RU2568910C2 - Anti-cd3*cd19 flexibody-based pharmaceutical compositions for treating b-cell diseases - Google Patents

Abstract

FIELD: medicine, pharmaceutics.

SUBSTANCE: group of inventions refers to medicine and pharmaceutics, particularly to a pharmaceutical composition containing an effective amount of anti-CD3*CD19 recombinant monoclonal antibody specified in a group containing the antibody SEQ ID No. 1 or the antibody SEQ ID No. 2, as well as phosphate-buffer saline, surfactant and mannitol in the concentration of 1.5-3.0%, as well as to using these compositions for treating B-cell malignancies or B-cell depletion.

EFFECT: group of inventions provides a longer shelf life of the composition.

5 cl, 3 ex, 10 tbl, 6 dwg

Description

The invention relates to medicine, in particular to pharmaceutical compositions containing an effective amount of a recombinant anti-CD3 * CD19 monoclonal antibody of the Flexibody format and suitable for long-term storage, as well as the use of such compositions for the treatment of malignant diseases of B cells or depletion of B cells.

Bispecific antibodies (BAPs) against CD3 * CD19 are currently being developed primarily for the treatment of malignant diseases of B cells or depletion of B cells, including non-Hodgkin’s lymphoma (WO 2010037835, RU2228202), B-cell leukemia (RU 2008129080), pediatric acute lymphoblastic leukemia (WO 20100052013), etc.

In Russia alone, more than 20,000 new cases of Non-Hodgkin lymphoma (NHL) are registered annually. Less than 5% of patients survive for 5 years, despite the fact that the incidence of non-Hodgkin lymphomas in Russia is 4: 100,000 people per year. The standard of treatment for NHL in Russia is the combination of rituximab and chemotherapeutic drugs. However, more than 20% of the initially diagnosed patients and about 50% of patients with recurrent lymphoma stop responding to rituximab therapy, and in the initially responding patients, the effectiveness of rituximab decreases with subsequent courses of therapy due to the development of resistance (Br. J. Haematol. 2011, v.155 , 426-437).

Current studies (e.g., J Clin Oncol, 2011, 29: 2493-8; Cancer, 2010, 116: 5568-74; Science, 2008, 321: 974-7) give every reason to believe that drugs based on bispecific antibodies against CD3 * CD19 promise to become more effective in the treatment of the above diseases.

The increased efficiency is explained by the fact that BAPs act as adapters that physically connect T cells with tumor cells and trigger a powerful signaling cascade of the T-cell receptor complex by binding to the invariant component of the CD3 T-cell receptor. In this case, the second (target) antigen-specific fragment recognizes CD19. Those. BAP can temporarily connect a T cell and a cancer cell by simultaneously binding to CD3 and the target antigen. In this case, the activation and proliferation of T cells, the formation of a cytolytic synapse with the release of cytotoxic granules, and the secretion of cytokines occur. Targeted lysis of cancer cells includes perforation of the target cell membrane involving perforin and subsequent granzyme-induced apoptosis. It should be noted that T cells activated with BAP recognize antigens on the surface of a cancer cell in the same way as ordinary monospecific antibodies. Thus, it is possible to redirect the cytotoxic function of T cells against tumor cells, and BAP activity is already observed at relatively low (picomolar) concentrations. Binding of BAP only to a T cell in the absence of a target cell does not cause T cell activation.

Examples of bispecific antibodies known from the prior art (Blood, (ASH Annual Meeting Abstracts), 2009, 114: 840) indicate that the effective therapeutic concentration of drugs derived from the use of multivalent antibodies is significantly lower compared to classic anti-cancer antibodies drugs. This contributes to good tolerance, the possibility of implementing long-term courses of treatment and reducing the risk of side effects. In addition, the volume and cost of producing bispecific antibodies is lower. Therefore, the development of a new composition based on such antibodies can help significantly reduce production costs and, most importantly, make these drugs available to all patients in need of such therapy.

Currently, the most successful in the BAT clinic is the aura-CD19 / anti-CD3 (CD19 * CD3) blinatumomab preparation (blinatumomab, MT103; a product from Micromet), whose action is based on the mobilization of unstimulated primary T cells against CD19 ⁺ lymphoma cells. Blinatumomab consists of two domains: the anti-CB19 scFv domain from the HD37 hybridoma and the anti-CD3 scFv domain from the TR66 hybridoma, which form the polypeptide of the following structure: VLCD19-VHCD19-VHCD3-VLCD3 (patent RU 2228202).

But blinatumomab is relatively small and quickly eliminated from the bloodstream. Therefore, to achieve stable concentrations of the drug in the blood, prolonged intravenous administration of the drug for 4-8 weeks per cycle is used, which is quite difficult for the patient.

To overcome these shortcomings, a new antibody format has been proposed called flexibody, the technology of which is described in detail in patent EP1293514. Flexibody consists of two pairs of variable domains VH and VL, which are located on the same polypeptide chain and connected by linkers, the length of which determines the possibility of the formation of multimeric structures: dimers, trimers and tetramers. The most promising option is when the resulting polypeptide is two non-covalently bound molecules of a bispecific antibody. In this case, flexibody has a molecular weight of about 120 kDa and is capable of divalently binding to both the target cell and the effector T cell, which allows increasing the therapeutic potential of the molecule (Kratz F, Senter P, Kiprijanov S. Bispecific Antibodies and Immune Therapy Targeting in Drug Delivery in Oncology: From Basic Research to Cancer Therapy. Wiley, 2011) and increase elimination time from the body. Such a flexibody molecule has a modular structure in which the diabody module serves to dimerize a single chain molecule. By pairing complementary VH and VL domains located on different peptide chains, functional Fv modules of the same specificity are formed, and a stable non-covalent protein dimer is obtained. Flexibody has been shown to be effective against B-cell diseases.

However, a pharmaceutical composition based on the use of anti-CD19 * CD3 antibodies of the Flexibody format has not yet been developed, which is useful for treating diseases associated with B cells and suitable for long-term storage.

It was the creation of such a pharmaceutical composition that was the object of the present invention.

To solve this problem, the inventors based on their previously obtained two antibodies against CD19 * CD3 format "flexibody" created pharmaceutical compositions that are effective against b-cell diseases and suitable for long-term storage.

Thus, the technical result of the present invention is to expand the arsenal of funds for the treatment of b-cell diseases and create a composition with a long shelf life.

Disclosure of invention

The present invention relates to pharmaceutical compositions comprising, as an active principle, an anti-CD19 * CD3 flexibody antibody selected from the following polypeptides:

Bis1 with orientation VLCD19-L1-VHCD19-L2-VHCD3-L3-VLCD3 (SEQ ID No. 1) or

Bis2 with orientation VHCD19-L1-VLCD19-L2-VLCD3-L3-VHCD3 (SEQ ID No. 2), in an effective amount, as well as the use of such pharmaceutical compositions for the treatment of B-cell diseases, depletion of B-cells or slowing the development of pathological conditions that are caused by B-cell disorders.

The spatial structure of flexibody formed by the Bis1 and Bis2 polypeptides is shown in FIG. The following peptides were used as linkers (L1, L2, and L3):

L1: GGSGGSGGS,

L2: GGSGGS,

L3: GGSGGSGGSGGSGGS.

To obtain these antibodies, the appropriate expression vectors were created at the MBC “Generium” company, and an industrial method for producing antibodies in CHO cells was developed, which includes the stages of cultivation, isolation and purification. As a result, we received a sufficient amount of antibodies for preclinical studies.

For therapeutic drugs, it is important to maintain stability and activity over the shelf life. Therefore, another task that the present invention solves is the creation of a drug capable of preserving its properties during storage for a long period of approximately 1.5-2 years.

In a most general embodiment, the present invention discloses pharmaceutical compositions comprising, as an active component, an effective amount of a recombinant anti-CD3 * CD19 monoclonal antibody in flexibody format selected from the group consisting of Bis1 antibody or Bis2 antibody; buffer providing pH = 6.8-7.8; surfactant and stabilizer suitable for long-term storage.

The following components are preferred: sodium phosphate buffer, Tween 80 as a surfactant, mannitol as an isotonic agent, and histidine as a stabilizer. We found that the greatest stability of the composition while maintaining biological activity can be achieved by using these components in the following concentrations:

flexibody Bis1 or Bis2 0.1-10.0 mg / ml sodium phosphate 15-25 mm sodium chloride 100-200 mm Twin 80 0.01% mannitol 1.5-3.0% histidine 15-25 mm

with pH = 6.8-7.8.

The most preferred is the following composition:

flexibody Bis1 or Bis2 0.1-1.0 mg / ml sodium phosphate 20 mm sodium chloride 150 mm Twin 80 0.01% mannitol 1.8-2.8% histidine 20 mm

with pH = 6.8-7.8.

Formulations containing Flexibody anti-CD3 * CD19 antibodies for injection may further include isotonic agents, for example, various sugars, as well as additional stabilizers, such as amino acids, such as arginine, glycine, methionine, and pharmacologically compatible preservatives and other targeted additives, as shown, for example, in the Handbook of Pharmaceutical Excipients (2 "ed. London: The Pharmaceutical Press; 1994).

The pharmaceutical compositions of the invention can also be prepared in lyophilized form, and in this case further include pharmaceutically acceptable solvents and diluents, for example, water, saline and other commonly used solvents.

These excipients can be used in combination with other active ingredients (for example, anti-cancer or anti-inflammatory drugs), provided that they do not cause unwanted effects.

The dosage of the pharmaceutical compositions of the invention in patients can be adjusted depending on the weight, age, gender of the patient and the presence of concomitant diseases.

The proposed mechanism of action of antibodies against CD19 * CD3 of the Flexibody format is similar to that described for the anti-CD19 / anti-CD3 antibody, the preparation blinatumomab (MT103); and consists in the activation of a multispecific T-cell cytological response against CD19 + lymphoma cells. Therefore, pharmaceutical compositions based on anti-CD19 * CD3 antibodies of the flexibody format according to the present invention can be used to treat all hematological cancers (lymphomas and leukemia) of B-cell nature. Such as B-cell tumors from precursors of B-lymphocytes, including: B-lymphoblastic lymphoma / B-cell acute lymphoblastic leukemia from progenitor cells; B-cell tumors from peripheral (mature) B-lymphocytes, including: B-cell chronic lymphocytic leukemia / lymphoma from small lymphocytes (lymphocytic lymphoma); B-cell prolymphocytic leukemia; lymphoplasmacytic lymphoma; splenic lymphoma of the marginal zone; hairy cell leukemia; plasma cell myeloma / plasmacytoma; extranodal B-cell lymphoma of the marginal zone of the MALT type; nodal B-cell lymphoma of the marginal zone; follicular lymphoma; mantle cell lymphoma; diffuse B-large cell lymphoma; mediastinal diffuse B-large cell lymphoma; primary exudative lymphoma; Burkitt’s lymphoma / leukemia, as well as for the treatment of autoimmune diseases caused by pathological regulation of B cells, such as rheumatoid arthritis, systemic lupus erythematosus and glomerulonephritis.

Description of figures

Figure 1. Schematic representation of the genetic structure and folding pathway leading to the formation of tetravalent double-stranded BAT “flexibody”.

Figure 2. The change in chromatographic purity of the drug Bis1 in the study of stability at 3 temperatures.

Figure 3. The change in chromatographic purity of the drug Bis2 in the study of stability at 3 temperatures.

Figure 4. Dynamics of changes in plasma concentrations of rats of MT103, Bis1 and Bis2 preparations after their single intravenous administration at a dose of 1.15 μg / kg.

Figure 5. The change in animal mass (g) during the study.

6. Change in the average tumor volume (mm ³ ) during the study.

Description of sequences

SEQ ID NO: 1 — amino acid sequence of Bis1 anti-CD3 * CD19 antibody in flexibody format with orientation V _L ^αCD19 -L1-V _H ^αCD19 -L2-V _H ^αCD3 -L3-V _L ^αCD3

SEQ ID NO: 2 — amino acid sequence of Bis2 anti-CD3 * CD19 antibody flexibody format with orientation V _H ^αCD19 -L1-V _L ^αCD19 -L2-V _L ^αCD3 -L3-V _H ^αCD3

The invention is illustrated by the following examples.

Example 1. Development of a pharmaceutical composition and analysis of its stability.

When selecting a buffer for storing the solutions of the Bis1 and Bis2 preparations, the sodium phosphate buffer (hereinafter referred to as the NPF) was taken as a basis, to which various stabilizers were added, necessary for the long-term stability of the protein. The studied compositions contained Tween80 as a surfactant and solutions of mannitol, sucrose or trehalose, as well as the amino acids arginine, glycine and histidine as stabilizers. The studied compositions are presented in table 1.

Table 1 Variants of compositions with Bis1 / Bis2 preparations at a concentration of 0.5 mg / ml Composition number Composition 0 NSE one NSE + 0.01% Tween80 2 NSE + 0.05% Tween80 3 NSE + 2.8% mannitol four NFB + 5% sucrose 5 NFB + 5% trehalose 6 NFB + 20 mM arginine 7 NFB + 20 mm glycine 8 NBF + 20 mM histidine 9 NFB + 20 mM histidine + 1.8% mannitol + 0.01% tween80 10 NFB + 20 mM arginine + 5% sucrose + 0.01% Tween80 eleven NFB + 20 mM glycine + 5% trehalose + 0.01% tween80

The concentration of the drug in each composition was 0.5 mg / ml. As a characteristic of stability, gel filtration HPLC was used, which made it possible to quantify both the presence of oligomeric forms of the protein and the fragments formed during the decay of the molecule. A study of the stability of the drug in a series of repeated freezing / thawing showed the importance of the presence of stabilizers in the final formulation. As can be seen from the above data (Table 2), phosphate-buffered saline with the addition of various amounts of surfactants did not prevent the decomposition of the molecule during freezing. Adding sugars or amino acids to the phosphate-buffered saline allowed us to achieve sufficient stability of the composition, in which the purity of the product did not change significantly during two series of freezing / thawing.

table 2 The stability of the compositions of the drug Bis1 when freezing / thawing Composition number Original cleanliness 1st freeze 2nd freeze 0 - 86.5 ± 0.5% one 95.4 ± 0.5% 83.4 ± 0.5% 76.4 ± 0.5% 2 95.0 ± 0.5% 81.6 ± 0.5% 72.8 ± 0.5% 3 95.2 ± 0.5% 95.1 ± 0.5% 94.7 ± 0.5% four 96.5 ± 0.5% 96.3 ± 0.5% 96.3 ± 0.5% 5 96.4 ± 0.5% 96.3 ± 0.5% 96.2 ± 0.5% 6 95.3 ± 0.5% 95.4 ± 0.5% 94.8 ± 0.5%

Composition number Original cleanliness 1st freeze 2nd freeze 95.2 ± 0.5% 95.3 ± 0.5% 94.5 ± 0.5% 8 95.3 ± 0.5% 95.4 ± 0.5% 94.9 ± 0.5% 9 95.2 ± 0.5% 95.2 ± 0.5% 94.6 ± 0.5% 10 96.6 ± 0.5% 96.0 ± 0.5% 96.2 ± 0.5% eleven 96.2 ± 0.5% 95.9 ± 0.5% 96.1 ± 0.5%

The study of the stability of the drug during accelerated storage (at a temperature of 25 ± 2 ° C) allows you to extrapolate the data obtained for a longer period and evaluate the approximate shelf life of the drug. Table 3 presents the storage data of the drug Bis1 (the percentage of the purity of the basic substance estimated using gel filtration HPLC) is shown at a temperature of 25 ± 2 ° C for 1 month. Similar results were obtained for the drug Bis2.

Table 3 The stability of the drug Bis1 when stored at 25 ± 2 ° C Composition number Source 1 Week 2 week 3 week 4 week * 0 - 95.1 ± 0.5% 95.1 ± 0.5% 96.0 ± 0.5% 96.8 ± 0.5% one 95.4 ± 0.5% 95.1 ± 0.5% 95.6 ± 0.5% 95.8 ± 0.5% 96.5 ± 0.5% 2 95.0 ± 0.5% 95.0 ± 0.5% 93.7 ± 0.5% 95.8 ± 0.5% 96.5 ± 0.5% 3 95.2 ± 0.5% 95.4 ± 0.5% 95.5 ± 0.5% 95.9 ± 0.5% 96.8 ± 0.5% four 96.5 ± 0.5% 96.6 ± 0.5% 91.3 ± 0.5% 85.6 ± 0.5% 89.3 ± 0.5% 5 96.4 ± 0.5% 96.5 ± 0.5% 84.3 ± 0.5% 83.2 ± 0.5% 72.1 ± 0.5% 6 95.3 ± 0.5% 95.3 ± 0.5% 95.8 ± 0.5% 96.0 ± 0.5% 96.9 ± 0.5% 7 95.2 ± 0.5% 95.1 ± 0.5% 95.6 ± 0.5% 96.0 ± 0.5% 96.7 ± 0.5% 8 95.3 ± 0.5% 95.5 ± 0.5% 95.6 ± 0.5% 96.0 ± 0.5% 96.8 ± 0.5% 9 95.2 ± 0.5% 95.2 ± 0.5% 95.4 ± 0.5% 96.0 ± 0.5% 96.9 ± 0.5% 10 96.6 ± 0.5% 96.4 ± 0.5% 92.1 ± 0.5% 88.6 ± 0.5% 66.2 ± 0.5% eleven 96.2 ± 0.5% 96.4 ± 0.5% 96.3 ± 0.5% 84.8 ± 0.5% 82.8 ± 0.5% * Slightly overestimated rates at week 4 are associated with a change in the chromatographic column.

As can be seen from the data presented, there is a sharp drop in the purity of the drug when using sucrose or trehalose in the formulation of the drug (compositions No. 4, 5, 10 and 11). The use of mannitol in the composition of the buffer did not lead to a significant change in the purity of the drug.

Based on the data obtained, compositions were selected in which there were no noticeable changes, or where such changes were the smallest when compared with the beginning of the study. Tween80 (surfactant) and mannitol as a stabilizer were selected as components of the final buffer for the antibody composition. Histidine was also included in the final formulation to further stabilize the drug. The compositions of the present invention have a pH in the range of 6.8-7.8.

The stability studies of the Bis1 and Bis2 preparations were carried out in a buffer containing: 150 mM sodium chloride, 0.01% tween 80, 1.8% mannitol, 20 mM histidine, 20 mM sodium phosphate. The concentration of antibodies was 0.5 mg / ml.

In the stability study, stability indicators were determined such as chromatographic and electrophoretic purity, as well as the biological activity of the drug by the method of ADCC (antibody-dependent cell-mediated cytotoxicity), which allows in vitro simulating the immune response process that occurs in a living organism. As a result of this analysis, a parameter was determined for antibodies such as the concentration of the active substance at which a half biological effect is observed, which is a qualitative characteristic of antibody-like structures specific for cell receptors.

For long-term tests, compositions containing Bis1 and Bis2 were laid down for storage at temperatures of 5 ± 3 ° C, 25 ± 2 ° C and minus 80 ° C. The duration of long-term testing of these compositions was 12 months. The results are presented in Table 4 and in figures 2 and 3.

Table 4 The stability indicators of the drugs Bis1 and Bis2 during long-term storage Preparation, storage conditions Indicator Baseline: March 2013 Summary: March 2014 Bis1 solution Authenticity. Electrophoresis in page Confirmed * Confirmed *

Preparation, storage conditions Indicator Baseline: March 2013 Summary: March 2014 5 ± 3 ° C Chromatographic purity: Main substance > 95.0% 97.66% Related Impurities: - aggregates not determined 1.46% - fragments not determined 0.88% Specific Activity, EC50 3 · 10 ^-4 4 · 10 ^-4 Bis1 solution 25 ± 2 ° C Authenticity. Electrophoresis in page Confirmed * Confirmed *. Impurities with a molecular weight of less than 55 kDa are determined in the sample. Chromatographic purity: Main substance > 95.0% 81.92% Related Impurities: - aggregates not determined 1.88% - fragments not determined 16.20% Specific Activity, EC50 3 · 10 ^-4 7 · 10 ^-2 Bis1 solution -80 ° C Authenticity. Electrophoresis in page Confirmed * Confirmed * Chromatographic purity: Main substance > 95.0% 98.78% Related Impurities: - aggregates not determined 0.97% - fragments not determined 0.26% Specific Activity, EC50 3 · 10 ^-4 1 · 10 ^-4 Bis2 solution 5 ± 3 ° C Authenticity. Electrophoresis in page Confirmed * Confirmed * Chromatographic purity: Main substance > 95.0% 94.50% Related Impurities: - aggregates not determined 4.98% - fragments not determined 0.53% Specific Activity, EC50 1.9 · 10 ^-5 2.5 · 10 ^-4

Preparation, storage conditions Indicator Baseline: March 2013 Summary: March 2014 Bis2 solution 25 ± 2 ° C Authenticity. Electrophoresis vPAAG Confirmed * Confirmed *. Impurities with a molecular weight of less than 55 kDa are determined in the sample Chromatographic purity: Main substance > 95.0% 78.95% Related Impurities: - aggregates not determined 3.31% - fragments not determined 17.75% Specific Activity, EC50 1.9 · 10 ^-5 1 · 10 ^-3 Bis2 solution -80 ° C Authenticity. Electrophoresis vPAAG Confirmed * Confirmed * Chromatographic purity: Main substance > 95.0% 94.85% Related Impurities: - aggregates not determined 5.09% - fragments not determined 0.08% Specific Activity, EC50 1.9 · 10 ^-5 8.3 · 10 ^-4 * The main band on the electrophoregram of the test sample is between the molecular weight markers of 55 and 70 kDa.

The value of the biological activity of the drug remained stable throughout the entire period of the study of drugs Bis1 and Bis2 at temperatures of 5 ± 3 ° C and -80 °. At a temperature of 25 ± 2 ° C after 12 months of the study, a significant decrease in the biological activity of both Bis1 and Bis2 was detected.

When determining the authenticity by polyacrylamide gel electrophoresis in samples Bis1 and Bis2 stored at 25 ± 2 ° С, impurities with a molecular mass of less than 55 kDa are detected, and these impurities are first detected in samples only after 6 months of accelerated storage. The extrapolation of the obtained stability data of the drug according to the instructions I42-2-82 ("Temporary instructions for the work to determine the shelf life of drugs based on the method of accelerated storage at elevated temperatures" M .: Ministry of Health of the Russian Federation, 1982) allows us to calculate the estimated shelf life of 2 years at a temperature of 5 ± 3 ° C. The results obtained are consistent with the data obtained when determining the chromatographic purity of the drug at a given temperature.

Thus, the composition found allows you to get a drug that is stable for at least 1.5 years when stored at 4 ° C (household refrigerator). These conditions satisfy the criteria for obtaining commercially available products suitable for the end user.

For the obtained compositions, pharmacokinetic parameters and antitumor activity were evaluated in comparison with the composition based on MT-103, used as a control.

Example 2. A comparative assessment of the pharmacokinetic parameters of the drugs Bis1 and Bis2.

The following pharmaceutical compositions were used to evaluate pharmacokinetic parameters:

Table 5 a drug Volume ml Concentration, mg / ml Osmolality, mOsm / kg pH The content of endotoxin, EE / ml Bis1 four 0.54 436.3 7.3 Less than 2 Bis2 four 0.59 412.7 7.3 Less than 2 MT103 four 0.52 390 7.4 Less than 1

The composition of both drugs, in addition to the main active ingredient, included the following components: for pharmaceutical compositions containing Bis1 or Bis2 antibodies: 150 mm sodium chloride, 0.01% polysorbate 80, 1.8% mannitol, 20 mm histidine, 20 mm sodium phosphate ;

for pharmaceutical compositions containing MT103: 150 mm sodium chloride, 20 mm sodium phosphate.

For preclinical studies, outbred CD rats (Sprauge Dowley), females weighing about 400 g, were obtained from the Pushchine nursery of the FIBH RAS. A total of 21 rats were used in the experiment.

Table 6 The distribution of animals in groups Group number A drug Dose (mcg / kg) Concentration (μg / ml) Number of animals The mass of the animal, g Group 1 Bis1 1.15 1.15 7 410 ± 21 Group 2 Bis2 1.15 1.15 7 41b ± 23 Group 3 MT103 1.15 1.15 7 396 ± 18

Animal conditions were in accordance with the Guide for the Care and Use of Laboratory Animals, 8th edition, 2011, NRC, USA (Laboratory Animal Care and Use Manual, 8th edition, 2011, National Research Committee, USA).

An intravenous route of administration of the test drug to the caudal caudal vein was chosen. This route of administration is due to the fact that the intravenous route of administration is expected in the clinical use of the drug.

Table 7 Study design Study day Events Recorded Indicators -55-0 Quarantine and adaptation Assessment of animal health, identification of signs of infectious pathology, social adaptation

Study day Events Recorded Indicators 0 The introduction of the study drug. Blood sampling at specific time points for the study of the pharmacokinetics of Bis1 drugs 0 min, 2 min, 30 min, 1 h, 2 h, 4 h, 8 h, 10 h one The introduction of the study drug. Blood sampling at specific time points to study the pharmacokinetics of Bis2 drugs 0 min, 2 min, 30 min, 1 h, 2 h, 4 h, 8 h, 10 h four The introduction of the study drug. Blood sampling at specific time points to study the pharmacokinetics of MT103 drugs 0 min, 2 min, 30 min, 1 h, 2 h, 4 h, 8 h, 10 h 5-28 Quantitative determination of the concentration of drugs in the blood plasma of rats. The concentration of drugs in rat plasma samples 29- Analysis of the data. C, C _max , T _1/2 , AUC _0-t , AUC _0-∞

Mass spectrometric method was used for quantitative measurement of Bis1, Bis2 and MT103 proteoforms in rat plasma. Synthetic peptides with the inclusion of the amino acid C ¹³ N ¹⁵ -Leu identical to the native peptides of the measured proteoforms obtained by the enzymatic trypsin cleavage reaction were used as the internal measurement standard.

The results of measuring the concentration of the studied drugs in the blood plasma of rats over time are presented in Figure 4. The results of calculating the pharmacokinetic parameters of the studied drugs are presented in table 8.

Table 8 Pharmacokinetic parameters of MT103, Bis1 and Bis2 preparations after their single intravenous administration at a dose of 1.15 μg / kg Indicator Bis1 Bis2 MT103 K _el , 1 / h 1.09 ± 0.14 1.45 ± 0.089 1.29 ± 0.10 T _1/2 h 25.56 ± 3.28 27.16 ± 1.66 30.51 ± 2.46 Cmax, ng / ml 115.73 ± 18.46 447.50 ± 156.00 130.52 ± 35.41 AUC _0-t 68.01 ± 7.27 266.03 ± 101.37 92.34 ± 26.12 AUC _0-∞ 68.47 ± 7.61 266.09 ± 101.39 92.51 ± 26.11

Thus, it was shown that the half-lives of the Bis2 and MT103 drugs do not significantly differ. The differences in the AUC and Cmax readings can be explained by the different volume of distribution of drugs with different spatial conformation.

Example 3. Comparison of the antitumor activity of Bis1, Bis2, and MT 103 in a Raji cell tumor xenograft model in NOD / SCID mice during reconstitution of human PBMC.

The aim of the study was to compare the efficacy of Bis1, Bis2, and MT103 in NOD / SCID mice at two dose levels (1 μg / animal or 10 μg / animal), on a Raja tumor cell xenograft model with simultaneous injection of peripheral blood mononuclear cells (PBMC) human immunodeficient mice NOD / SCID.

Cells of the Raja line (human Burkitt’s lymphoma) were pre-mixed with human PBMC and inoculated subcutaneously with subsequent intravenous administration of the studied drugs. For this, eight experimental groups of immunodeficient NOD / SCID mice (9 animals per group) on day 0 (d0) were either subcutaneously injected with either human Raji Burkita lymphoma cells or a suspension of pre-mixed Raji lymphoma cells and human PBMC. Three animals from each group received human PBMC from one of three separate healthy donors (cohorts 1, 2, and 3). Animals on day 0 (2 hours after cell inoculation), on days 1, 2, 3 and 4, were injected intravenously with either NFB (sodium phosphate buffer solution) or the studied preparations Bis1, Bis2 or MT 103 at a dose of 1 μg or 10 μg. The injection volume was 200 μl. In all groups, the incidence and volume of the tumor were evaluated over 39 days (days 3 through 41).

Injected solutions were prepared by diluting a basic solution of Bis1 (0.54 mg / ml), Bis2 (0.59 mg / ml) or MT103 (0.52 μg / ml) in 0.9% saline to obtain a concentration of 50 μg / ml (dose of 10 μg) or 5 μg / ml (dose of 1 μg). The experimental groups are described in table 9 below:

Table 9 Group Rat number Cells * inoculated by sc injection (d0) The studied drug (iv injection 1 time / day) 1-3 Raji Cells NSE one 4-6 Raji Cells 7-9 Raji Cells 10-12 Cells of the Raji line + MCPC (Donor 1) NSE 2 13-15 Cells of the Raji line + MCPC (Donor 2) 16-18 Cells of the Raji line + MCPC (Donor 3) 19-21 Cells of the Raji line + MCPC (Donor 1) Bis1 1 mcg / animal 3 22-24 Cells of the Raji line + MCPC (Donor 2) 25-27 Cells of the Raji line + MCPC (Donor 3) 28-30 Cells of the Raji line + MCPC (Donor 1) Bis1 10 mcg / animal four 31-33 Cells of the Raji line + MCPC (Donor 2) 34-36 Cells of the Raji line + MCPC (Donor 3) 37-39 Cells of the Raji line + MCPC (Donor 1) Bis2 1 mcg / animal 5 40-42 Cells of the Raji line + MCPC (Donor 2) 43-45 Cells of the Raji line + MCPC (Donor 3) 46-48 Cells of the Raji line + MCPC (Donor 1) Bis2 10 mcg / animal 6 49-51 Cells of the Raji line + MCPC (Donor 2) 52-54 Cells of the Raji line + MCPC (Donor 3) 7 55-57 Cells of the Raji line + MCPC (Donor 1) MT103

Group Rat number Cells * inoculated by sc injection (d0) The studied drug (iv injection 1 time / day) 58-60 Cells of the Raji line + MCPC (Donor 2) 1 mcg / animal 61-63 Cells of the Raji line + MCPC (Donor 3) 64-66 Cells of the Raji line + MCPC (Donor 1) MT103 10 mcg / animal 8 67-69 Cells of the Raji line + MCPC (Donor 2) 70-72 Cells of the Raji line + MCPC (Donor 3) * 2.5 × 10 ⁶ cells of the Raji line, 1 × 10 ⁷ MCPC.

In experimental animals, the following indicators were determined: survival, body weight, tumor volume.

Body weight was determined on day 0 and subsequently recorded three times a week until day 41. Tumor volume was monitored 3 times a week from day 3 to day 41 by measuring the large and small diameter of the tumor with a pair of compasses. Based on the diameters, the tumor volume was calculated using the following formula:

Volume = (small diameter) ² × large diameter × 0.5

In control group 1 receiving NFB (only Raji cells), 44.4% of the animals survived until day 41 (completion of the study). Survival decreased by 0% in animals of group 2 treated with cells of the Raji line and human PBMC. In both groups, mortality was due solely to killing due to the large tumor volume. The administration of Bis1, Bis2 and MT 103 increased survival in groups 3-8 to 89% or 100%, which is shown in Table 10, and with a higher dose, the studied drugs are more effective. than drug comparison:

Table 10 Mortality and survival of animals Group number Injectable drug Dose (mcg / animal) Animals (n) Survival (n) (%) one NSE - 9 four 44.4 2 NSE - 9 0 0.0 3 Bis1 one 9 8 88.9 four Bis1 10 9 9 100.0 5 Bis2 one 9 8 88.9 6 Bis2 10 9 9 100.0 7 MT103 one 9 8 88.9 8 MT103 10 9 8 88.9

Ending criteria: tumor volume> 10% of body weight; tumor ulceration; a dying animal has been euthanized (for ethical reasons); the animal is found dead with signs of auolysis; weight loss> 20%.

The average body weight of animals that were inoculated with a mixture of Raji cell line and human PBMC and the studied drugs Bis1, Bis2 or MT 103 were injected increased during the study. In this case, the relative average body weight in the groups receiving the study drug was lower than in group 2 receiving the NPS (see Figure 5).

Tumor growth was significantly inhibited or slowed down in all study groups treated with Bis1, Bis2 or MT 103 (groups 3-8) compared with control group 2 treated with NPF. Moreover, in all the studied groups treated with Bis1, Bis2 or MT103, inhibition of tumor growth was statistically significant (p <0.05). Upon completion of the study, a dose of 10 μg of all three studied drugs Bis1, Bis2, and MT 103 significantly inhibited tumor growth in NOD / SCID mice to 4% and 5% of tumor volume compared to control group 2. The antitumor effect at this dose was similar for all three studied drugs. A dose of 1 μg was almost equally effective for all drugs except Bis2. The results are presented in Fig.6.

Claims (5)

1. A pharmaceutical composition having antitumor activity against diseases of a B-cell nature, comprising an effective amount of a recombinant anti-CD3 * CD19 monoclonal antibody selected from the group consisting of an antibody characterized by the sequence of SEQ ID No. 1 or an antibody characterized by the sequence of SEQ ID No. 2; as well as phosphate-buffered saline, surfactant and mannitol in a concentration of 1.5-3.0%. 2. The pharmaceutical composition according to claim 1, characterized in that it is a composition of the following composition:
antibody 0.1-10.0 mg / ml sodium phosphate 15-25 mm sodium chloride 100-200 mm Twin 80 0.01% mannitol 1.5-3.0% histidine 15-25 mm
with pH = 6.8-7.8. 3. The pharmaceutical composition according to p. 2, characterized in that it is a composition of the following composition:
antibody 0.1-1.0 mg / ml sodium phosphate 20 mm sodium chloride 150 mm Twin 80 0.01% mannitol 1.8-2.8% histidine 20 mm
with pH = 6.8-7.8. 4. The use of the composition according to any one of paragraphs. 1-3 for the treatment of malignant diseases of b-cells or depletion of b-cells. 5. The use of claim 4, wherein the malignant disease of the B cells is non-Hodgkin's lymphoma.

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