EA036905B1 - Bispecific antibodies against cd3*cd19 - Google Patents

Abstract

The invention relates to biotechnology and, in particular, can be used in medicine and in the pharmaceutical industry for preparing a drug for treating a B-cell disorder, depleting B-cells or slowing the development of a pathological condition associated with B-cell abnormalities. A bispecific antibody specifically binding to CD3 and CD19 comprises two chains connected by disulphide bonds. Each chain contains light and heavy chain variable domains of antibodies against CD3 and CD19, linker sequences, an immunoglobulin IgG hinge region and an immunoglobulin IgG Fc domain constant region. The antibody is produced with the aid of recombinant DNA technology using a nucleic acid that codes for the antibody and/or using a vector containing said nucleic acid, which are introduced into a host cell. A method of producing the antibody includes at least cultivating a host cell and isolating the target product. A method of purifying the antibody includes at least a chromatography stage. The antibody is used to treat a B-cell disorder, deplete B-cells or slow the development of a pathological condition associated with B-cell abnormalities. A corresponding pharmaceutical composition contains said antibody and a pharmaceutically acceptable carrier. A method of treating disorders includes administering to a patient in need thereof a therapeutically effective amount of the antibody. The invention provides for more convenient anti-CD3*CD19 therapy as a result of an anti-CD3*CD19 antibody with an increased half-life and an enhanced cytotoxic effect, which has high bioavailability even when administered subcutaneously.

Description

Technology area

The invention relates to biotechnology and, in particular, can be used in medicine and the pharmaceutical industry for the manufacture of a drug for the treatment of B-cell disease, depletion of B-cells or slowing the development of a pathological condition associated with B-cell disorders.

Prior art

Bispecific antibodies (BAPs) against CD3 * CD19 are currently being developed mainly for the treatment of malignant diseases of B cells or depletion of B cells, including non-Hodgkin's lymphoma (WO 2010037835, RU 2228202), B-cell leukemia (RU 2008129080), pediatric acute lymphoblastic leukemia (WO 20100052013), etc.

Several completed and ongoing studies (e.g. J Clin Oncol, 2011, 29: 2493-8; Cancer, 2010, 116: 5568-74; Science, 2008, 321: 974-7; BLINCYTO, AmGen Recearch GmbH (NCT00560794 , NCT00274742, NCT01207388, NCT01209286, NCT01471782, NCT01466179,

NCT01741792); AFM11, Affimed Therapeutics AG (NCT02106091)) strongly suggest that anti-CD3 * CD19 bispecific antibodies have the potential to be more effective in treating the aforementioned 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, i.e. BAP can connect a T cell and a cancer cell through simultaneous binding to CD3 and a target antigen. In this case, activation and proliferation of T cells occurs, the formation of a cytolytic synapse with the release of cytotoxic granules and the secretion of cytokines. Targeted lysis of cancer cells involves perforation of the target cell membrane with perforin and subsequent granzyme-induced apoptosis. It should be noted that, after binding to T cells, BAPs recognize antigens on the surface of a cancer cell in the same way as conventional monospecific antibodies. Thus, it is possible to redirect the cytotoxic function of T cells against tumor cells, and BAP activity is observed even at relatively low (picomolar) concentrations. Binding of BAP only with 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 preparations based on the use of multivalent antibodies is significantly lower than that of classical anti-cancer antibodies. drugs. This contributes to good tolerability, the possibility of carrying out long courses of treatment and reducing the risk of side effects. In addition, the required volume and cost of bispecific antibody production is lower. Therefore, the creation of such antibodies will significantly reduce the scale and cost of manufacturing drugs based on them and, most importantly, make these drugs available to all patients in need of such therapy.

To date, quite a few anti-CD3 * CD19 bispecific antibodies have been developed and produced. The most promising of them are single-stranded molecules as such or non-covalent dimers consisting of single-stranded molecules.

One of the single-chain antibodies against CD3 * CD19 is blinatumomab (Blincyto, MT103). Blinatumomab consists of two domains: the anti-CD19 scFv domain from the HD37 hybridoma and the anti-CD3 scFv domain from the OKT3 hybridoma. To obtain an antibody, a DNA fragment VLCD19-VHCD19-VHCD3-VLCD3 is expressed in DHFR-negative CHO cells (patent RU 2228202).

Blinatumomab, in comparison with full-length antibodies, has a relatively small molecular weight (55 kDa versus 155 kDa), which is why it is quickly eliminated from the bloodstream. Therefore, to achieve stable concentrations of the drug in the blood, continuous intravenous administration of the drug is used for 2-4 weeks in one cycle (in the regime of two weeks - a dropper, two weeks - a break), which is quite difficult for the patient to tolerate.

In addition, blintomumab is difficult to manufacture on an industrial scale, since homo- and heterodimers can be formed during the production of single-chain antibodies, which complicates the isolation and purification of such antibodies. On the other hand, the opposite process is possible - undesirable dissociation of dimers, as was shown, for example, in the article Glockshuber et al., Biochemistry, 1990, vol 29: 1362-1367. To avoid this, antibodies stabilized by disulfide bonds have been proposed (Cell Oncol. (2012), 35: 423-434), which increased both the stability of the antibody in solution and the therapeutic efficacy in comparison with the single-chain antibody.

Despite the lack of ease of use and complexity of preparation, blinatumomab is one of the most successful antibodies against CD3 * CD19.

Blinatumomab belongs to BiTE® antibodies. BiTE® antibodies demonstrate a high efficiency of redirected lysis of cells expressing the target tumor antigen. EC50 values

- 1,036905 for BiTE® antibodies generally range from 0.1 to 50 pM (Bauerle P. A. and Reinhardt C. (2009) Cancer Res. 69 (12) pp. 4941-4944). Powerful cytolysis of tumor cells is required in order to produce a pharmacologically desirable effect. In a review, Rader C. reports that the powerful lytic capacity of the BiTE® format is due to (1) its small size, which brings the target and effector cells into close proximity, allowing cytolytic synapses to form, and (2) its monovalent activation of the TCR complex, which prevents systemic activation of effector cells in the absence of target cells (Rader C. (2011), Blood, 117 (17), pp. 4403-4404). In addition, the prior art discloses that the BiTE® class of bispecific antibodies is typically 100 to 10,000 times more efficient in lysis of tumor cells relative to other bispecific formats and total IgG1 monoclonal antibodies (Wolf et al. (2005) Drug Discov Today 10 (18), pp. 1237-1244).

Thus, for a person skilled in the art, it follows that small monovalent formats of anti-CD3 * CD19 bispecific antibodies of the BiTE® format exhibit a more potent redirected T cell lytic activity than large bivalent CD3 * CD19 bispecific formats, and the use of large bivalent CD3 * CD19 bispecific antibodies are inappropriate.

Disclosure of invention

The present invention solves the problem of obtaining a bivalent anti-CD3 * CD19 antibody that is convenient in use and preparation in comparison with blinatumomab and, at the same time, is highly effective in the treatment of malignant diseases of B cells, depletion of B cells or slowing down the development of pathological conditions associated with B- cellular disorders.

The work of the team of the authors of the present invention to improve the pharmacokinetic characteristics of antibodies against CD3 * CD19 led to the creation of a new antibody construct, which is a bispecific double-stranded molecule, each chain of which contains the Fc part of normal IgG and four binding domains connected by linkers and located in the following order from N -end to C-terminus: VL (CD3) -L1-VH (CD19) -L2-VL (CD19) -L3-VH (CD3) -H-CH2-CH3 (IgG), where VL (CD3), VL ( CD19), VH (CD3) and VH (CD19) are the variable domains of the light and heavy chains of antibodies against CD3 and CD19, respectively, L1, L2, L3 are linker sequences, H is the hinge region of IgG immunoglobulin and CH2-CH3 (IgG) - constant region of the Fc domain of an IgG immunoglobulin. The two chains are connected by disulfide bonds.

It has surprisingly been found that such bispecific molecules, in comparison with the blinatumomab antibody (MT-103), have an increased half-life and are easy to obtain. At the same time, even more unexpected for the team of authors were such properties of this antibody as an enhanced cytotoxic effect and high bioavailability even with subcutaneous administration at the level of 60%. The latter characteristic is especially important, since until now, due to low bioavailability, antibody preparations have been administered mainly by intravenous route, which is more difficult and less comfortable for the patient. The increased half-life, high efficacy and bioavailability after subcutaneous administration will reduce the frequency of drug administration and increase the ease of use.

Thus, the first aspect of the present invention is a recombinant anti-CD3 * CD19 monoclonal bispecific antibody comprising two disulfide-linked chains, each of which consists of the following domains: VL (CD3) -L1-VH (CD19) -L2VL (CD19) - L3-VH (CD3) -H-CH2-CH3 (IgG), where VL (CD3), VL (CD19), VH (CD3) and VH (CD19) are the variable domains of the light and heavy chains of antibodies against CD3 and CD19, respectively , L1, L2, L3 - linker sequences, H - hinge region of IgG immunoglobulin and CH2-CH3 (IgG) - constant region of the Fc domain of IgG immunoglobulin.

Moreover, the specified constant region of the Fc domain of immunoglobulin IgG is preferably a constant region of the Fc domain of immunoglobulin IgG2 and contains the sequence shown in SEQ ID NO: 5. Linker sequences L1, L2, L3 preferably have a length of 5-15 amino acids and have sequences according to essentially corresponding to the sequences shown in SEQ ID NO: 6, 7, 8, respectively. The hinge region of the IgG immunoglobulin essentially corresponds to the sequence shown in SEQ ID NO: 9.

The specified antibody preferably has a half-life in blood, increased compared with the half-life of blinatumomab, measured under substantially similar conditions. In this case, the half-life in blood of the antibody of the invention may be more than 40 hours. Moreover, the specified antibody preferably, but not necessarily, has an amino acid sequence substantially corresponding to the sequence shown in SEQ ID NO: 10.

Further objects of the present invention are a nucleic acid encoding a chain sequence of said anti-CD3 * CD19 bispecific antibody of the invention and a vector containing it. The specified vector can be a vector for ensuring the expression of the bispecific antibody against CD3 * CD19 in the host cell according to the invention, containing the above

- 2,036905 nucleic acid operably linked to nucleotide sequences allowing heterologous expression in the host cell. The specified vector can be a vector for cloning, containing the above nucleic acid, and restriction sites flanking it, allowing you to cut out the specified nucleic acid using suitable restriction enzymes. Preferably, but not necessarily, the specified vector is the expression plasmid pRA1451, characterized by the following features:

consists of 11308 bp, has a molecular weight of 6.99 MDa, contains a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 10, provides resistance to mammalian cells transfected with the specified plasmid to puromycin;

contains the following elements:

CMVe / EF1a pro - enhancer of CMV virus and promoter of transcription of elongation factor alpha gene,

BGH рА - bovine growth hormone polyadenylation signal, sv40 enh - SV40 virus enhancer,

SGE 1 - element of attachment to the nuclear matrix,

Amp - β-lactamase gene, sv40 pro - SV40 virus promoter, puro - puromycin resistance gene, pA - synthetic polyadenylation signal, contains unique recognition sites for the following restriction endonucleases:

AccI (6490 bp), Agel (460 bp), Apal (995 bp), BmgBI (3270 bp), BsmI (7269

bp), EcoRV (6457 bp), Hindlll (1590 bp), Notl (11146 bp), Pfol (11021 bp),

Pmll (5699 bp), Spel (4622 bp), Swal (6466 bp), Tthl111 (10585 bp).

Another object of the present invention is a host cell containing a nucleic acid according to the invention or a vector for allowing expression of the anti-CD3 * CD19 bispecific antibody of the invention in a host cell. The specified cell can be both prokaryotic and eukaryotic cells. Preferably, but not necessarily, said cell can be a mammalian cell, in particular a Chinese hamster cell.

A further object of the present invention is a method for producing an anti-CD3 * CD19 bispecific antibody of the invention, comprising at least culturing a host cell of the invention under conditions enabling expression of an anti-CD3 * CD19 antibody of the invention and isolating the resulting product.

It is also an object of the present invention to provide a method for purifying an anti-CD3 * CD19 antibody of the invention, comprising carrying out at least one chromatography step under conditions such that an anti-CD3 * CD19 antibody of the invention is substantially free of host cell contaminants. The method may further comprise a centrifugation step, preferably carried out prior to the chromatography step, and another chromatography step. Moreover, the first stage of chromatography is preferably, but not necessarily, performed using a sorbent based on protein A, and the second stage of chromatography is a stage of cation exchange chromatography.

A further object of the present invention is the use of an anti-CD3 * CD19 antibody of the invention for the treatment of B-cell diseases, depletion of B-cells or retarding the development of pathological conditions associated with B-cell disorders.

Said use involves administering an antibody of the invention to a patient, preferably, but not necessarily, intravenously or subcutaneously. In this case, the antibody is administered to the patient in a therapeutically effective amount.

Another object of the invention is a pharmaceutical composition for the treatment of B-cell disease, depletion of B-cells or slowing down the development of pathological conditions associated with B-cell disorders, containing as an active component the anti-CD3 * CD19 antibody of the invention in a therapeutically effective amount and a pharmaceutically acceptable carrier.

Another object of the present invention is a method of treating a B-cell disease, depletion of B-cells or slowing the development of pathological conditions associated with B-cell disorders, comprising administering to a patient in need thereof an effective dose of an anti-CD3 * CD19 antibody according to the invention or an effective amount of a pharmaceutical composition according to invention.

In this case, the specified B-cell disease, depletion of B-cells or slowing down the development of a pathological condition associated with B-cell disorders is one of the following: B-lymphoblastic lymphoma / B-cell acute lymphoblastic leukemia from cells

- 3 036905 predecessors; B-cell tumors from peripheral (mature) B-lymphocytes, including B-cell chronic lymphocytic leukemia / small lymphocyte lymphoma (lymphocytic lymphoma); B-cell prolymphocytic leukemia; lymphoplasmacytic lymphoma; splenic lymphoma of the marginal zone; hairy cell leukemia; plasma cell myeloma / plasmacytoma; MALT-type extranodal B-cell lymphoma of the marginal zone; nodal B-cell lymphoma of the marginal zone; follicular lymphoma; lymphoma from cells of the mantle zone; diffuse large B-cell lymphoma; diffuse mediastinal large B-cell lymphoma; primary exudative lymphoma; Burkitt's lymphoma / leukemia, and for the treatment of autoimmune diseases caused by abnormal regulation of B cells such as rheumatoid arthritis, systemic lupus erythematosus, glomerulonephritis, multiple sclerosis and myasthenia gravis.

Brief Description of Drawings

FIG. 1. Schematic representation of the construct of the GNR-047 antibody.

FIG. 2. Scheme for constructing an expression vector.

FIG. 3. Map of the expression vector pRA1451.

FIG. 4. Analysis of the ability of the GNR-047 antibody and MT-103 antibody to bind the native CD3 receptor on the Jurkat cell line and the CD19 receptor on the Raji cell line.

FIG. 5a. Study of the pharmacokinetic characteristics of the antibody GNR-047 when administered intravenously at a dosage of 0.5 mg / kg.

FIG. 5 B. A study of the pharmacokinetic characteristics of the MT-103 antibody when administered intravenously at a dosage of 0.5 mg / kg.

FIG. 6a. Study of the pharmacokinetic characteristics of the antibody GNR-047 when administered subcutaneously at a dosage of 2.0 mg / kg.

FIG. 6b. Study of the pharmacokinetic characteristics of the MT-103 antibody when administered subcutaneously at a dosage of 2.0 mg / kg.

FIG. 7. Change in the average tumor volume during the study.

Best Mode for Carrying Out the Invention

The following describes the most preferred embodiments of the present invention, discloses the semantic content of the terms used in the present invention, and presents examples demonstrating the implementation of the present invention and the results achieved thereby.

The term recombinant anti-CD3 * CD19 monoclonal bispecific antibody used in accordance with the present invention includes antibodies that specifically bind to both CD3 and CD19, which are obtained using recombinant technologies.

The anti-CD3 * CD19 antibody of the invention can be obtained from murine and / or human sequences and / or camel sequences and / or llama sequences and / or other suitable sequences, including artificial ones, including those created by modifying mouse and / or human sequences, for example, by introducing mutations. Preferably, the anti-CD3 * CD19 antibody of the invention is chimeric. More preferably, the anti-CD3 * CD19 antibody of the invention is fully human.

The recombinant anti-CD3 * CD19 monoclonal bispecific antibody of the invention contains the variable domains of the light and heavy chains of the anti-CD3 antibody, the variable domains of the light and heavy chains of the anti-CD19 antibody, linker sequences, the hinge region, the Fc sequence, and has two chains interconnected by disulfide bonds. Moreover, each chain preferably has the following structure: VL (CD3) -L1-VH (CD19) -L2-VL (CD19) -L3VH (CD3) -H-CH2-CH3 (IgG), where VL (CD3), VL ( CD19), VH (CD3) and VH (CD19) are the variable domains of the light and heavy chains of antibodies against CD3 and CD19, respectively; L1, L2, L3 - linker sequences; H is the hinge region of an IgG immunoglobulin; and CH2CH3 (IgG) is an IgG immunoglobulin Fc domain constant region.

Anti-CD3 antibodies are well known in the art. Examples of anti-CD3 antibodies include, but are not limited to, OKT3, G4.18, 145-2C11, Leu4, HIT3a, BC3, SK7, SP34, RIV-9, and UCHT1. Preferably, the antu-CD3 antibody binds to the same epitopes as OKT3, G4.18, 145-2C11, Leu4, HIT3a, BC3, SK7, SP34, RIV-9 and UCHT1.

A person skilled in the art, without undue experimentation, can obtain additional anti-CD3 antibodies and, inter alia, determine if antibodies are specific for the same epitopes as anti-CD3 antibodies OKT3, G4.18, 145-2C11, Leu4, HIT3a, BC3, SK7, SP34, RTV-9 and UCHT1, determining whether the former prevent the latter from binding to the antigenic CD3 polypeptide.

In general, any variable domains of the light and heavy chains of anti-CD3 antibodies that exhibit high binding specificity and affinity to the antigenic CD3 polypeptide can be used within the framework of the present invention. However, it is most preferred in accordance with the present invention to use the variable domains of the light and heavy chains of anti-CD3 antibodies VL (CD3) and VH (CD3), characterized by the sequences shown in SEQ ID NO: 1 and SEQ ID NO: 2.

- 4 036905

Ahtu-CD19 antibodies are also well known in the art as are the methods for their preparation. Examples of aHTu-CD19 antibodies include, without limitation, HD37, HIB19, SJ25-C1, 4G7-2E3. In addition, these methods and antibodies are disclosed in the Eurasian application EA 200800094 published 06/30/2008. In general, any variable domains of the light and heavy chains of anti-CD19 antibodies that exhibit high specificity and strong binding to the antigenic CD19 polypeptide can be used within the framework of the present invention. However, it is most preferred in accordance with the present invention to use the variable domains of the light and heavy chains of anti-CD19 antibodies VL (CD3) and VH (CD3), characterized by the sequences shown in SEQ ID NO: 3 and SEQ ID NO: 4.

The term Fc sequence is a term well known to those skilled in the art — the terminal portion of an immunoglobulin molecule that interacts with an Fc receptor on the cell surface and with certain proteins of the complement system. Depending on the amino acid sequence of the constant region of heavy chains, immunoglobulins are subdivided into classes: IgA, IgD, IgE, IgG and IgM, and some of them can be further subdivided into subclasses (isotypes), for example, IgG1, IgG2, IgG3 and IgG4, IgA1 and IgA2. According to the constant regions of the heavy chains, the various classes of immunoglobulins are named [alpha], [delta], [epsilon], [gamma] and [mu], respectively. The four isotypes of human IgG bind different receptors, such as the neonatal Fc receptor, activating Fc gamma receptors, FcgRI, FcgRIIa, and FcgRIIIa, inhibiting the FcgRIIb and C1q receptor with different affinities, producing different activities. However, the term Fc sequence in the framework of the present invention refers, inter alia, to Fc sequences, modified to change the affinity for the corresponding receptor.

An antibody of the present invention contains an Fc portion to prolong the half-life. Such an Fc sequence is preferably of human origin, more preferably a human Fc sequence of an IgG antibody, more preferably an IgG2, even more preferably the sequence shown in SEQ ID NO: 5. The selection of an IgG2 type Fc sequence allows effector loading due to the Fc sequence and thus reduce the targeted toxicity of the antibody of the invention.

The term linker as used in accordance with the present invention refers to peptide linkers. The length and / or sequence of the linker affects the stability and / or solubility of the bispecific molecule. The linker can increase the flexibility of the resulting binding molecule and / or can improve its binding to the target antigen by reducing steric hindrances. The length and sequence of the linker depends on the sequence and length of the sequences to be linked. Methods for testing the suitability of various linkers are well known to those skilled in the art. For example, the properties of a binding molecule can be easily tested by analyzing its binding affinity using various types of linkers. The stability of the resulting molecule can be measured by methods known in the art, such as, for example, high performance liquid chromatography, which separates the test protein material into fractions, depending on the molecular weight of the protein and hydrodynamic characteristics.

Particularly preferred are linkers, which are peptides consisting of at least 50%, at least 60%, at least 70%, at least 80%, at least 90 or 100% of small amino acids such as glycine , serine and alanine. Linkers consisting only of glycine and serine molecules are particularly preferred.

Preferably, the linker sequences of the invention consist of 5-15 amino acids. More preferably, the linkers L1, L2, L3 of the anti-CD3 * CD19 antibody of the invention are selected from the following sequences:

L1: (XXS) k, where k = 2-3, L2: (XXS) n, where η = 4-5, L3: (XXS) m, where m = 2-3, X = G.

It is even more preferable to use in the antibody according to the invention L1, L2, L3 with the sequences shown in SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8.

The term hinge region, as used in accordance with the present invention, refers to a sequence that, in natural immunoglobulin, binds the CH1 domain to the CH2CH3 region of an Fc fragment. The hinge region used in the antibody of the invention is preferably homologous to the native immunoglobulin region and typically includes cysteine residues linking the two heavy chains via disulfide bonds, as in natural immunoglobulins. Typical sequences of the hinge regions of human and murine immunoglobulins can be found in ANTIBODY ENGINEERING, a PRACTICAL GUIDE, (Borrebaeck, ed., W.H. Freeman and Co., 1992). Suitable hinge regions of the present invention may be derived from IgG1, IgG2, IgG3, IgG4, and other immunoglobulin isotypes. Most preferably, an IgG1 hinge region with the sequence shown in SEQ ID NO: 9 is used in the antibody construct of the invention.

The ordering of the variable regions in the antibody construct of the invention is fundamental. The authors carried out work to assess in vitro toxicity for the release of proinflammatory cytokines and CD3 mediated cytotoxicity of the antibody in relation to

- 5 036905

CD19 + tumor cells (hereinafter, cytotoxic biological activity) of various variants of bispecific antibodies of the BiMS format:

VL (CD3) -L1-VH (CD19) -L2-VL (CD19) -L3-VH (CD3) -H-CH2-CH3 (IgG) (target),

VH (CD19) -L1- VL (CD3) -L2- VH (CD3) -L3- VL (CD19) -H-CH2-CH3 (IgG),

VH (CD3) -Ll-VH (CD19) -L2-VL (CD19) -L3-VL (CD3) -H-CH2-CH3 (IgG),

VH (CD19) -L1- VH (CD3) -L2- VL (CD3) -L3- VL (CD19) -H-CH2-CH3 (IgG), as a result of which a high cytotoxic biological activity and a low toxic activity of the bispecific antibodies VL (CD3) -L1-VH (CD19) -L2-VL (CD19) L3-VH (CD3) -H-CH2-CH3 (IgG), which may be hypothetically associated with high antibody affinity due to the conformation of variable domains according to the invention in relation to CD3 specificity.

In the most preferred embodiment of the present invention, the anti-CD3 * CD19 antibody has the sequence SEQ ID NO: 10. However, the person skilled in the art will appreciate that without undue experimentation and the cost of inventive creativity, guided by the knowledge disclosed above, he can create an antibody of the invention with a different sequence , but highly effective in the treatment of malignant B-cell diseases, depletion of B-cells or retarding the development of pathological conditions associated with B-cell disorders, while being easy to use and obtain compared to blinatumomab. As a criterion for the selection of suitable sequences of antibodies according to the invention can be used the specificity / efficiency of binding to CD3 and CD19, characteristic of the antibody with the sequence shown in SEQ ID NO: 10 (see example 3), the half-life of the antibody in the blood from about 40 hours , more preferably from about 65 hours.

The anti-CD3 * CD19 antibody of the invention is encoded by a nucleic acid, the structure of which can be deduced by methods known in the art from the structure of said antibody. In particular, in the most preferred embodiment of the present invention, the nucleic acid according to the invention has a nucleotide sequence deduced from the amino acid sequence shown in SEQ ID NO: 10. The codon composition of the nucleic acid sequence according to the invention is selected in such a way that the selected host cell maximizes levels of its transcription and subsequent translation. The term “nucleic acid” as used in accordance with the present invention preferably refers to DNA. However, this term can also refer to RNA.

In one embodiment of the present invention, a host cell may comprise a nucleic acid sequence of the invention stably inserted into the cellular genome. In another embodiment of the present invention, the host cell comprises a nucleic acid sequence of the invention operably inserted for expression purposes in a construct such as an expression vector or linear expression element.

In a most preferred embodiment of the present invention, the expression of an anti-CD3 * CD19 antibody of the invention in a host cell is provided by an expression vector.

The term expression vector used in accordance with the present invention refers to any suitable vector, including chromosomal, extrachromosomal and synthetic nucleic acid vectors containing a suitable set of expression control elements.

The vector for providing expression of the anti-CD3 * CD19 bispecific antibody of the invention is preferably designed so as to allow expression of the nucleic acid of the invention in mammalian cells. However, the specified vector, without limitation, can also be designed to ensure the expression of the nucleic acid according to the invention in the cells of plants, bacteria, yeast, animals other than mammals. Examples of such vectors include SV40 derivatives, bacterial plasmids, phage DNA, baculoviruses, yeast plasmids, vectors derived from combinations of plasmids and phage DNA, and vectors from viral nucleic acid (RNA or DNA). Suitable immunoglobulin expression vectors are also disclosed, for example, in McLean et al., Mol. Immunol. 37: 837-45 (2000); Walls et al., Nucleic Acids Res. 21: 2921-9 (1993) and Norderhaug et al., J. Immunol. Meth. 204: 77-87 (1997).

The vector for ensuring the expression of the anti-CD3 * CD19 bispecific antibody of the invention can simultaneously be a vector for cloning and have the ability to autonomously replicate in bacterial, yeast and other cells, as well as be suitable for obtaining a nucleic acid encoding an anti-CD3 * CD19 antibody of the invention, by treating the specified cloning vector with suitable restriction enzymes. However, it is more preferable that the cloning vector containing the nucleic acid encoding the anti-CD3 * CD19 antibody of the invention does not contain additional elements to ensure the expression of said nucleic acid to increase the efficiency of its amplification in the host cell.

In one embodiment of the present invention, the vector for providing expression

- 6 036905 is a vector that is suitable for the expression of antibodies in bacterial cells. Examples of such vectors include BlueScript vectors (Stratagene), pIN vectors, pET vectors (Novagen,

Madison WI) and others.

In another embodiment, the expression vector is a vector that is suitable for expression in a yeast system. Suitable vectors include, for example, vectors containing constitutive or inducible promoters such as alpha factor, alcohol oxidase and PGH.

In a further embodiment of the present invention, the expression vector is a vector that is suitable for expression of an antibody in a plant. Suitable vectors include, for example, the vectors disclosed in RF patent RU 2412251 published 02/20/2011.

In a further embodiment of the present invention, the expression vector is a vector that is suitable for expression of an antibody in an insect. Suitable vectors include, for example, the vectors disclosed in international application publication number WO 2010/025764 dated 03/11/2010.

In a most preferred embodiment of the present invention, the expression vector is a vector that is suitable for expression of an antibody in Chinese hamster cells.

In an expression vector of the invention, a nucleic acid encoding an antibody of the invention can be linked to any suitable promoter, enhancer, and other expression-enhancing elements. Moreover, these elements are selected based on the type of host cell.

In particular, the site of replication initiation from plasmid pBR322 (Product No. 303-3s, New England Biolabs, Beverly, Mass) is applicable to most gram-negative bacteria, while the various sites of replication initiation from SV40, polyomavirus, adenovirus, vesicular stomatitis virus (VSV) or papillomaviruses (such as HPV or BPV) can be used to clone vectors in mammalian cells.

The transcription termination sequence is usually located 3 '(right) from the end of the coding regions of the polypeptide and serves to terminate transcription. Transcription termination sequences in prokaryotic cells often contain a G-C-rich moiety followed by a poly T sequence. Transcription termination sequences can be cloned from a commercially purchased library as part of a vector.

The selectable marker gene element encodes a protein necessary for the survival and growth of a host cell grown in a selective culture medium. Common selectable marker genes encode proteins that (a) confer resistance to antibiotics or other toxins such as ampicillin, tetracycline, or kanamycin for prokaryotic host cells, (b) complement auxotrophic cell deficiencies, or (c) provide essential nutrients. Preferred selectable markers are the kanamycin resistance gene, the ampicillin resistance gene and the tetracycline resistance gene. The neomycin resistance gene can also be used for selection in prokaryotic and eukaryotic host cells.

Examples of selectable markers for mammalian cells include dihydrofolate reductase (DHFR) and thymidine kinase. The mammalian cell transformants are placed under selection pressure conditions to which only the transformants are adapted for survival due to the marker present in the vector. Selection pressure is applied by culturing the transformed cells under conditions in which the concentration of the selection agent in the medium is sequentially changed, resulting in the amplification of both the selection gene and the target nucleic acid encoding the antibody of the invention. As a result, the amount of nucleic acid encoding an antibody of the invention thus amplified will allow for increased amounts of said antibody.

A ribosome binding site is usually present to initiate translation of mRNA. For example, such a site is characterized by a Shine-Dalgarno sequence (prokaryotes) or a Kozak sequence (eukaryotes). This element is usually located 3 '(to the right) of the promoter and 5' (to the left) of the coding sequence of the expressed polypeptide. The Shine-Dalgarno sequence varies but is usually polypurine (having a high A-G content). Many Shine-Dalgarno sequences have been identified, each of which can be easily synthesized using the methods presented above and used in a prokaryotic vector.

An enhancer sequence can be inserted into a vector to increase transcription in eukaryotic host cells. Several enhancer sequences of mammalian genes are known (eg, globin, elastase, albumin, alpha-fetoprotein and insulin). However, a viral enhancer should usually be used. The SV40 enhancer, cytomegalovirus early promoter enhancer, polyomavirus enhancer and adenovirus enhancers are examples of enhancer elements for activating eukaryotic promoters.

Vector components can be homologous (from the same species and / or strain as the host cell), heterologous (from a species other than the host cell species or strain), hybrid

- 7,036905 (a combination of different sequences from more than one source), synthetic or native sequences, which usually act as regulators of immunoglobulin expression. The sources of the vector components can be any prokaryotic or eukaryotic organism, any vertebrate or invertebrate organism, or any plant, provided that these components are functional in the host cell machinery and can be activated by the host cell machinery.

In the most preferred embodiment of the present invention, the vector for providing expression of the anti-CD3 * CD19 bispecific antibody of the invention is a vector for providing heterologous expression in Chinese hamster cells (CHO).

The vector for providing expression of the anti-CD3 * CD19 bispecific antibody of the invention may further comprise a nucleic acid sequence encoding a secretion / localization sequence that can direct the polypeptide to the periplasmic space or culture medium. Such sequences are known in the art and include secretion leaders or signal peptides, sequences targeting organelles (e.g., nuclear localization sequences, ER retention signals, mitochondrial transit sequences, chloroplast transit sequences), membrane localization / anchor sequences (e.g., stop sequences translocations, GPI anchor sequences), etc., which are well known in the art.

Recombinant expression of an anti-CD3 * CD19 antibody of the invention can be performed in any suitable host cells known in the art. In this case, the conditions for expression are selected individually for the type of host cell and the elements used for heterologous expression.

The term "host cell" in the present invention is intended to mean cells into which an expression vector of the invention has been introduced. It should be borne in mind that this term serves both to refer to the transformants themselves and their offspring. In this case, despite the fact that in subsequent generations the appearance of modifications due to environmental influences or artificial mutagenesis is possible, the term host cell in accordance with the present invention should also be applied to such offspring. A host cell according to the invention can be eukaryotic and prokaryotic. The prokaryotic host cell may be a bacterial cell. The eukaryotic host cell can be, inter alia, a yeast cell, a mammalian cell, a non-mammalian animal cell, eg, an insect cell, a plant cell. In particular, the eukaryotic host cells of the present invention can be CHO cells, HEK293 cells, HEK294 cells, PER.C6 cells, NS0 cells and lymphocyte cells, as well as prokaryotic cells of the E. coli type.

Some embodiments of the present invention comprise administering a vector to provide expression of the invention into a host cell in vivo, i. E. into a cell that is part of a living multicellular organism. The specified organism is preferably a human.

According to the present invention, various combinations of expression host cell / expression vector can be used to express an antibody. In this case, it is necessary to select a vector taking into account its preservation of functionality in the host cell in which the specified vector will be introduced. The vector must be compatible with the host cell machinery so that amplification and expression is possible. For example, expression vectors suitable for eukaryotic hosts include SV40, bovine papilloma virus, adenovirus, adeno-associated virus, cytomegalovirus, and retrovirus. Expression vectors that can be used in bacterial hosts include bacterial plasmids such as pBluescript, pGEX2T, pUC, pCR1, pBR322, pMB9 and their derivatives, plasmids like RP4, characterized by a wide list of possible hosts, λgt10 and λ11, phage DNA represented by various lambda phage derivatives such as NM989; and other phage DNAs such as M13 and single-stranded filamentous phage. Expression vectors that can be used with yeast cells include plasmid 2 μ and its derivatives. The vector that can be used in insect cells is pVL941.

In the most preferred embodiment of the present invention, to provide expression of the anti-CD3 * CD19 bispecific antibody of the invention, a vector combination is used to provide heterologous expression in Chinese hamster cells with cells of a commercially available CHO-M strain (Selexis).

A host cell expressing the anti-CD3 * CD19 bispecific antibody of the invention can be obtained by transfecting the parent cell with a vector to induce expression of the anti-CD3 * CD19 bispecific antibody of the invention using any method known in the art, including electroparating, transduction, calcium phosphate transfection, transfection with cationic lipids, scrape-loading, and infection.

The method of obtaining antibodies against CD3 * CD19 according to the invention includes culturing the specified

- 8 036905 host cell under conditions ensuring expression of the anti-CD3 * CD19 bispecific antibody of the invention and isolation of the resulting product. In this case, the specified expression can be carried out both under the conditions of a small-scale or large-scale fermenter, and under conditions of incubation in flasks, on a rocking chair. The incubation is carried out in a suitable nutrient medium containing carbon and nitrogen sources and inorganic salts by one of the known methods. Suitable media are commercially available, but can also be prepared yourself using components described, for example, in the catalog of the American Type Culture Collection (ATCC). In general, as used in accordance with the present invention, the term medium includes any culture medium, solution, solid, semi-solid or solid support that can support or contain any host cell, including bacterial host cells, yeast host cells, insect host cells, plant host cells, mammalian host cells, including CHO cells.

An antibody of the invention can be isolated by any method known in the art. However, when choosing a method for isolating an antibody, it is necessary to take into account the characteristics of the expression system used (expression host cell / expression vector). Thus, if the antibody preferentially accumulates inside the host cell, it is necessary to choose a method for isolating the product that will ensure its extraction from the host cell. If the antibody accumulates predominantly in the culture fluid, then the target product can be isolated from the culture fluid by methods for extracting antibodies from the culture fluid. For example, an antibody of the invention can be recovered from the culture broth by standard methods including, but not limited to, chromatography, centrifugation, filtration, extraction, drying, nebulization, evaporation, or precipitation. An antibody of the invention may be further purified using a variety of techniques known in the art, including chromatography (e.g., ion exchange, affinity, hydrophobic, or size exclusion), electrophoresis, separation (e.g., ammonium sulfate precipitation), SDS-PAGE, or extraction.

Preferably, the method for purifying an anti-CD3 * CD19 antibody of the invention comprises at least one chromatographic purification step. More preferably, a method for purifying an anti-CD3 * CD19 antibody of the invention comprises centrifugation, affinity chromatography using a protein A sorbent, and high performance cation exchange chromatography.

Purified in accordance with the requirements of regulatory authorities, the anti-CD3 * CD19 antibody of the invention is included in the pharmaceutical composition of the invention, preferably together with pharmaceutically acceptable carriers and / or diluents.

The mechanism of action of the proposed antibodies against CD3 * CD19 is similar to that described for anti-CD3 / anti-CD19 antibodies - the drug blinatumomab (MT103); and consists in activating a multispecific T-cell cytological response against CD19 + lymphoma cells. Therefore, pharmaceutical compositions based on antibodies against CD3 * CD19 according to the invention can be used to treat all hematological cancers (lymphomas and leukemias) of a B-cell nature, for example, 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 / small lymphocyte lymphoma (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; lymphoma from cells of the mantle zone; diffuse large B-cell lymphoma; diffuse mediastinal large B-cell lymphoma; primary exudative lymphoma; Burkitt's lymphoma / leukemia, as well as for the treatment of autoimmune diseases caused by abnormal regulation of B cells, such as rheumatoid arthritis, systemic lupus erythematosus, glomerulonephritis, multiple sclerosis and myasthenia gravis.

The pharmaceutical composition according to the present invention can be applied directly to animals and humans (for example, locally by injection, including subcutaneous injection). If the composition according to the present invention is administered parenterally, for example intravenously, intradermally, subcutaneously, intraperitoneally, intramuscularly, buccal, intraorbital, intracerebral, intraspinal, intraventricular, intrathecally, intracerebrally, then the composition preferably comprises a portion of an aqueous or saline solution that is not liquid negative impact on the balance of electrolytes and / or fluids in patients when the target composition is delivered to patients.

According to a preferred embodiment, the pharmaceutical composition of the invention is prepared in a form suitable for parenteral administration, preferably for subcutaneous administration, including in the form of a sterile aqueous solution, suspension, emulsion, concentrate for the preparation of an aqueous solution, a lyophilized preparation for the preparation of an aqueous solution, ras

- 9 036905 non-aqueous solvent based solution.

Pharmaceutically acceptable agents for use in the pharmaceutical composition of the invention include carriers, excipients, diluents, antioxidants, preservatives, coloring, flavoring and diluting agents, emulsifying agents, suspending agents, solvents, fillers, bulking agents, buffers, delivery vehicles, tonicity agents, cosolvents , moisturizing agents, complexing agents, buffering agents, antimicrobial agents and surfactants, as shown, for example, in the Handbook of Pharmaceutical Excipients (2d ed. London: The Pharmaceutical Press; 1994).

In this case, in accordance with the present invention, for example, either Hank's solution or Ringer's solution, or a suitable buffer solution, such as physiological buffered saline, can be used as a carrier or solvent for preparing a sterile aqueous solution. Buffers can be conventional buffers such as acetate, citrate, phosphate, bicarbonate buffers, or Tris-HCl. The acetate buffer can have a pH of about 4-5.5 and the Tris buffer can have a pH of about 7-8.5. Additional pharmaceutical agents are presented in Remington's Pharmaceutical Sciences, 18th Edition, A.R. Gennaro, ed., Mack Publishing Company, 1990. The anhydrous solvent can be propylene glycol, polyethylene glycol, or olive oil, or an injectable ester such as ethyl oleate. Also an example of a suitable carrier for the purpose of preparing a pharmaceutical composition of the invention is neutral buffered saline or saline mixed with serum albumin.

As auxiliary substances, the pharmaceutical composition according to the invention may also contain ascorbic acid, low molecular weight polypeptides, hydrophilic polymers such as polyvinylpyrrolidone, amino acids such as glycine, glutamine, asparagine, arginine or lysine, monosaccharides, disaccharides and other carbohydrates including glucose or manno dextrins, chelating agents such as EDTA, sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and / or nonionic surfactants such as Tween, pluronics or polyethylene glycol (PEG), alkali metal halides (preferably sodium or potassium chloride), benzalkonium chloride, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorexidine, sorbic acid, hydrogen peroxide sorbitan esters, polysorbates such as polysorbate 80, tromethamine, lecithin, cholesterol, tyloxapal, high molecular weight structural additives (e.g. gum arabic, alginic acid, calcium phosphate (dibasic), cellulose, carboxymethylcellulose, sodium carboxymethylcellulose, hydroxymethylcellulose, hydroxymethylcellulose, hydroxymethylcellulose, hydroxymethylcellulose cellulose, dextran, dextrin, dextrates, sucrose, tylose, pregelatinized starch, calcium sulfate, amylose, glycine, bentonite, maltose, sorbitol, ethyl cellulose, disodium hydrogen phosphate, disodium phosphate, disodium pyrosulfate, glucose gel, polyvinyl chloride glucose, compressible sugar, magnesium aluminum silicate, maltodextrin, polyethylene oxide, polymethacrylates, povidone, sodium alginate, tragacanth, microcrystalline cellulose, starch and zein).

The most preferred excipients for use in the pharmaceutical composition of the invention are sodium phosphate buffer, polysorbate 80 as a surfactant, mannitol as an isotonic agent, and histidine as a stabilizer.

These excipients can be used in combination with other active ingredients (eg, anti-cancer or anti-inflammatory agents) provided they do not cause undesirable effects.

If the pharmaceutical composition of the invention is a lyophilized composition, it may further comprise one or more lyoprotectants disclosed, for example, in US patents 6685940, 6566329 and 6372716. In one embodiment of the present invention, a lyoprotectant is used in the pharmaceutical composition, which is a non-reducing sugar, such as sucrose, lactose, or trehalose. The amount of lyoprotectant is preferably selected so that, after reconstitution, the resulting formulation is isotonic. In this case, the simultaneously selected amount of lyoprotectant should be sufficient to prevent an unacceptable amount of protein degradation and / or aggregation after lyophilization. Examples of concentrations of lyoprotectants for sugars (eg, sucrose, lactose, trehalose) in the pre-lyophilized composition are from about 10 to about 400 mM.

The pharmaceutical composition of the invention, if obtained in lyophilized form, may additionally be completed with a pharmaceutically acceptable solvent and / or diluent, for example water, saline and other commonly used pharmaceutically acceptable solvents.

The pharmaceutical compositions may contain a therapeutically effective amount of an amount of the antibodies of the invention.

The term a therapeutically effective amount used in accordance with the present invention refers to that amount of an antibody of the invention that is effective in

- 10,036905 for the necessary periods of time to achieve the desired therapeutic result, with any toxic or deleterious effects of the antibody being outweighed by the therapeutically beneficial effects of the antibody, which in a preferred embodiment of the present invention is at least one of the following: relief of symptoms, recovery or an increase in the speed of recovery. The therapeutically effective amount of an antibody of the invention may vary according to factors such as the condition of the disease, age, sex and weight of the individual, the ability of the antibody to induce the desired response in a particular individual, the presence of comorbidities, the route of administration, the frequency of administration, the pretreatment regimen of the individual.

It will not be difficult for a person skilled in the art to determine the effective amount of an antibody of the invention based on known rules and patterns. Ultimately, the attending physician, based on personal experience and practice, can decide on the amount of antibody of the invention, which is appropriate to treat each patient individually. Moreover, at first, the attending physician can administer small doses of the antibody of the invention and adjust the amount according to the patient's response. Dose ranges are from about 0.5 μg / kg to about 2 mg / kg, depending on the above factors. In this case, preferably, the pharmaceutical composition according to the invention contains 10-100 μg of the antibody.

The pharmaceutical composition of the invention is prepared, dispensed and administered in accordance with the principles of good medical practice. Standard pharmaceutical formulation techniques are known to those skilled in the art (Remington 1995).

The method of treating a B-cell disease or depletion of B-cells or retarding the development of a pathological condition associated with a B-cell disorder according to the present invention comprises administering to a patient in need thereof an effective amount of an antibody of the invention or a pharmaceutical composition of the invention. However, in a preferred embodiment of the present invention, the antibody is administered to the patient subcutaneously. In this case, the anti-CD3 * CD19 antibody of the invention can be administered to a patient in combination with another drug, including at least one of the following purposes: to enhance the therapeutic effect against B-cell disease, to reduce side effects in the treatment of B-cell disease, to relieve symptoms of B-cell disease.

The duration of therapy using the combination of the present invention will vary depending on the severity of the disease and condition to be treated, as well as the potential response of each individual patient. It is assumed that the duration of therapy is determined by the severity of the symptoms. Ultimately, the attending physician decides on the appropriate duration of therapy using the combination of the present invention.

The present invention is further illustrated by the following examples. In particular, in confirmation of the above, the authors experimentally showed that the antibody of the invention can be obtained using technologically simple known recombinant methods, commercially available means (vector, host cell, medium) and traditional methods for the isolation and purification of antibodies (example 1);

Based on the anti-CD3 * CD19 antibody of the invention, a pharmaceutical composition can be obtained in a technologically simple way, including in a lyophilized form, suitable for subsequent reconstitution with traditional diluents (example 2).

Also, the authors of the present invention experimentally demonstrated in comparison with MT103 a significantly increased half-life (T1 / 2) and exposure (AUC ( _0-inf) ) of the anti-CD3 * CD19 antibody of the invention while maintaining the maximum peak concentration (Cmax) when administered intravenously, as well as increased bioavailability of the anti-CD3 * CD19 antibody of the invention, both intravenously and subcutaneously (example 4). Additionally, the authors of the present invention experimentally confirmed the higher affinity of the antibody according to the invention to CD3 and CD19 and its higher antitumor activity when administered subcutaneously in comparison with the preparation MT103 (examples 3 and 5).

Example 1. Obtaining antibodies against CD3 * CD19.

Construction of recombinant plasmid DNA.

For cloning, a commercially available vector from Selexis was chosen, containing the SGE1 genetic element, which enhances expression as a result of the close proximity of the target transgene to a highly active nuclear transcription complex.

The gene encoding the anti-CD3 * CD19 antibody chain was constructed by annealing overlapping chemically synthesized oligonucleotides. The sequence of the target gene was cloned into a commercially available vector pUC19, resulting in the vector pAPG_GNR047, which was used for further work. After treatment of the pAPG_GNR047 vector with HindIII, XbaI restriction endodeoxyribonucleases (New Ingland Biolabs) forming sticky ends, a 2235 bp fragment was isolated, which was then ligated with the pST101 expression vector linearized at the HindIII and XbaI sites. The resulting plasmid pRA1451 was

- 11 036905 verified by restriction mapping. The construction scheme for the expression vector pRA1451 is shown in FIG. 2.

This was followed by sequential double transfection followed by selection for puromycin resistance using standard methods.

The resulting plasmid pRA1451 encoding a polypeptide with the sequence shown in SEQ ID NO: 10 is characterized by the following features:

consists of 11308 bp, has a molecular weight of 6.99 MDa, encodes a polypeptide bispecific antibodies against CD3 * CD19, GNR-047, provides resistance to mammalian cells transfected with the specified plasmid to puromycin;

contains the following elements:

CMVe / EF1a pro - enhancer of CMV virus and promoter of transcription of elongation factor alpha gene,

BGH рА - bovine growth hormone polyadenylation signal, sv40 enh - SV40 virus enhancer,

SGE 1 - element of attachment to the nuclear matrix,

Amp, β-lactamase gene, sv40 pro, SV40 virus promoter, puro, puromycin resistance gene, pA, synthetic polyadenylation signal.

contains unique recognition sites for the following restriction endonucleases:

AccI (6490 bp), Agel (460 bp), Apal (995 bp), BmgBI (3270 bp), BsmI (7269 bp), EcoRV (6457 bp) o.), Hindlll (1590 bp), Notl (11146 bp), Pfol (11021 bp), Pmll (5699 bp), Spel (4622 bp), Swal (6466 bp), Tthl 1II (10585 bp).

The genetic construction of the expression vector is shown in FIG. 3.

Transfection was performed using a commercially available CHO M strain (Selexis). The choice of a producer cell line is dictated by the need to form an optimal glycosylation profile in the synthesis of human proteins, as well as their stability, safety, and the possibility of using suspension conditions for cultivating this cell line, which are the most important parameters for the production of therapeutic antibodies.

Cultivation was carried out in a fed-batch mode using the BalanCD CHO Growth A nutrient medium (Irvine Scientific) and the CellBoost 3 (HyClone) nutrient supplement for 10 days at 37 ° C, 5% CO2 in the gas phase of the incubator. After cultivation, the culture fluid was clarified by centrifugation and transferred for isolation.

For the isolation and purification of the target product, a scheme was developed that includes an affinity stage using a sorbent based on protein A (with a product yield of over 90%) and cation-exchange chromatography on a high-resolution sorbent, which ensures efficient separation of the target form of the protein and its oligomeric forms (the yield of the target fraction more than 80%). As a result, bispecific antibodies were obtained in quantities sufficient to study their pharmacokinetic and pharmacodynamic properties.

Example 2. Pharmaceutical composition based on antibodies against CD3 * CD19.

For experiments on animals, a pharmaceutical composition of the following composition was used: 0.7 mg / ml antibodies against CD3 * CD19 according to the invention (hereinafter GNR-047), 150 mM sodium chloride, 1.8% mannitol, 20 mM sodium phosphate, 0.01 % polysorbate 80.

The conditions for the purification of antibodies against CD3 * CD19 according to the invention at the last chromatographic stage of the process are selected in such a way that the target protein is eluted from the sorbent in the form of a concentrated substance containing all the required components of the final formulation. Thus, for the preparation of the finished dosage form, an antibody solution with a concentration of about 5 mg / ml (concentrated solution obtained in example 1 after chromatographic purification) was diluted to the required concentration of 0.7 mg / ml using the buffer of the finished dosage form with stirring. The resulting solution was subjected to sterilizing filtration using a filter with a pore diameter of 0.22 μm and poured into sterile vials for storing the composition and subsequent experiments.

To obtain a lyophilized composition, a solution containing antibodies against CD3 * CD19 according to the invention in a formulation buffer (150 mM sodium chloride, 1.8% mannitol, 20 mM sodium phosphate, 0.01% polysorbate 80) was lyophilized under standard conditions, including the stage of freezing and vacuum dehydration. In this case, sodium chloride and mannitol were used as crystallizing components, the presence of which makes it possible to form a tablet, and mannitol also played the role of a stabilizer during lyophilization. The resulting lyophilic preparation was stored at 4C in sealed vials.

Water was used as a solvent for reconstitution of the lyophilized composition.

- 12 036905 for injection. To reconstitute the preparation, 0.5 ml of solvent was added to the vial containing the lyophilized antibody composition at room temperature and gently stirred with shaking until complete dissolution. Bacteriostatic water for injection can also act as a solvent, dissolving in which allows the drug to be used repeatedly within 7 days.

Example 3. Study of biological activity.

The biological activity of the obtained antibody was assessed by determining the binding constants in a competitive assay on the cells of the Raji CD3- / CD19 + cell line (Raji, ATCC® CCL-86) and Jurkat CD3 + / CD19- (Jurkat, ATCC® TIB-152) from the ATCC collection.

The reference drug was MT-103 antibody (blinatumomab). After incubation with the target molecules in serial 10-fold dilutions from 100 to 0.1 nM, a competing antibody (HD37 or OKT3) at a concentration of 6 nM was added to the cells. The cells were washed with sodium phosphate buffered saline at pH 7.0 and stained with anti-Human-FITS antibodies. Stained cells were analyzed on FACS Calibur.

The binding results are shown in table. 1 and FIG. 4.

Table 1

EC50, cells Jurkat (CD3 + / CD19 -), hM EC50, Raji cells (CD3- / CD19 +), nM MT103 21.58 1.70 GNR-047 0.30 0.87

The table shows that the GNR-047 antibody binds to both CD3 and CD19, and in both cases it is more effective than the reference drug MT103. Such low IC50 values in comparison with the reference drug can be explained by the presence of 2x valencies for each specificity (CD3 and CD19).

Example 4. Comparison of pharmacokinetic characteristics for intravenous and subcutaneous administration.

In view of the fact that the preparation GNR-047 showed improved pharmacokinetic characteristics when administered intravenously as compared to MT103, it was expedient to determine the pharmacokinetic parameters with a more convenient subcutaneous administration, in which GNR-047 would be prolonged release from the depot into the bloodstream.

For the experiment, we used rats of the Sprague Dawley line (nursery of laboratory animals in Pushchino, a branch of the Institute of Bioorganic Chemistry named after academicians M.M. Shemyakin and Yu.A. Ovchinnikov, Russian Academy of Sciences) weighing about 400 g, 3 rats per group.

Each rat was injected with the test antibody GNR-047 or MT-103 intravenously at a dosage of 0.5 mg / kg or subcutaneously at a dosage of 2.0 mg / kg. Blood samples were taken at regular intervals after injection, starting at 5 minutes, and the concentration of drugs in blood plasma was determined using an enzyme-linked immunosorbent assay (ELISA).

The results are presented in table. 2 and in FIG. 5 and 6.

table 2

The nature of istika MT-103 Antibody GNR-047 Route of administration, dosage Intravenous, 0.5 mg / kg Possible, 2.0 mg / kg Intravenous, 0.5 mg / kg Possible, 2.0 mg / kg T1 / 2, hours 4.6 n / a * 58.7 70.1 Stax, μg / ml 14.4 0.68 16.6 8.1 AUQo-t), μg * h / ml 43.1 15.5 514 1112 AUC (O-inf), μg * h / ml 59.7 15.5 * * 560 1420 A (absolute bioavailability) * ** - 6% - 63%

* n / a - not reliably determined;

** - AUC ( _{0. In} f) in this case coincides with AUC ( _0-t ), since at the last point of the pharmacokinetic curve the concentration of MT103 is close to zero;

*** - parameter is calculated taking into account the difference in doses.

Where

T _1/2 - required to reduce the concentration of the drug in the blood plasma by 50% in

- 13 036905 as a result of elimination; Cmax - the highest value of drug concentration in blood plasma; AUC ( _{0. T} ) is the area bounded by the pharmacokinetic curve and the abscissa in the time interval from the moment of drug administration to the moment of taking the last blood sample; AUC ( _0-inf ) - the area bounded by the pharmacokinetic curve and the abscissa, extrapolated in time from the moment of drug administration to infinity; / a - part of the drug dose (in%) that reached the systemic circulation after extravascular administration.

As a result, a significant increase in the half-life (T _1/2 ) and exposure (AUQ ( _0-inf) - the area under the pharmacokinetic curve) of the GNR-047 preparation in comparison with the MT103 preparation was shown, while maintaining the maximum peak concentration (Cmax) after intravenous administration ...

When administered subcutaneously to rats, the absolute bioavailability of GNR-047 was about 60%, while MT-103 showed a very low bioavailability of 6% (see Fig. 6b).

Example 5. Comparison of the antitumor activity of GNR-047 and MT 103 in the Raji (Raji) cell line tumor xenograft model in NOD / SCID mice during the reconstitution of human PBMC.

The aim of the study was to compare the efficacy of GNR-047 and MT103 preparations in NOD / SCID mice (Taconic, Denmark) at several dose levels, in a Raji tumor cell xenograft model with simultaneous injections of human peripheral blood mononuclear cells (PBMC) into immunodeficient NOD / SCID mice ...

Cells of the Raji line (human Burkitt's lymphoma) were pre-mixed with human PBMC and inoculated subcutaneously, followed by intravenous or subcutaneous administration of the test drugs. For this, nine experimental groups of immunodeficient NOD / SCID mice (9 animals per group) were subcutaneously injected with either Raji cells of human Burkitt's lymphoma, or a suspension of premixed Raji cells and human PBMC. Three animals from each group received human PBMC from one of three separate healthy donors (cohorts 1, 2 and 3). Two hours after the inoculation of the cells, the animals were injected intravenously or subcutaneously with either NPB (sodium phosphate buffered saline), or the test preparations GNR-047 or MT 103 at various doses. The injected volume was 200 μl. In all groups, tumor volume was assessed for 45 days (days 3 to 48).

The injected solutions were prepared by diluting the solution (composition) GNR-047 obtained in example 2 (0.7 mg / ml) or MT103 (0.52 μg / ml) in 0.9% saline to obtain a concentration of 5 μg / ml (dose 1 μg) or 0.5 μg / ml (dose 0.1 μg). Experimental groups are described in table. 3.

Table 3

Group Mouse No. Cells * inoculated by s.c. injection (q 0) Study drug (injection once / day) one 1-3 4-6 7-9 Cells of the Raji line Cells of the Raji line Cells of the Raja line NSE 2 10-12 13-15 16-18 Raji cells + PBMC (Donor 1) Raji cells + PBMC (Donor 2) Raji cells + PBMC (Donor 3) NSE 3 19-21 22-24 25-27 Raji cells + PBMC (Donor 1) Raji cells + PBMC (Donor 2) Raji cells + PBMC (Donor 3) MT-103 1 μg / animal, intravenous 4 28-30 31-33 34-36 Raji cells + PBMC (Donor 1) Raji cells + PBMC (Donor 2) Raji cells + PBMC (Donor 3) MT-103 0.1 μg / animal, intravenous

- 14 036905

Group Mouse No. Cells * inoculated by s.c. injection (q 0) Study drug (injection 1 time / day) five 37-39 40-42 43-45 Raji cells + PBMC (Donor 1) Raji cells + PBMC (Donor 2) Raji cells + PBMC (Donor 3) GNR-047 1 μg / animal, IV 6 46-48 49-51 52-54 Raji cells + PBMC (Donor 1) Raji cells + PBMC (Donor 2) Raji cells + PBMC (Donor 3) GNR-047 0.1 μg / animal, IV 7 55-57 58-60 61-63 Raji cells + PBMC (Donor 1) Raji cells + PBMC (Donor 2) Raji cells + PBMC (Donor 3) GNR-047 4 μg / animal, IV 8 64-66 67-69 70-72 Raji cells + PBMC (Donor 1) Raji cells + PBMC (Donor 2) Raji cells + PBMC (Donor 3) GNR-047 1.7 μg / animal, subcutaneous 9 73-75 76-78 79-81 Raji cells + PBMC (Donor 1) Raji cells + PBMC (Donor 2) Raji cells + PBMC (Donor 3) GNR-047 0.17 mcg / animal, subcutaneous

* - 2.5 x 10 ⁶ Raji cells, 1 x 10 ⁷ PBMC.

The following parameters were determined in experimental animals: survival rate, body weight, tumor volume, tumor weight.

Body weights were determined on day 0 and subsequently recorded three times a week until day 48. Tumor volume was monitored 3 times a week from day 3 until the end of the study by measuring the large and small tumor diameters with a caliper. Based on the diameters, the tumor volume was calculated using the following formula:

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

In the control group 1, which received NPB (only Raji cells), 11% of the animals survived to the end of the study. The survival rate in animals of group 2, which received Raji cells and human PBMC, did not change. In both groups, mortality was due solely to euthanasia for ethical reasons (due to the large tumor volume).

The introduction of GNR-047 and MT 103 increased the survival rate in groups 3-9, which is reflected in table. 4, and at a higher dose, the study drug was more effective than the reference drug:

Table 4

Animal survival

Group no. Injected drug Dose (μg / animal) Animals (η) Survival (η) (%) one NSE - 9 one eleven 2 NSE - 9 4 44.4 3 MT 103 1.0 9 4 44.4 4 MT 103 ο, ι 9 7 77.8 five GNR-047 1.0 9 9 100 6 GNR-047 ο, ι 9 7 77.8 7 GNR-047 4.0 9 8 88.9 8 GNR-047 1.7 9 8 88.9 9 GNR-047 0.17 9 7 77.8

The criteria for removing experimental animals from the study: tumor volume> 10% of body weight; ulceration of the tumor; weight loss> 20%.

Tumor growth was significantly inhibited or suppressed in all study groups treated with GNR-047 or MT 103 (groups 3-9) compared with control group 2 treated with NPB. Moreover, in all study groups receiving GNR-047 or MT 103, the inhibition of tumor growth was statistically significant (p <0.05).

Table 5 shows the tumor sizes for groups 1, 2, 4, 8 and 9, and the corresponding comparative graphs, allowing to estimate the tumor size after intravenous and subcutaneous administration (for groups 4, 8 and 9), are shown in FIG. 7.

- 15 036905

Table 5

Group Exploration day 3 6 8 ten thirteen fifteen 17 one 0.0 10.0 14.6 28.9 47.9 95.2 199.9 2 2.2 6.0 13,7 28.4 58.8 122.3 236.6 4 6.1 10.2 19.6 27.1 38.2 64.3 112.7 8 1.9 3.4 10.9 15.0 19.0 28.6 34.2 9 4.8 7.3 12.0 15,8 22.7 37.4 48.9 Group Exploration day 20 22 24 27 29 31 one 387.0 561.5 781.5 1046.5 1214.8 1559.2 2 443.9 598.8 745.0 1038.8 1243.7 1465.2 4 149.8 176.4 254.5 377.1 465.4 589.6 8 37.2 33.5 45.1 92.3 108.4 187.9 9 75.9 94.8 190.8 259.8 320.3 409.9

Industrial applicability.

The present invention can be used in medicine to treat B-cell diseases or B-cell depletion, 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 / small lymphocyte lymphoma (lymphocytic lymphoma); B-cell prolymphocytic leukemia; lymphoplasmacytic lymphoma; splenic lymphoma of the marginal zone; hairy cell leukemia; plasma cell myeloma / plasmacytoma; MALT-type extranodal B-cell lymphoma of the marginal zone; nodal B-cell lymphoma of the marginal zone; follicular lymphoma; lymphoma from cells of the mantle zone; diffuse large B-cell lymphoma; diffuse mediastinal large B-cell lymphoma; primary exudative lymphoma; Burkitt's lymphoma / leukemia, as well as for the treatment of autoimmune diseases caused by abnormal regulation of B cells, such as rheumatoid arthritis, systemic lupus erythematosus, glomerulonephritis, multiple sclerosis and myasthenia gravis. The present invention can also be used in the pharmaceutical industry to obtain pharmaceutical compositions, finished dosage forms and kits containing the antibody of the invention for the treatment of the above diseases.

Free text sequence listing.

SEQ ID No. 1 - variable domain of the light chain of anti-CD3 antibodies,

SEQ ID No. 2 - variable domain of the heavy chain of anti-CD3 antibody,

SEQ ID No. 3 - variable domain of the light chain of anti-CD19 antibody,

SEQ ID No. 4 - variable domain of the heavy chain of anti-CD19 antibody,

SEQ ID No. 5 - constant domain CH2 CH3 IG2,

SEQ ID No. 6 - linkers,

SEQ ID No. 7 - linker L2,

SEQ ID No. 8 - linker L3,

SEQ ID No. 9 - Hinge region H,

SEQ ID No. 10 — GNR-047 antibody.

- 16 036905

Sequence Listing <110> Limited Liability Company, International Biotechnology Center Generium <120> BISPECIFIC ANTIBODIES AGAINST CD3 * CD19 <160> 10 <170> Patentin version 3.5 <210> 1 <211> 106 <212> PRT <213> mouse < 223> anti-POP antibody light chain variable domain <400> 1

Asp lie Val Leu Thr Gln Ser Pro Ala He Met Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Arg Ala

10 15 2025

Ser Ser Ser Val Ser Tyr Met Asn Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro Lys Arg Trp He Tyr Asp Thr

35 40 4550

Ser Lys Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr He Ser

60 65 7075

Ser Met Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Phe Thr Phe Gly Ala Gly

85 90 95 100

Thr Lys Leu Glu He Lys

105 <110> Limited Liability Company, Generium International Biotechnology Center <120> BISPECIFIC ANTIBODIES AGAINST CD3 * CD19 <160> 10 <170> Patentin version 3.5 <210> 2 <211> 123 <212> PRT <213> mouse <223 > variable domain of the heavy chain anti-POP antibody <400> 2

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

10 15 2025

Gly Tyr Thr Phe Thr Arg Tyr Thr Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp lie Gly Tyr

35 40 4550

He Asn Pro Ser Arg Gly Tyr Thr Asn Tyr Asn Gln Lys Phe Lys Asp Lys Ala Thr Leu Thr Thr Asp Lys Ser

60 65 7075

Ser Ser Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala Arg Tyr Tyr

85 90 95 100

Asp Asp His Tyr Ser Leu Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser Ala Ser Ala Ala 105 110 115 120 <110> Limited Liability Company, Generium International Biotechnology Center <120> SPECIFIC ANTIBODIES AGAINST CD3 * CD19 <160> 10 <170> Patentin version 3.5 <210> 3 <211> 111 <212> PRT <213> mouse <223> light chain variable domain of anti-C019 antibody <400> 3

Asp lie Gln Leu Thr Gln Thr Pro Ala Ser Leu Ala Val Ser Leu Gly Gln Arg Ala Thr He Ser Cys Lys Ala

10 15 2025

Ser Gln Ser Val Asp Tyr Asp Gly Asp Ser Tyr Leu Asn Trp Tyr Gln Gln He Pro Gly Gln Pro Pro Lys Leu

35 40 4550

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

60 65 7075

Thr Leu Asn He His Pro Val Glu Lys Val Asp Ala Ala Thr Tyr His Cys Gln Gln Ser Thr Glu Asp Pro Trp

85 90 95 100

Thr Phe Gly Gly Gly Thr Lys Leu Glu He Lys 105110 <110> Limited Liability Company, International Biotechnology Center Generium <120> BISPECIFIC ANTIBODIES AGAINST CD3 * CD19 <160> 10 <170> Patentin version 3.5 <210> 4

- 17 036905 <211> 125 <212> PRT <213> mouse <223> variable domain of the heavy chain of anti-CO19 antibody <400> 4

Gln Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Pro Gly Ser Ser Val Lys lie Ser Cys Lys Ala

10 15 2025

Ser Gly Tyr Ala Phe Ser Ser Tyr Trp Met Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp He Gly

35 40 4550

Gln Ue Trp Pro Gly Asp Gly Asp Thr Asn Tyr Asn Gly Lys Phe Lys Gly Lys Ala Thr Leu Thr Ala Asp Glu

60 65 7075

Ser Ser Ser Thr Ala Tyr Met Gln Leu Ser Ser Leu Ala Ser Glu Asp Ser Ala Val Tyr Phe Cys Ala Arg Arg

85 90 95 100

Glu Thr Thr Thr Val Gly Arg Tyr Tyr Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser

105 110 115 120 125 <110> Limited Liability Company, International Biotechnology Center Generium <120> BISPECIFIC ANTIBODIES AGAINST CD3 * CD19 <160> 10 <170> Patentin version 3.5 <210> 5 <211> 216 <212> PRT <213> human <223> constant domain CH2 CH3 IG2 <400> 5

Ala Pro Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met He Ser Arg Thr

10 15 2025

Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly

35 40 4550

Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Val Val Ser Val Leu

60 65 7075

Thr Val Val His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ala Pro

85 90 95 100

He Glu Lys Thr lie Ser Lys Thr Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu

105 110 115 120 125

Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp He Ala Val Glu Trp

130 135 140 145150

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu

155 160 165 170 175

Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala

180 185 190 195 200

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

205 210 215 220225 <110> Limited Liability Company, International Biotechnology Center Generium <120> BISPECIFIC ANTIBODIES AGAINST CD3 * CD19 <160> 10 <170> Patentin version 3.5 <210> 6 <211> 6 <212> PRT <220> artificial sequence <223> linker L1 <400> 6

Gly Gly Ser Gly Gly Ser 5 <110> Limited Liability Company, International Biotechnology Center Generium <120> BISPECIFIC ANTIBODIES AGAINST CD3 * CD19 <160> 10 <170> Patentin version 3.5 <210> 7 <211> 12 <212> PRT <220> artificial sequence <223> linker L2 <400> 7

Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser

ten

- 18 036905 <1Y> Limited Liability Company, International Biotechnological Center Generium <120> BISPECIFIC ANTIBODIES AGAINST CD3 * CD19 <160> 10 <170> Patentin version 3.5 <210> 8 <211> 6 <212> PRT <220> artificial sequence <223> linker L3 <400> 8

Gly Gly Ser Gly Gly Ser 5 <110> Limited Liability Company, International Biotechnology Center Generium <120> BISPECIFIC ANTIBODIES AGAINST CD3 * CD19 <160> 10 <170> Patentin version 3.5 <210> 9 <211> 12 <212> PRT <220> artificial sequence <223> hinge region H <400> 9

Glu Arg Lys Cys Val Glu Cys Pro Pro Cys Pro 5 10 <110> Limited Liability Company, Generium International Biotechnology Center <120> BISPECIFIC ANTIBODIES AGAINST CD3 * CD19 <160> 10 <170> Patentin version 3.5 <210> 10 < 211> 720 <212> PRT <220> artificial sequence <223> antibody GNR-047 <400> 10

Asp lie Val Leu Thr Gln Ser Pro Ala lie Met Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Arg Ala

10 15 2025

Ser Ser Ser Val Ser Tyr Met Asn Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro Lys Arg Trp He Tyr Asp Thr

35 40 4550

Ser Lys Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr lie Ser

60 65 7075

Ser Met Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Phe Thr Phe Gly Ala Gly

85 90 95 100

Thr Lys Leu Glu He Lys Gly Gly Ser Gly Gly Ser Gln Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val

105 110 115 120 125

Arg Pro Gly Ser Ser Val Lys He Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Ser Tyr Trp Met Asn Trp Val

130 135 140 145150

Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp He Gly Gln He Trp Pro Gly Asp Gly Asp Thr Asn Tyr Asn Gly

155 160 165 170 175

Lys Phe Lys Gly Lys Ala Thr Leu Thr Ala Asp Glu Ser Ser Ser Thr Ala Tyr Met Gln Leu Ser Ser Leu Ala

180 185 190 195 200

Ser Glu Asp Ser Ala Val Tyr Phe Cys Ala Arg Arg Glu Thr Thr Thr Val Gly Arg Tyr Tyr Tyr Ala Met Asp

205 210 215 220225

Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly

230 235 240 245250

Gly Ser Asp He Gln Leu Thr Gln Thr Pro Ala Ser Leu Ala Val Ser Leu Gly Gln Arg Ala Thr lie Ser Cys

255 260 265 270275

Lys Ala Ser Gln Ser Val Asp Tyr Asp Gly Asp Ser Tyr Leu Asn Trp Tyr Gln Gln He Pro Gly Gln ProPro

280 285 290 295300

Lys Leu Leu He Tyr Asp Ala Ser Asn Leu Val Ser Gly He Pro Pro Arg Phe Ser Gly Ser Gly Ser Gly Thr

305 310 315 320325

Asp Phe Thr Leu Asn lie His Pro Val Glu Lys Val Asp Ala Ala Thr Tyr His Cys Gln Gln Ser Thr Glu Asp

330 335 340 345350

Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu He Lys Gly Gly Ser Gly Gly Ser Gln Val Gln Leu Gln Gln

355 360 365 370375

Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Arg

380 385 390 395400

Tyr Thr Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp He Gly Tyr He Asn Pro Ser Arg Gly

405 410 415 420425

Tyr Thr Asn Tyr Asn Gln Lys Phe Lys Asp Lys Ala Thr Leu Thr Thr Asp Lys Ser Ser Ser Thr Ala Tyr Met

430 435 440 445450

Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala Arg Tyr Tyr Asp Asp His Tyr Ser Leu

- 19 036905

455 460 465 470475

Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser Ala Ser Ala Ala Glu Arg Lys Cys Cys Val Glu Cys

480 485 490 495500

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

505 510 515 520525 lie Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Gln Phe Asn Trp

530 535 540 545550

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Val

555 560 565 570575

Val Ser Val Leu Thr Val Val His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly

580 585 590 595600

Leu Pro Ala Pro He Glu Lys Thr He Ser Lys Thr Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

605 610 615 620625

Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp He

630 635 640 645650

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

655 660 665 670675

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

680 685 690 695700

Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

705 710 715720

Claims (31)

1. Bispecific antibody that specifically binds to CD3 and CD19, comprising two disulfide-linked chains, each of which consists of the following domains: VL (CD3) -L1VH (CD19) -L2-VL (CD19) -L3-VH (CD3) -H-CH2-CH3 (IgG), where VL (CD3), VL (CD19), VH (CD3) and VH (CD19) are the variable domains of the light and heavy chains of antibodies against CD3 and CD19, respectively, L1, L2, L3 - linker sequences essentially corresponding to SEQ ID NOs: 6, 7 and 8, respectively, H - the hinge region of an IgG immunoglobulin, essentially corresponding to the sequence of SEQ ID NO: 9, CH2-CH3 (IgG) - constant region of the Fc-domain of IgG immunoglobulin. 2. An antibody according to claim 1, characterized in that said constant region of the Fc domain of an IgG immunoglobulin is a constant region of the Fc domain of an IgG2 immunoglobulin. 3. The antibody according to claim 2, characterized in that the constant region of the Fc domain of the IgG2 immunoglobulin contains the sequence shown in SEQ ID NO: 5. 4. Antibody according to claims 1 to 3, characterized in that it has an amino acid sequence substantially corresponding to the sequence shown in SEQ ID NO: 10. 5. An antibody according to claim 1, characterized in that it has a blood half-life that is increased compared to the half-life of the blinatumomab when measured under substantially similar conditions. 6. An antibody according to claim 1, characterized in that it has a blood half-life of more than 40 hours. 7. A nucleic acid encoding an antibody according to any one of claims 1-6. 8. A vector for ensuring expression of the anti-CD3 * CD19 bispecific antibody in a host cell, comprising a nucleic acid encoding a light or heavy chain bispecific antibody sequence of a bispecific antibody specifically binding to CD3 and CD19 according to any one of claims 1-6. 9. The vector according to claim 8, characterized in that it is the expression plasmid pRA1451, characterized by the following features: consists of 11308 bp, has a molecular weight of 6.99 MDa, contains a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 10, provides resistance to mammalian cells transfected with the specified plasmid to puromycin; contains the following elements: CMVe / EFla pro - enhancer of the CMV virus and promoter of transcription of the gene for elongation factor alpha, BGH рА - bovine growth hormone polyadenylation signal, sv40 enh - SV40 virus enhancer, SGE 1 - element of attachment to the nuclear matrix, Amp - β-lactamase gene, sv40 pro - SV40 virus promoter, puro - puromycin resistance gene, pA - synthetic polyadenylation signal, contains unique recognition sites for the following restriction endonucleases: - 20 036905 Accl (6490 i.o.), Agel (460 no), Apal (995 no), BmgBI (3270 no), Bsml (7269 no), EcoRV (6457 i.o.), Hindlll (1590 no), Notl ( 11146 no), Pfol (1 1021 no), Pmll (5699 no), Spel (4622 no), Swal (6466 no), Tthl 111 (10585 no). 10. A host cell for expressing a bispecific antibody specifically binding to CD3 and CD19 according to any one of claims 1 to 6, comprising a nucleic acid according to claim 7 or a vector according to claim 8. 11. A cell according to claim 10, wherein it is a eukaryotic cell. 12. A cell according to claim 11, wherein it is a mammalian cell. 13. A cage according to claim 12, characterized in that it is a Chinese hamster cage. 14. A method for producing an antibody according to any one of claims 1-6, comprising at least culturing a cell according to any one of claims 10 to 13 under conditions ensuring expression of an antibody according to any one of claims 1 to 6, and isolating the target product. 15. The method of claim 14, wherein the cell is a CHO cell. 16. A method for purifying an antibody according to any one of claims 1 to 6, comprising at least one chromatography step carried out under conditions such that an antibody preparation according to any one of claims 1 to 6 is substantially free of host cell impurities. 17. The method of claim 16, further comprising a centrifugation step. 18. A method according to claim 17, characterized in that the centrifugation step is performed prior to the chromatography step. 19. The method of claim 17, further comprising another chromatography step. 20. A method according to claim 19, characterized in that the first chromatography step is performed using a protein A sorbent, and the second chromatography step is a cation exchange chromatography step. 21. Use of an antibody according to claims 1-6 for the treatment of a B-cell disease, depletion of B-cells or slowing the development of a pathological condition associated with B-cell disorders. 22. The use according to claim 21, characterized by said B-cell disease, B-cell depletion or delayed development of a pathological condition associated with B-cell disorders, is one of the following: 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 / small lymphocyte lymphoma (lymphocytic lymphoma); B-cell prolymphocytic leukemia; lymphoplasmacytic lymphoma; splenic lymphoma of the marginal zone; hairy cell leukemia; plasma cell myeloma / plasmacytoma; MALT-type extranodal B-cell lymphoma of the marginal zone; nodal B-cell lymphoma of the marginal zone; follicular lymphoma; lymphoma from cells of the mantle zone; diffuse large B-cell lymphoma; diffuse mediastinal large B-cell lymphoma; primary exudative lymphoma; Burkitt's lymphoma / leukemia, as well as for the treatment of autoimmune diseases caused by abnormal regulation of B cells, such as rheumatoid arthritis, systemic lupus erythematosus, glomerulonephritis, multiple sclerosis and myasthenia gravis. 23. A pharmaceutical composition comprising a therapeutically effective amount of an antibody according to any one of claims 1 to 6 and a pharmaceutically acceptable carrier. 24. A pharmaceutical composition according to claim 23, characterized in that it is intended for a B-cell disease, depletion of B-cells, or retarding the development of a pathological condition associated with B-cell disorders. 25. A pharmaceutical composition according to claim 24, wherein said B-cell disease, B-cell depletion or retardation of the development of a pathological condition associated with B-cell disorders is one of the following: 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 / small lymphocyte lymphoma (lymphocytic lymphoma); B-cell prolymphocytic leukemia; lymphoplasmacytic lymphoma; splenic lymphoma of the marginal zone; hairy cell leukemia; plasma cell myeloma / plasmacytoma; MALT-type extranodal B-cell lymphoma of the marginal zone; nodal B-cell lymphoma of the marginal zone; follicular lymphoma; lymphoma from cells of the mantle zone; diffuse large B-cell lymphoma; diffuse mediastinal large B-cell lymphoma; primary exudative lymphoma; Burkitt's lymphoma / leukemia, as well as for the treatment of autoimmune diseases caused by abnormal regulation of B cells, such as rheumatoid arthritis, systemic lupus erythematosus, glomerulonephritis, multiple sclerosis and myasthenia gravis. 26. A pharmaceutical composition according to any one of claims 23-25, characterized in that it additionally contains at least one of the following: sodium chloride, mannitol, sodium phosphate, polysorbate 80. - 21 036905 27. A method of treating a B-cell disease, depletion of B-cells or retarding the development of a pathological condition associated with B-cell disorders, comprising administering to a patient in need thereof a therapeutically effective amount of the antibody according to claims 1-6. 28. The method of claim 27, wherein the antibody is administered to the patient subcutaneously. 29. The method of claim 27, wherein the antibody is administered intravenously to the patient. 30. The method of claim 27, wherein the antibody is administered to the patient in a therapeutically effective amount. 31. The method of claim 27, wherein said B-cell disease, depletion of B-cells, or retardation of a pathological condition associated with B-cell disorders is one of the following: 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 / small lymphocyte lymphoma (lymphocytic lymphoma); B-cell prolymphocytic leukemia; lymphoplasmacytic lymphoma; splenic lymphoma of the marginal zone; hairy cell leukemia; plasma cell myeloma / plasmacytoma; MALT-type extranodal B-cell lymphoma of the marginal zone; nodal B-cell lymphoma of the marginal zone; follicular lymphoma; lymphoma from cells of the mantle zone; diffuse large B-cell lymphoma; diffuse mediastinal large B-cell lymphoma; primary exudative lymphoma; Burkitt's lymphoma / leukemia, as well as for the treatment of autoimmune diseases caused by abnormal regulation of B cells, such as rheumatoid arthritis, systemic lupus erythematosus, glomerulonephritis, multiple sclerosis and myasthenia gravis.