CD138 as an antigen [11].

Known murine antibodies to syndecan-1 - B-B4 (IgG1) [13], B-B2 (IgG2b), as well as chimeric [8, 14] - MP5 (IgG1κ) and conjugates thereof with labels [12] [3]. Such compounds are used for research or diagnostic purposes, and the antibody performs only a targeting function [8, 14]. Effector molecules, usually conjugated to an antibody, perform a cytotoxic function [16, 17, 18]. The mouse and rabbit Mab perceives the human immune system as foreign, due to differences in the constant part of antibodies (C _H , C _L ), and triggers an immune response against them. For use in humans, chimeric antibodies are developed, the constant part of which is human, and the variable (V _H , V _L ) - mice. Using this modification, it is possible to obtain safer Mabs.

A recombinant anti-syndecan-1 antibody (OC-46F2) is known that was created by in vitro selection of a library for phage display (ETH-2-Gold) of human antibodies on human melanoma cells and further expression of the resulting genes in mammalian cells [26]. This antibody has been shown to inhibit vascular maturation and tumor growth in an experimental model of human melanoma (in mice), and is also therapeutic in terms of an experimental model of human ovarian cancer carcinoma.

OC-46F2 is a single chain variable fragment (scFv) - a hybrid protein of variable fragments of the heavy (V _H ) and light (V _L - lambda) chains of immunoglobulins connected by a short linker peptide.

This compound does not contain constant antibody fragments, variable fragments of the molecule provide only binding to the antigen, which causes the inhibitory effect shown by the authors. However, to eliminate the tumor, the use of additional active substances is necessary, possibly the creation of conjugates. We observe the same drawback as when using a chimeric antibody.

The prototype of the invention is a chimeric monoclonal antibody to CD138 of the IgG4 subclass [15]. However, this Mab only provides targeting of the cytotoxic agent conjugated to it (DM4).

Thus, the mouse antibodies against CD138 described today are unsuitable for use in humans due to their high immunogenicity, they are used for laboratory purposes, chimeric MAb do not have an independent therapeutic effect - they are used only as a targeting agent, and can also be immunogenic. We cannot exclude the individual reaction of the patient to a particular Mab. In addition, the active agent that is targeted using antibodies, or the products of its breakdown, can be a stress factor for the body, due to their structure, ability and breakdown period of this substance, the nature of the compounds into which it breaks down. The exact structure of natural human monoclonal antibodies to syndecan-1 has not been established.

Given the functional role of syndecan-1 in the invasive growth and metastasis of tumors, the creation of antibodies to it, which have independent high cytotoxicity, along with higher affinity and lower immunogenicity, is an urgent task.

This problem is solved by the proposed invention.

The technical result from the use of the proposed antibodies to CD138 is expressed, as a minimum, in an increase in the spectrum of MAb to CD138 used in the treatment of tumor diseases, which allows them to be treated for individual intolerance or poor tolerance of analogues.

The technical result from the use of the proposed antibodies to CD138 is expressed in an increase in affinity for the antigen, and, accordingly, the effectiveness of the antibody. The indicated technical result is achieved by antibody humanization: the use of frame sections of a human antibody allows more precise orientation of hypervariable regions, which increases the correspondence of the epitope and paratope and, accordingly, affinity.

The technical result from the use of the proposed antibodies to CD138 is also expressed in the induction of cytotoxicity. The specified technical result is achieved by the fact that sections of IgG3 chains mediating antibody- and complement-dependent cytotoxicity are introduced into the constant fragment of the heavy chain of an IgG4 type antibody.

The technical result from the use of the proposed antibodies to CD138 is also expressed in a decrease in immunogenicity due to the humanization of the antibody.

The technical result from the use of the proposed anti-CD138 antibody is also expressed in the reduction of the stress effect on the body of the antitumor drug, due to the fact that the active substance is the monoclonal antibody molecule itself - by nature a protein that subsequently degrades to amino acids, i.e. when using it, any consequences of a possible non-elimination from the body of an active agent (non-protein chemical substance) or its decomposition products from the body for any time are excluded.

The technical result from the use of the proposed antibodies to CD138 is also expressed in increasing the half-life of the antibody, which is achieved by the introduction of amino acid substitutions at the C-end of the constant part of the heavy chain. This allows you to reduce the dosage, to implement a smaller number of introductions of antibodies, which can reduce the cost of treatment.

Also, the technical result is to obtain a high level of antibody expression in mammalian cells in a short time due to codon optimization of coding sequences, as well as the introduced signal secretory sequence at the N-end of the heavy and light chains, which allows to reduce the cost and speed up the production.

SUMMARY OF THE INVENTION

The objective of the invention was the creation of a universal monoclonal antibody to CD138, suitable for the treatment of tumor diseases, with high affinity for the antigen (CD138), low immunogenicity and high cytotoxicity, as well as a long half-life that can be obtained in large quantities in a short period of time .

This problem is solved by the fact that a monoclonal humanized antibody to syndecan-1 (CD138) is proposed, based on IgG4, with IgG3 sites introduced into the C _H antibody fragment, causing the formation of antibody-dependent and complement-dependent cytotoxicity, with an introduced signal secretory sequence of N -the end of the heavy and light chains, cleaved after secretion of proteins from producer cells, as well as amino acid substitutions that increase the half-life of this antibody; having the heavy chain sequence of SEQ ID NO: 1 and light chain SEQ ID NO: 2 encoded in the nucleotide sequences of SEQ ID NO: 3 and SEQ ID NO: 4, codon optimized for increased expression in mammalian cells (CHO).

The proposed humanized anti-CD138 antibody binds highly affinely with syndecan-1 overexpressed on tumor cells due to the humanization of variable fragments. Hypervariable regions of mouse monoclonal antibodies obtained independently as a result of immunization with syndecan-1 were transferred to human acceptor antibodies that are most structurally homologous with respect to key positions; their skeleton regions allow more precise orientation of hypervariable regions in space, which increases the correspondence of the epitope and paratope and, therefore, provides a higher Compared to chimeric antibodies, affinity for the target. At the N-terminus of the heavy and light chains, a signal secretory sequence is introduced that cleaves after the secretion of proteins from producer cells.

The design of the proposed antibody is able to provide its independent cytotoxic effect by antibody-dependent and complement-dependent mechanisms by optimizing the sequence of the constant fragment (IgG4-IgG3 shifting), which ensures the involvement of cells of the immune system. This eliminates the use of a cytotoxic agent, i.e. from procedures for choosing the nature of its use in conjunction with Mab (accession or sharing), procedures for its conjugation with an antibody, maintaining the activity of both components and other ensuing consequences.

IgG4 antibodies do not activate complement, while IgG3 can activate a cascade of proteolytic reactions carried out by the complement system.

The proposed monoclonal antibody to syndecan-1 (CD138) contains IgG3 sites - amino acid substitutions in the heavy chain are introduced, which provides the induction of antibody-dependent and complement-dependent cytotoxicity, as well as amino acid substitutions to increase the half-life of the antibody at the C-terminus of the constant part of the heavy chain .

Codon optimization of nucleotide sequences (SEQ ID NO: 3, SEQ ID NO: 4) encoding the antibody of the invention (heavy and light chains), along with the introduction of a signal secretory sequence, allowed to obtain up to 2 grams of the target protein per liter with three days of cell cultivation mammalian producers (CHO).

The resulting humanized monoclonal antibody is characterized by a Kd value of 8.0 * 10 ^-9 .

BALB / C Nude mice were used to test the monoclonal antibody, in which the allergic reaction may be more pronounced than in standard laboratory mice. However, an allergic reaction was not observed, which suggests that the developed drug will be quite safe in terms of allergic side effects.

The results of testing the drug on in vivo models showed that in the laboratory animals tumor regression was observed, lack of metastasis; animals treated with the drug had a longer life span.

The results of these drug tests can be interpreted as preliminary toxicity data.

The ability to induce antiproliferative and antimetastatic activity is shown on cells of the A431 line.

Thus, the proposed humanized anti-CD138 antibody has an antitumor effect by blocking syndecan-1-mediated functions (adhesion, migration of tumor cells) and the induction of a cytotoxic immune response, i.e. not only inhibits tumor growth and cell migration, but also contributes to their elimination in a natural way.

Thus, it becomes possible not only to diagnose but also treat cancer with the monoclonal anti-CD138 antibody claimed in the invention by inducing antibody- and complement-dependent cytotoxicity against tumors in which syndecan-1 is overexpressed due to the introduction of corresponding amino acid substitutions in constant sections of human antibodies (IgG4-IgG3 shifting). When using this invention, high therapy efficiency is achieved by increasing the affinity of the antigen-binding portion of the monoclonal antibody to syndecan-1. By introducing appropriate amino acid substitutions, the half-life of the antibody is also increased after administration to the patient. Codon optimization of the heavy and light chains, as well as the introduction of a signal secretory sequence at the N-terminus of the heavy and light chains, allows the production of antibodies in large quantities for short periods in mammalian cells.

The proposed Mab is safe for humans in terms of allergic side effects due to humanization.

A brief description of the graphic materials

Figure 1. Electrophoregram showing the expression of the gene encoding syndecan-1 in the used myeloma cell line SP 2.0

1) quality control of cDNA synthesis, the result of PCR with KG3PDH primers;

2) positive control, PCR with primers specific for the DNA sequence encoding CD138, using plasmid DNA containing the cloned syndecan-1 gene as a template;

3) PCR for cDNA from myeloma cells using primers specific for DNA encoding syndecan-1;

4) expression control - PCR using primers specific for DNA encoding syndecan-1 on total RNA from myeloma cells;

5) molecular weight marker 1 kb DNA Ladder (Fermentas).

Figure 2. Dynamics of the development of SP 2.0 myeloma in BALB / C mice in three groups of animals. The black bar is the indices of the control group, the gray one is the group of mice injected with 1 mg of the preparation of a humanized monoclonal antibody, the shaded one is the group of mice injected with 0.1 mg of the preparation of a humanized monoclonal antibody.

MODES FOR CARRYING OUT THE INVENTION

Example 1. Obtaining a monoclonal humanized antibody to syndecan-1.

1.1. Getting syndecan-1

The gene encoding syndecan-1 was synthesized by means of chemical-enzymatic synthesis [20] of the corresponding gene by the two-step method. The resulting gene was cloned in the pET151 / D-TOPO vector, which provides a high level of synthesis of a recombinant protein crosslinked with a polyhistidine sequence, for the convenience of further purification using metal chelate chromatography, as a result of which the protein can be obtained as a separate amino acid sequence, without polyhistidine and any or other impurities, due to the presence of a site for the TEV protease, in E. coli BL21 (DE3) Star cells, which contain the mutated rne gene ( rne131 ), encoding a truncated form of RNase E, which reduces the intracellular the in vivo destruction of mRNA, leading to an increase in its enzymatic stability and, accordingly, protein production. This gene was expressed in E. coli BL21 (DE3) Star cells.

Recombinant protein was isolated from cells of strain BL21 [DE3] Star pET151sind-1 by dissolving the cells in a lysis buffer. Purification of syndecan-1 was carried out using metal chelate chromatography.

The recombinant protein preparation had a purity of 95-97% and was used to obtain a hybridoma producing mouse monoclonal antibodies to syndecan-1 and to test the resulting monoclonal antibodies.

1.2. Immunization of Balb / c Sydecan-1 Mice

Immunization of Balb / c mice was carried out; purified recombinant syndecan-1 protein was used as antigen. During primary immunization, mice were injected subcutaneously with 50 μg of antigen in complete Freund's adjuvant (PAF). After 28 days, the level of the immune response to the introduced antigen was analyzed, evaluating the titer of specific serum antibodies using an enzyme-linked immunosorbent assay (ELISA). Animals with the maximum value of the immune response were re-injected subcutaneously with 30 μg of antigen in incomplete Freund's adjuvant (NAF) and the level of the secondary immune response was evaluated after 21 days, then 4 μg before somatic hybridization was administered 30 μg of antigen in saline (RF) intraperitoneally .

1.3. Obtaining a hybridoma synthesizing monoclonal antibodies specific for syndecan-1

Splenocytes of immunized mice with a titer of serum antibodies to the syndecan-1 protein of approximately 1: 30,000 and murine myeloma cells sp2 / 0-Ag14 (ATCC, CRL-1581) in a 2: 1 ratio were used as fusion partners. Hybridization was carried out using a solution of polyethylene glycol (Sigma) in accordance with standard methods. The selection of stable hybrid cells was performed by culturing on IMDM medium (Iscove-s Modified Dulbecco Medium, Sigma) containing 10% FCI serum (HyClone) and HAT (Gibco). Clones of hybrid cells secreting antibodies specific for specific antigens were selected using enzyme-linked immunosorbent assay (ELISA). As a result of the initial screening, 10 clones of hybrid cells producing monoclonal antibodies to syndecan-1 were selected. After five clonings, 4 stable producing clones were obtained. An mRNA encoding the heavy and light chains of an anti-syndecan-1 antibody was isolated from each of the cell lines.

1.4. Obtaining the nucleotide sequences of the heavy and light chains of a humanized antibody to syndecan-1

RNA was isolated from stable clones of hybrid cells producing murine monoclonal antibodies to syndecan-1 using the TRI Reagent kit manufactured by Sigma, USA, according to the manufacturer's instructions. On 5-10x10 ⁶ cells, 1 ml of TRI Reagent lysis solution was used. CDNA synthesis was performed using the Revert Aid® First Strand cDNA Synthesis Kit (manufactured by Fermentas, Lithuania).

Amplification of genes encoding variable fragments of murine antibodies was performed by PCR using specific primers (5 'degenerate primers for annealing at unknown VH and VL sites) and as a matrix of synthesized cDNA. The production of DNA fragments for cloning was carried out by PCR using high-precision, heat-resistant DNA polymerase Pfx. The obtained PCR products were cloned in the pGMT-easy vector using the Fast Ligation Kit, Fermentas and sequenced by the Sanger enzymatic method.

Sequencing results were used to obtain a humanized monoclonal antibody.

Antibodies were humanized by transferring the detected (calculated using computer algorithms on the obtained nucleotide sequences) hypervariable regions (CDRs) of the variable fragment of murine antibodies responsible for complementarity to the antigen onto the human variable fragment; CDRs were placed between the corresponding framework regions according to the results of database analysis and bioinformatic modeling of structural similarity (analogy) between antibodies. The resulting humanized antibody has the specificity of a maternal mouse antibody, but increased affinity due to the more appropriate spatial arrangement of the CDRs due to the frame regions.

For molecular graphics used programs: Insight II; Accelrys, CA. Conformational homology between a donor mouse antibody and a human acceptor antibody was simulated using WAM (http://antibody.bath.ac.uk), SWISS-MODEL (http://www.expasy.org), INSIGHT-HOMOLOGY (Accelrys) , COMPOSER (Tripos, MO) and the GCG Wisconsin Package. The sequences of variable fragments of murine antibodies were annotated and numbered using the Kabat and Chothia databases.

Information on the structure of variable fragments is taken into account in the numbering developed by Chothia [21]; however, this numbering does not consider amino acid residues in variable fragments of antibodies. In accordance with the above data, the Abinandan KR and Martin AC algorithms modified by Kabat and Chothia were used for numbering and annotation of variable fragments [22, 23]. When numbering, this took into account not only corrected data of multiple alignments of Kabat antibody sequences and data on the Chothia CDR structure taking into account the FR structure, but also data on V _H / V _L folding angles in donor and acceptor antibodies. For automatic annotation, programs with an interactive web interface were used: SeqTest (AbCheck) http://www.bioinf.org.uk/abs/seqtest.html, Abnum http://www.bioinf.org.uk/abs/abnum/ , manual adjustment and verification were carried out using the local version of the Kabat database http://www.kabatdatabase.com/index.html and the KabatMan program http://www.bioinf.org.uk/abs/simkab.html, IMGT http tools : //imgt.cines.fr/ and data on multiple sequence alignment of variable antibody fragments using MUSCULE http://www.ebi.ac.uk/Tools/muscle/index.html and the FASTA software package http: //fasta.bioch .virginia.edu / fasta_www2 / fasta_http: //down.shtml, whose structures presented in the PDB for 2012, as well as several Perl scripts developed by the inventors to automate data analysis. V _H / V _L folding angles were calculated and predicted based on the algorithm proposed by Abhinandan [22]. All necessary calculations related to modeling the structure of protein molecules were performed using Modeller and Swiss-Model.

The next step in the humanization of antibodies was the selection of acceptor variable fragments of human antibodies for transfer. The correct choice of a human acceptor antibody almost 100% guarantees the successful humanization of antibodies, with Kd sufficient for high-strength binding to the antigen. Using molecular modeling methods and the approaches described above, a structural model of the variable fragment of the mouse monoclonal anti-syndecan-1 antibody was constructed. Using the obtained three-dimensional models, the most homologous (especially in key positions) sequences of human acceptor antibodies were determined, while only antibodies with the value of the “humanity” coefficient H> 1 were used (which could potentially reduce the ANAS response). Based on the obtained model, the amino acid and, correspondingly, nucleotide sequences of the variable fragments of the heavy and light (kappa) chains of the antibody were calculated.

1.5. Obtaining humanized monoclonal antibodies to syndecan-1

To obtain the desired properties of antibodies, IgG3 regions were introduced into the constant fragment of the heavy chain, which has the highest degree of activation of the complement system of all isotypes, as well as high affinity for binding to the Fc receptor of phagocytic cells, into the structure of the constant fragment of IgG4, which informed the antibody of the ability induction of antibody-dependent and complement-dependent cytotoxicity. The amino acid substitutions 1M252Y, S254T, T256E, H433K, N434F were also introduced at the C-terminus of the constant IgG4 fragment to increase the half-life of the antibody.

The sequences of the calculated variable and constant fragments of the heavy chain were combined into one amino acid sequence by adding a signal secretory sequence at the N-terminus (SEQ ID NO: 1). The sequences of the calculated variable and constant fragments of the light kappa chain were combined into one amino acid sequence by adding a signal secretory sequence (SEQ ID NO: 2) at the N-terminus.

The amino acid sequences of the protein were converted to nucleotide sequences, optimizing the latter to increase the production of this antibody in mammalian cells (CHO) using the program at http://www.encorbio.com/protocols/Codon.htm.

The result was the final version of the nucleotide sequences (SEQ ID NO: 3, SEQ ID NO: 4) encoding a humanized monoclonal antibody to syndecan-1 (SEQ ID NO: 1, SEQ ID NO: 2), and synthesized using a chemical synthesis. The synthesized fragments were cloned into pUC57 plasmid vector for production, then cloned into pcDNA3.1 + vector for transfection of mammalian cells.

Transfection of mammalian cells with the created plasmids was carried out by the method of calcium phosphate precipitation.

To carry out the transformation of mammalian cells (CHO) by plasmid DNA, the cells were seeded in 12-well plates (Costar, USA) with a culture density of 5 × 10 ⁴ cells / cm ² . The next day, to synchronize cell divisions, the culture medium was replaced. After three hours, plasmid DNA precipitated with calcium phosphate was added to the cells. To prepare a pellet, 250 μl of a solution containing 50 μg of DNA in 250 mM CaCl ₂ was slowly mixed with 250 μl of a solution (1.64% NaCl, 1.13% HEPES pH 7.12 and 0.04% Na ₂ HPO ₄ ). After 24 hours of incubation at 37 ° C in an atmosphere of 5% CO _{2, the} medium was replaced with a similar but containing 2 antibiotics: 80 μg / ml zeocin and 2 μg / ml blasticidin S to select clones containing both transformed plasmids and, therefore, expressing full-sized humanized antibodies, selection was carried out for 20 days, in the wells containing living cells, the medium was changed (the previous culture medium was not poured, but was used to determine the amount of secreted antibodies by ELISA), and after another day the cells were removed from spoons and analyzed for expression of the transformed gene. The transfection efficiency analysis was carried out on an EPICS XL Beckman Coulter flow cytometer (Beckman Coulter, USA).

The level of humanized antibodies in the culture medium obtained by stable transfect of the CHO line was evaluated using standard solid-phase ELISA.

As a result of 6 clonings, stable CHO transfectomas were obtained, which were accumulated for cryopreservation and development of an experimental batch of antibodies. The productivity of the transfect SNO expressing antibodies to syndecan-1 was 500 μg / 10 ⁷ cells / day.

1.6. The cultivation of producer cells of a humanized monoclonal antibody to syndecan-1

The producer cells were cultured using BIOSTAT® Bplus bioreactors and an IMDM autoclaved medium supplemented with 45 g of DFBS (0.5%) and 25.8 g (100 mM) of zinc sulfate seven-water (ZnSO ₄ x 7H ₂ O) per 9 l of medium . The operating mode was set: temperature 37ºC, pH 6.9-7.2, oxygen concentration 50% air saturation. After reaching the given regime, the bioreactor was inoculated, for which, under aseptic conditions, seed was introduced into it. The cultivation time was 3 days.

At the end of cultivation, the culture fluid was filtered through a sterile capsule "Sartopure" ("Sartorius", Germany), with a pore diameter of 1.2 μm, at a speed of 1 l / min. Then, the clarified liquid was concentrated on a Viva Flow 200 system (Sartorius, Germany) using a 50 kDa filter. Concentration was carried out until a total volume of 200 ml was reached.

1.7. Purification of the resulting humanized monoclonal anti-syndecan-1 antibody

Chromatographic purification was carried out in two stages using sterile solutions. At the first stage, we used the BioLogic DuoFlow Pathfinder (Bio-Rad) system with an automatic BioFrac fraction collector and a YMC TriArt semi-preparative chromatographic column, 250x4.6 mm, С18 sorbent. Before starting work, the column was balanced using 200 ml of buffer (1 kg of water for injection and 1 g of trifluoroacetic acid) in manual mode through a chromatograph pump at a speed of 2 ml / min.

The prepared material in a volume of 200 ml was introduced into the chromatograph through a chromatograph pump at a speed of 0.5 ml / min. Elution was performed with a buffer (2 kg of acetonitrile, 2 g of trifluoroacetic acid) at a rate of 0.5 ml per minute. The fraction was collected at the maximum absorption at 260 nm. The volume of the fraction was approximately 500 ml.

The second purification step was carried out using a BioSil SEC 125-5, 300x7.8 mm gel chromatography column. The column was pre-equilibrated with 0.02 M PBS buffer. The resulting material was introduced into the chromatograph through a chromatograph pump at a speed of 0.5 ml / min. Elution was performed with a buffer (0.6 M NaCl solution) with a concentration gradient from 0.1 to 0.6 M. A fraction was collected that had an absorbance at A280 nm of at least 3.4 optical units. The fraction was collected in vials. The volume of the resulting solution was approximately 1 L with an antibody concentration of 2 mg per 1 ml.

The Kd of the obtained antibody was measured, its effect on model cell lines (antiproliferative activity) and xenograft tumor models (inhibition of tumor development) was studied. It turned out that the created antibody has a sufficiently high affinity (Kd = 8.0 * 10 ^-9 ).

Example 2. The study of the antitumor effect of a humanized monoclonal antibody to syndecan-1 in the myeloma model Sp2.0

As a model used the myeloma cell line SP2.0, syngeneic with BALB / C mice, previously showing expression of the gene encoding syndecan-1 in it (Figure 1). For this, RNA was isolated from cell culture and cDNA synthesis and analysis was performed.

SP2.0 tumor cells were grafted into BALB / c NUDE mice: 500,000 SP2.0 tumor cells per animal were injected subcutaneously into the lower back. Used 3 groups of animals: 2 experimental and 1 control.

After the animals were injected monoclonal antibody 1 time per week for 5 weeks at a dose of 1 mg and 0.1 mg starting from 6 days after the introduction of tumor cells, when the tumor size was> 200 +/- 23 mm ³ . The size of the tumor (spherical shape) was determined twice a week for 36 days using a caliper according to the formula: V (tumor) = π / 6 * length * width ² , where length> width.

The following results were obtained: with the introduction of a humanized monoclonal antibody to syndecan-1 in mice with Sp2.0 myeloma grafted cells, there was a significant suppression of tumor growth (Figure 2). The use of a monoclonal antibody preparation for syndecan-1 allowed to reduce the tumor volume by 50% at the 5th week of the experiment.

Example 3. The study of the antitumor effect of a humanized monoclonal antibody to syndecan-1 on the example of the study of cytotoxicity on human cells of the line A431

The anti-tumor effect of the humanized monoclonal anti-syndecan-1 antibody was studied. BALB / c NUDE mice were subcutaneously injected with 10 ⁷ (300 μl) human A431 cells in the flank area. After the animals, a monoclonal antibody was administered at a dose of 50 mg / kg body weight and a control monoclonal antibody against ovalbumin at a dose of 50 mg / kg body weight.

The monoclonal antibody was injected intraperitoneally twice a week in a volume of 200 μl starting from 6 days after the introduction of tumor cells, when the tumor size was> 250 +/- 23 mm ³ . The size of the tumor (spherical shape) was determined twice a week for 36 days using a caliper according to the formula: V (tumor) = π / 6 * length * width ² , where length> width. The survival rate of animals was also estimated, which is the percentage of surviving animals relative to the initial group size at a given time point.

The following indicators were obtained: monoclonal antibody activation of complement dependent cytolysis of A431 cells occurs at a concentration of 0.3 μg / ml, which is a good indicator, and the maximum antiproliferative activity on A431 cells is carried out at a concentration of 0.2 μg / ml.

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Claims (2)

1. A monoclonal antibody of the IgG4 isotype specific for syndecan-1 (CD138), represented by two heavy and two light chains, each of which contains a variable fragment and a constant fragment, for the treatment of tumor diseases, characterized in that it contains IgG3 sites in a constant fragment, the variable fragment is represented by murine, hypervariable, regions and human, framework, regions, the constant fragment is completely human and contains amino acid substitutions at the C-terminus to increase the half-life, the first chain additionally contains a signal secretory sequence at the N-terminus that is cleaved by secretion from producer cells, the heavy chain is represented by the amino acid sequence of SEQ ID NO: 1, encoded by the nucleotide sequence of SEQ ID NO: 3, the light chain is kappa and is represented by the amino acid sequence of SEQ ID NO: 2 encoded by the nucleotide sequence of SEQ ID NO: 4, the nucleotide sequences being codon optimized for expression in mammalian cells, the antibody is used as a self oyatelnogo active ingredient. 2. The antibody according to claim 1, characterized in that it has an independent antibody-dependent and complement-dependent cytotoxicity.

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