WO2017095267A1 - Bispecific antibodies against cd3*cd19 - Google Patents

Abstract

The invention relates to biotechnology and, in particular, can be used 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

 SPECIFIC ANTIBODIES AGAINST CD3 * CD19

FIELD OF TECHNOLOGY

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

BACKGROUND OF THE INVENTION

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 (WO2010037835, RU2228202), B-cell leukemia (RU2008129080), and acute lymphoblastic childhood leukemia (WO20100052013) H T.fl.

 A number of studies completed and ongoing (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)) give reason to believe that drugs based on bespecifically antibodies against CD3 * CD 19 promise to become more effective in the treatment.

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 CD 19, i.e. BAP can 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 after binding to T cells, BAPs recognize antigens on the surface of the cancer cell in the same way as conventional monospecific antibodies. Thus, it is possible to redirect cytotoxic function of T cells against tumor cells, and BAP activity is already observed at relatively low (picomolar) concentrations. Binding of BAT 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 required volume and production cost of bespecifically antibodies is lower. Therefore, the creation of such antibodies will significantly reduce the scale and cost of producing drugs based on them and, most importantly, make these drugs available to all patients in need of such therapy.

 At present, quite a few bispecific antibodies against CD3 * CD19 have been developed and obtained. The most promising of them are single-chain molecules as such, or non-covalent dimers consisting of single-chain molecules.

 One of the single chain antibodies against CD3 * CD1 is Blinatumomab (Blincyto, MT103). Blinatumomab consists of two domains: the anti-C019 scFv domain from the HD37 hybridoma and the anra-CD3 scFv domain from the OKTZ hybridoma. To obtain the antibody, the VLCD 19-VHCD 19-VHCD3-VLCD3 DNA fragment is expressed in DHFR-negative CHO cells (patent RU2228202).

 Blinatumomab, in comparison with full-sized antibodies, has a relatively small molecular weight (55 kDa versus 155 kDa), which is why it is rapidly 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 per cycle (in a two-week regimen - an dropper, two weeks - a break), which is quite difficult for the patient to tolerate.

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

 Despite the lack of ease of use and the difficulty of obtaining blinatumomab is one of the most successful antibodies against CD3 * CD19.

 Blinatumomab refers to BiTE® antibodies. BiTE® antibodies demonstrate high efficacy of redirected lysis of cells expressing the tumor target antigen. EC50 values 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 necessary in order to cause a pharmacologically desired effect. In a review, Rader C. reports that the powerful lytic ability of the BiTE® format is explained by (1) its small size, which brings the target and effector cells in close proximity, allowing the formation of cytolytic synapses, and (2) its monovalent involvement 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 describes that the BiTE® class of bispecific antibodies typically has 100-10000 times higher tumor cell lysis efficiencies relative to other bispecific formats and full IgGl 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 bispecific anti-CD3 * CD19 antibodies of the BiTE® format exhibit more powerful redirected T-cell lytic activity than large bivalent CD3 * CD19 bispecific formats, and the use of large bivalent CD3 * CD19 bispecific antibodies impractical.

SUMMARY OF THE INVENTION

The present invention solves the problem of obtaining a bivalent antibody against CD3 * CD19, convenient to use and obtain compared with blinatumomab and at the same time highly effective in treating malignant diseases of B cells, depletion of B cells or slowing 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 has led to the creation of a new antibody design, 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 arranged in the following order from the конца-end to the C-end: VL (CD3) -L 1 - VH (CD 19) -L2-VL (CD 19) -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 CD 19, respectively, LI, L2, L3 are linker sequences, N - the hinge region of IgG immunoglobulin and CH2-CH3 (IgG) is the constant region of the Fc domain of IgG immunoglobulin. The two chains are connected by disulfide bonds.

 It was unexpectedly discovered that such bispecific molecules, in comparison with the blinatumomab antibody (MT-103), are characterized by an increased half-life and are easy to obtain. Moreover, such properties of the indicated antibody as enhanced cytotoxic effect and high bioavailability even with subcutaneous administration at the level of 60% were even more unexpected for the team of authors. The latter characteristic is especially important, since until now, due to the low bioavailability, antibody preparations have been administered mainly via the intravenous route, which is more complex and less comfortable for the patient. The increased half-life, high efficiency and bioavailability with subcutaneous administration will reduce the frequency of administration of the drug and increase the convenience of its use.

 Thus, the first object of the present invention is a recombinant monoclonal bispecific anti-CD3 * CD19 antibody comprising two disulfide-linked chains, each of which consists of the following domains: VL (CD3) -Ll-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 CD 19, respectively, LI, L2, L3 are linker sequences, H is the hinge region of IgG immunoglobulin and CH2-CH3 (IgG) is the constant region of the Ig immunoglobulin Fc domain G.

 Moreover, said constant region of the IgG immunoglobulin Fc domain is preferably a constant region of the IgG2 immunoglobulin Fc domain and contains the sequence shown in SEQ ID NO: 5. The linker sequences LI, L2, L3 preferably have a length of 5-15 amino acids and have sequences according to essentially corresponding to the sequences presented 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 pancreatitis, measured in essentially similar conditions. Moreover, the half-life in blood of an antibody of the invention may be more than 40 hours. Moreover, the specified antibody preferably, but not necessarily has an amino acid sequence essentially corresponding to the sequence presented in SEQ ID NO: 10.

 The following objects of the present invention are a nucleic acid encoding a chain sequence of the specified bispecific anti-CD3 * CD19 antibody of the invention, and a vector containing it. The vector may be a vector for expressing a bispecific anti-CD3 * CD19 antibody in a host cell of the invention, comprising the aforementioned nucleic acid operably linked to nucleotide sequences enabling heterologous expression in the host cell. The specified vector may be a cloning vector containing the above nucleic acid, and flanking its restriction sites, allowing you to cut the specified nucleic acid using suitable restriction enzymes. Preferably, but not necessarily, the vector is an expression plasmid pRA1451 characterized by the following features:

 - consists of 11308 bp

 - has a molecular weight of 6.99 MDa,

 contains the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 10,

- provides resistance of mammalian cells transfected with the indicated 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 pA - signal polyadenylation of bovine growth hormone,

 - sv40 enh- enhancer of the SV40 virus,

 - SGE 1 - element of attachment to the nuclear matrix,

 - Amp - β-lactamase gene,

 - sv40 pro - promoter of the virus S V40,

 - rigo — puromycin resistance gene,

 - pA - synthetic polyadenylation signal,

- contains unique recognition sites for the following restriction endonucleases: Accl (6490 IO), Agel (460 IO), Apal (995 IO), BmgBI (3270 IO), Bsml (7269 IO), EcoRV (6457 i. O.), Hindlll (1590 I.O.), Notl (11146 I.O.), Pfol (11021 I.O.), Pmll (5699 I.O.), Spel (4622 I.O.), Swal (6466 acting), Tthl 111 (10585 acting).

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

A further object of the present invention is a method for producing a bispecific anti-CD3 * CD19 antibody of the invention, comprising at least culturing a host cell of the invention under conditions that express the anti-CD3 * CD1 antibody of the invention and isolating the resulting product.

Another object of the present invention is a method for purifying an anti-CD3 * CD19 antibody of the invention, comprising at least one chromatography step under conditions providing a preparation of an anti-CD3 * CD19 antibody of the invention, substantially free of host cell impurities. The specified method may further include a centrifugation step, preferably performed before the chromatography step, and another chromatography step. Moreover, the first chromatography step is preferably, but not necessarily, performed using a protein A sorbent, and the second chromatography step is a cation exchange chromatography step.

The next object of the present invention is the use of antibodies against CD3 * CD19 according to the invention for the treatment of b-cell diseases, depletion of b-cells or slow the development of pathological conditions associated with b-cell disorders.

Said use comprises 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 treating B-cell disease, depleting B-cells or slowing down the development of pathological conditions associated with B-cell disorders, comprising, as an active component, an anti-CD3 * CD19 antibody of the invention in a therapeutically effective amount and a pharmaceutically acceptable carrier.

 The next object of the present invention is a method of treating 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 of the invention, or an effective amount pharmaceutical compositions of the invention.

 At the same time, the indicated B-cell disease, depletion of B-cells or a slowdown in the development of the 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 / 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, glomerulonephritis, multiple sclerosis and myasthenia gravis.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Schematic representation of the construction of antibodies GNR-047

 FIG. 2. The design scheme of the expression vector

 FIG. 3. Map of 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 CD 19 receptor on the Raji cell line

FIG. 5a. Study of the pharmacokinetic characteristics of the GNR-047 antibody when administered intravenously at a dose of 0.5 mg / kg FIG. 56. The study of the pharmacokinetic characteristics of the MT-103 antibody when administered intravenously at a dose of 0.5 mg / kg

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

 FIG. 66. A study of the pharmacokinetic characteristics of the MT-103 antibody when administered subcutaneously at a dose 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 are the most preferred embodiments of the present invention, the semantic content of the terms used in the framework of the present invention is disclosed, and examples are presented that demonstrate the implementation of the present invention and the results achieved.

 The term "recombinant monoclonal bispecific anti-CD3 * CD19 antibody" used in accordance with the present invention includes antibodies that specifically bind to both CD3 and CD 19, which are obtained using recombinant technologies.

 Antibody against CD3 * CD19 according to the invention can be obtained on the basis of 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 modification of mouse and / or human sequences, for example, through the introduction of 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 monoclonal bispecific anti-CD3 * CD19 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-CD 19 antibody, the linker sequence, the hinge region, the Fc sequence and has two chains connected by disulfide bonds . Moreover, each chain preferably has the following structure: 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 CD 19, respectively;

LI, L2, L3 — linker sequences; H is a hinge region immunoglobulin IgG; and CH2-CH3 (IgG) is a constant region of the Fc domain of an IgG immunoglobulin.

 AHTH-CD3 antibodies are well known in the art. Examples of anti-POP antibodies include, but are not limited to, OKTZ, G4.18, 145-2C11, Leu4, NGH3, SCZ, SK7, SP34, RIV-9, and UCHT1. Preferably, the anti-POP antibody binds to the same epitopes as OCTZ, G4.18, 145-2C11, Leu4, NGH3a, SCZ, SK7, SP34, RIV-9 and UCHT1.

 One of ordinary skill in the art can obtain additional anti-CD3 antibodies without undue experimentation and, inter alia, determine whether antibodies to the same epitopes are specific as anti-POP antibodies OKTZ, G4.18, 145-2C11, Leu4, NGGZa, VSZ , SK7, SP34, RTV-9, and UCHT1, finding out if the former prevent the binding of the latter to the CD3 antigenic polypeptide.

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

 AHTH-CD19 antibodies are also well known in the art, as are methods for their preparation. Examples of anti-C019 antibodies include, but are not limited to, HD37, HIB19, SJ25-C1, 4G7-2E3. In addition, these methods and antibodies are disclosed in Eurasian application EA200800094, published June 30, 2008. In general, any variable domains of the anti-CD 19 antibody light and heavy chains that exhibit high specificity and binding strength to the CD 19 antigenic polypeptide can be used within the scope of the present invention. However, it is most preferred to use the variable light and heavy chain domains of the antibody in accordance with the present invention. anti-CD19 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 — this is the terminal portion of an immunoglobulin molecule that interacts with the Fc receptor on the cell surface and with some proteins of the complement system. Depending on the amino acid sequence of the constant region of the heavy chain, immunoglobulins are divided into classes: IgA, IgD, IgE, IgG and IgM, and some of them can be further divided into subclasses (isotypes), for example, IgGl, IgG2, IgG3 and IgG4, IgAl and IgA2. In accordance with the constant regions of the heavy chains, the various classes of immunoglobulins are called [alpha], [delta], [epsilon], [gamma] and [mu], respectively. Four isotypes of human IgG bind various receptors, such as the neonatal Fc receptor, activating gamma Fc receptors, FcgRI, FcgRIIa and FcgRIIIa, inhibiting the FcgRIIb receptor and Clq with different affinities, obtaining different types of activity. 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.

 The antibody of the present invention contains an Fc portion to extend the half-life. Such an Fc sequence is preferably of human origin, more preferably is the human Fc sequence of an IgG antibody, more preferably IgG2, even more preferably, is the sequence shown in SEQ ID NO: 5. In this case, the selection of an IgG2-type Fc sequence allows one to reduce the effector load due to the Fc sequence and thus reduce the directed 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 interference. The length and sequence of the linker depends on the sequence and length of the linked sequences. Methods for testing the suitability of various linkers are well known to the skilled person. 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 obtained molecule can be measured by methods known in the art, such as, for example, high-performance liquid chromatography, dividing the studied protein material into fractions, depending on the molecular weight of the protein and hydrodynamic characteristics.

Linkers that 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, series and alanine. Linkers consisting solely of glycine and serine molecules are particularly preferred. Yu Preferably, the linker sequences of the invention are comprised of 5-15 amino acids. More preferably, the LI, L2, L3 linkers of the anti-CD3 * CD19 antibody of the invention are selected from the following sequences:

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

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

 The term “hinge region”, used in accordance with the present invention, refers to a sequence that in a natural immunoglobulin binds the CH1 domain to the region of the CH2-CH3 Fc fragment. The hinge region used in the antibody of the invention is preferably homologous to the natural region of the immunoglobulin and typically includes cysteine residues linking the two heavy chains via disulfide bonds, as in natural immunoglobulins. Typical hinge sequences of human and mouse 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 IgGl, IgG2, IgG3, IgG4 and other immunoglobulin isotypes. Most preferably, the IgGl hinge region with the sequence shown in SEQ ID NO: 9 is used in the antibody construct of the invention.

 The arrangement of the variable regions in the construction of the antibody of the invention is of principle principle. The authors carried out work to assess in vitro toxicity for the release of pro-inflammatory cytokines and CD3-mediated antibody cytotoxicity against CD 19+ tumor cells (hereinafter, cytotoxic biological activity) of various variants of the bispecific BiMS format antibody:

 VL (CD3) -Ll-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 high cytotoxic biological activity and low toxicity of the bispecific variant were established antibodies VL (CD3) -L1-VH (CD19) -L2-VL (CD19) -L3-VH (CD3) -H-CH2-CH3 (IgG), which may be hypothetically associated with high affinity of the antibody due to the conformation of variable domains according to the invention in relation to CD3 specificity. In a most preferred embodiment of the present invention, an anti-CD3 * CD19 antibody is characterized by the sequence of SEQ ID NO: 10. However, the specialist in this field it is clear that without undue experimentation and the cost of inventive creativity, guided by the above disclosure, he can create an antibody according to the invention with a different sequence, but with high efficiency in the treatment of malignant diseases of b-cells, depletion of b-cells or slow development of pathological conditions associated with B-cell disorders, and at the same time convenient in use and preparation compared with blinatumomab. As a criterion for selecting suitable antibody sequences of the invention, specificity / binding efficiency of CD3 and CD 19, characteristic of an antibody with the sequence shown in SEQ ID NO: 10 (see Example 3), blood half-life from about 40, can be used 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 derived by methods known in the art from the structure of the indicated antibody. In particular, in the most preferred embodiment of the present invention, the nucleic acid of 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 of the invention is selected so that in the selected host cell maximum levels of its transcription and subsequent translation were achieved. The term "nucleic acid" as used in accordance with the present invention preferably refers to DNA. However, this term may also refer to RNA.

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

In a most preferred embodiment of the present invention, expression of an anti-CD3 * CD19 antibody of the invention in a host cell is provided using a vector for expression. The term “expression vector” as used in accordance with the present invention refers to any suitable vector, including chromosomal, extrachromosomal vectors and synthetic nucleic acid vectors containing a suitable set of expression control elements.

 The vector for expressing the bispecific anti-CD3 * CD19 antibody of the invention is preferably constructed to allow expression of the nucleic acid of the invention in mammalian cells. However, the specified vector without limitation can also be constructed to ensure 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 derivatives of SV40, 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).

 A vector for expressing a bispecific anti-CD3 * CD19 antibody of the invention may simultaneously be a cloning vector and have the ability to autonomously replicate in bacterial, yeast and other cells, and also be suitable for the production of a nucleic acid encoding an anti-CD3 * CD19 antibody of the invention, by treating said cloning vector with suitable restriction enzymes. However, it is more preferable that the cloning vector containing a nucleic acid encoding an anti-CD3 * CD19 antibody of the invention does not additionally contain elements for expressing said nucleic acid to enhance its amplification in the host cell.

 In one embodiment of the present invention, the expression vector is a vector that is suitable for expression of an antibody in bacterial cells. Examples of such vectors include BlueScript vectors (Stratagene), pIN vectors, pET vectors (Novagen, Madison WI), and others.

In another embodiment of the present invention, the expression vector is a vector that is suitable for expression in the 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 invention, the expression vector is a vector that is suitable for expression of an antibody in a plant. Suitable vectors include, for example, vectors disclosed in RF patent RU2412251, published on 02.20.2011.

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

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

 In the expression vector of the invention, the nucleic acid encoding the antibody of the invention may be coupled to any suitable promoter, enhancer and other expression promoting elements. Moreover, these elements are selected based on the type of host cell.

 In particular, the replication initiation site from plasmid pBR322 (Product No. 303-3s, New England Biolabs, Beverly, Mass) is applicable to most gram-negative bacteria, while the different replication initiation sites are from SV40, polyomavirus, adenovirus, and 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 '(to the right) from the end of the coding regions of the polypeptide and serves to terminate the transcription. Transcription termination sequences in prokaryotic cells often contain a G-C-rich fragment, followed by a poly T-sequence. Transcription termination sequences can be cloned from a library purchased commercially as part of a vector.

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

 Examples of breeding markers for mammalian cells include dihydrofolate reductase (DHFR) and thymidine kinase. Mammalian cell transformants are placed under selection pressure conditions, to which only transformants are adapted for survival, due to the marker present in the vector. The selection pressure is superimposed by culturing the transformed cells under conditions in which the concentration of the selection agent in the medium changes sequentially, which leads to 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 the antibody of the invention amplified in this way will produce increased amounts of the antibody.

 The 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 '(right) from the promoter and 5' (left) from 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 the 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 (e.g., globin, elastase, albumin, alpha-fetoprotein and insulin). However, usually a virus enhancer should be used. The SV40 enhancer, cytomegalovirus early promoter enhancer, polyomavirus enhancer and adenovirus enhancers are examples of enhancer elements for activating eukaryotic promoters.

The components of the vector can be homologous (from the same species and / or strain as the host cell), heterologous (from a species other than the species or strain of the host cell), hybrid (a combination of different sequences from more than one source ), synthetic or native sequences that typically act as regulators of immunoglobulin expression. The sources of the components of the vector can be any prokaryotic or eukaryotic organism, any a vertebrate or invertebrate organism, or any plant, provided that these components are functional in the mechanism of the host cell and can be activated by the mechanism of the host cell.

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

 A vector for expressing a bispecific anti-CD3 * CD19 antibody of the invention may further comprise a nucleic acid sequence encoding a secretion / localization sequence that can direct the polypeptide into the periplasmic space or into the culture medium. Such sequences are known in the art and include secretion leaders or signal peptides, organelle targeting sequences (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 field of the present invention.

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

The term "host cell" in the present invention is used to refer to cells into which the 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. Moreover, although modifications may occur in subsequent generations due to environmental influences or artificial mutagenesis, the term “host cell” in accordance with the present invention should also be applied to such progeny. A host cell of the invention may be eukaryotic and prokaryotic. A prokaryotic host cell may be, inter alia, a bacterial cell. The eukaryotic host cell may be, inter alia, a yeast cell, a mammalian cell, a non-mammalian animal cell, for example, an insect cell, a plant cell. In particular, eukaryotic host cells of the present invention may be CHO cells, HEK-293 cells, HEK-294 cells, PER.C6 cells, NS0 cells and lymphocyte cells, as well as prokaryotic cells such as E. coli.

 Some embodiments of the present invention include the introduction of a vector to allow expression of the invention in an in vivo host cell, i.e. into a cell that is part of a living multicellular organism. The specified organism is preferably a person.

 According to the present invention, various expression host / vector combinations for expression can be used to express the antibody. In this case, it is necessary to choose a vector taking into account the preservation of its functionality in the host cell in which the specified vector will be introduced. The vector must be compatible with the mechanism of the host cell, so that amplification and expression are possible. For example, expression vectors suitable for eukaryotic hosts include SV40, cattle 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, pCRl, pBR322, pMB9 and their derivatives, plasmids like RP4, characterized by a wide list of possible hosts, gtlO and λΐ 1, phage DNA, represented by various lambda phage derivatives, such as ΝΜ989, and other phage DNAs, such as M13 and single-stranded filamentous phage. Expression vectors that can be used with yeast cells include plasmid 2μ and its derivatives. A vector that can be used in insect cells is pVL941.

 In a most preferred embodiment of the present invention, to combine the expression of the bispecific anti-CD3 * CD19 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 a bispecific anti-CD3 * CD19 antibody of the invention can be obtained by transfection of a parent cell with a vector to allow expression of a bispecific anti-CD3 * CD19 antibody of the invention using any method known in the art, including electroporation, transduction, calcium -phosphate transfection, transfection with cationic lipids, scrape-loading (scraping load) and infection.

A method for producing an anti-CD3 * CD19 antibody of the invention comprises culturing said host cell under conditions providing expression of a bispecific anti-CD3 * CD19 antibody of the invention and isolating the resulting product. Moreover, this expression can be carried out both under conditions of a small-scale or large-scale fermenter, and under incubation conditions in flasks, on a rocking chair. Incubation is carried out in a suitable nutrient medium containing sources of carbon and nitrogen and inorganic salts, one of the known methods. Suitable media are commercially available, but can also be prepared independently using components described, for example, in the catalog of the American Type Culture Collection (ATCC). In general, the term “medium” as used in accordance with the present invention 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, cells - insect hosts, plant host cells, mammalian host cells, including CHO cells.

 An antibody of the invention may be isolated by any method known in the art. However, when choosing a method for isolating the antibody, it is necessary to take into account the features of the expression system used (host cell for expression / vector for expression). So, if the antibody mainly accumulates inside the host cell, it is necessary to choose a method of isolating the product that will ensure its extraction from the host cell. If the antibody accumulates predominantly in the culture fluid, the target product can be isolated from the culture fluid by methods designed to extract antibodies from the culture fluid. For example, an antibody of the invention can be isolated from the culture fluid by standard methods, including, without limitation, chromatography, centrifugation, filtration, extraction, drying, spraying, evaporation or precipitation. An antibody of the invention can be further purified using various techniques known in the art, including chromatography (e.g., ion exchange, affinity, hydrophobic or 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 resolution cation exchange chromatography.

The anti-CD3 * CD19 antibody of the invention purified according to regulatory requirements 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 anti-CD3 * CD19 antibody is similar to that described for the anti-POP / anti-C019 antibody, Blinatumomab preparation (MT103); and consists in the activation of a multispecific T-cell cytological response against CD 19+ 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 leukemia) of B-cell nature, for example, such as B-cell tumors from precursors of B-lymphocytes, including: 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, 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 in the form of injections, including subcutaneous injections). If the composition according to the present invention is administered parenterally, for example intravenously, intradermally, subcutaneously, intraperitoneally, intramuscularly, buccally, intraorbitally, intracerebrally, intraspinally, intraventricularly, intrathecally, intracerebrally, the composition preferably comprises a portion of an aqueous or physiologically compatible solution or suspension of liquid does not adversely affect the balance of electrolytes and / or liquids in patients upon delivery of the target composition 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 sterile aqueous a solution, suspension, emulsion, concentrate for the preparation of an aqueous solution, a lyophilized preparation for the preparation of an aqueous solution, a solution based on a non-aqueous solvent.

 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 , wetting agents, complexing agents, buffering agents, antimicrobial agents and surfactants, as shown, for example, in the "Handbook of Pharmace utical Excipients ”(2" d ed. London: The Pharmaceutical Press; 1994).

 In this case, as a carrier or solvent to obtain a sterile aqueous solution, in accordance with the present invention, for example, either Hank's solution, or Ringer's solution, or a suitable buffer solution, for example, physiological buffer solution, can be used. The buffers may be conventional buffers such as acetate, citrate, phosphate, bicarbonate buffers or Tris-HCl. Acetate buffer may have a pH of about 4-5.5, and Tris buffer may 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 may be propylene glycol, polyethylene glycol or olive oil or an ester suitable for injection, such as ethyl oleate. Also an example of a suitable carrier for the purpose of preparing the pharmaceutical composition of the invention is a neutral buffered saline or saline mixed with serum albumin.

As adjuvants, the pharmaceutical composition of 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, mannose 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, pluronic or polyethylene glycol (PEG), alkali metal halides (preferably sodium or potassium chloride), benzalkonium chloride, thimerosal, phenethyl alcohol, methyl paraben, propyl paraben, chlororexidine, sorbic acid, peroxide hydrogen, sorbitan esters, polysorbates such as polysorbate 80, tromethamine, lecithin, cholesterol, tyloxapal, high molecular weight structural additives (for example, gum arabic, alginic acid, calcium phosphate (dibasic), cellulose, carboxymethyl cellulose, hydroxyloxy cellulose cellulose cellulose cellulose cellulose microcrystalline cellulose, dextran, dextrin, dextrates, sucrose, tylose, pregelatinized starch, calcium sulfate, amylose, glycine, bentonite, maltose, sorbitol, ethyl cellulose, disodium hydrogen phosphate, disodium phosphate, disodium pyrosulphite, polyvinyl alcohol, gelatin, glucose, guar gum, liquid glucose, pressed sugar, magnesium-aluminum silicate, maltodextrin polyethylene glycol polyethylene glycol polyethylene glycol polyethylene glycol polyethylene glycol polyethylene glycol , starch and zein).

 The most preferred adjuvants 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 (for example, anti-cancer or anti-inflammatory drugs), provided that they do not cause unwanted 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 Pat. Nos. 6,659,540, 6,566,329 and 6,373,716. In one embodiment of the present invention, a lyoprotectant, which is a non-reducing sugar, such as sucrose, lactose or trehalose. The amount of lyoprotectant is preferably chosen so that after recovery, the resulting finished form is isotonic. At the same time, the selected amount of lyoprotectant should be sufficient to prevent an unacceptable amount of protein degradation and / or aggregation after lyophilization. Examples of sugar lyoprotectant concentrations (e.g., sucrose, lactose, trehalose) in a pre-lyophilized composition are from about 10 mM to about 400 mM.

The pharmaceutical composition according to the invention, if obtained in lyophilized form, can be optionally completed with a pharmaceutically acceptable solvent and / or diluent, for example, physiological water solution and other commonly used pharmaceutically acceptable solvents.

 Pharmaceutical compositions may contain a therapeutically effective amount of an antibody of the invention.

 The term “therapeutically effective amount” as used in accordance with the present invention refers to that amount of an antibody of the invention that is effective for the necessary periods of time to achieve the desired therapeutic result, and any toxic or harmful effects of the antibody should be outweighed by therapeutically beneficial effects. from this antibody, which in a preferred embodiment of the present invention is at least one of leduyuschego: symptom relief, healing or increase the speed of recovery. The therapeutically effective amount of an antibody of the invention can vary according to factors such as the condition of the disease, age, gender, and weight of the individual, the ability of the antibody to induce the desired response specifically in the individual, the presence of concomitant diseases, route of administration, frequency of administration, individual treatment regimen.

 It will not be difficult for a person skilled in the art to determine an 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 number of antibodies according to the invention, which it is advisable to treat each patient individually. Moreover, the attending physician may first administer small doses of the antibody of the invention and, according to the patient’s response, adjust its amount. Dosage 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 antibody.

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

A method of treating B-cell disease or B-cell depletion, or slowing the development of a pathological condition associated with B-cell disorders of the present invention includes administering to a patient in need thereof an effective amount of an antibody of the invention or a pharmaceutical composition of the invention. Moreover, in a preferred embodiment of the present of the invention, the antibody is administered subcutaneously to the patient. Moreover, the anti-CD3 * CD19 antibody of the invention can be administered to a patient in combination with another drug, including for at least one of the following purposes: to enhance the therapeutic effect against B-cell disease, to reduce side effects during treatment B-cell disease, to alleviate the symptoms of B-cell disease.

 The duration of therapy using the combination of the present invention varies depending on the severity of the disease to be treated and the condition, and also depending on 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 foregoing, the authors in the experiment showed that:

 • an antibody according to the invention can be obtained using technologically simple known recombinant methods, commercially available agents (vector, host cell, medium) and traditional methods for the isolation and purification of antibodies (Example 1);

 • based on antibodies against CD3 * CD19 according to 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).

The authors of the present invention also experimentally demonstrated, in comparison with MT103, a significantly increased half-life (T1 / 2) and exposure (AUC (O-inf)) of the anti-CD3 * CD19 antibody of the invention, while maintaining the maximum peak concentration (Cxx) when administered intravenously, and also increased bioavailability of antibodies against CD3 * CD19 according to the invention with both intravenous and subcutaneous administration (Example 4). Additionally, the authors of the present invention experimentally confirmed a higher affinity of the antibodies of the invention for CD3 and CD 19 and its higher antitumor activity when administered subcutaneously in comparison with MT103 (Examples 3 and 5).

Example 1. Obtaining antibodies against CD3 * CD19

Construction of recombinant plasmid DNA For cloning, a commercially available Selexis vector containing the SGE1 genetic element was selected to enhance expression as a result of the close proximity of the target transgene with a highly active nuclear transcription complex.

 The gene encoding the anti-CD3 * CD19 antibody chain was constructed by annealing the overlapping chemically synthesized oligonucleotides. The sequence of the target gene was cloned into the commercially available vector pUC 19, whereby the vector pAPG_GNR047 was obtained, which was used for further work. After treating the pAPG_GNR047 vector with Hindlll, Xbal (New Ingland Biolabs) restriction endodesoxyribonucleases forming sticky ends, a 2235 bp fragment was then ligated, which was then ligated with the Hindlll and Xbal linearized pSTlOl expression vector. The resulting plasmid pRA1451 was verified by restriction mapping. The construction scheme of the expression vector pRA1451 is shown in FIG. 2.

 After that, sequential double transfection was carried out, 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 bespecifically antibodies against CD3 * CD19, GNR-047,

- provides resistance of mammalian cells transfected with the indicated 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 pA - signal polyadenylation of bovine growth hormone,

 - sv40 enh- enhancer of the SV40 virus,

 - SGE 1 - element of attachment to the nuclear matrix,

 - Amp - β-lactamase gene,

 - sv40 pro - SV40 virus promoter,

 - puro - puromycin resistance gene,

 - pA is a synthetic polyadenylation signal.

- contains unique recognition sites for the following restriction endonucleases: Accl (6490 no), Agel (460 no), Apal (995 no), BmgBI (3270 acting), Bsml (7269 acting), EcoRV (6457 no), Hindlll (1590 no), Notl ( 11146 no), Pfol (11021 no), Pmll (5699 no), Spel (4622 no), Swal (6466 no), Tthl 1 II (10585 no).

 The genetic design of the expression vector is shown in Fig.Z.

 Transfection was performed using a commercially available strain of CHO-M (Selexis). The selection of the producer cell line is due to 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 culture conditions for this cell line, which are the most important parameters for the production of therapeutic antibodies.

Cultivation was carried out in fed-batch mode using BalanCD CHO Growth A nutrient medium (Irvine Scientific) and CellBoost 3 (HyClone) nutrient supplement for 10 days at 37 ° С, 5% С0 ₂ in the gas phase of the incubator. After cultivation, the culture fluid was clarified by centrifugation and transferred to the selection.

To isolate and purify the target product, a scheme was developed that included the affinity step using a sorbent based on protein A (with a yield of more than 90%) and cation exchange chromatography on a high-resolution sorbent that effectively separated the target form of the protein and its oligomeric forms (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 animal experiments, a pharmaceutical composition of the following composition was used: 0.7 mg / ml anti-CD3 * CD19 antibody of the invention (hereinafter GNR-047), 150 mM sodium chloride, 1.8% mannitol, 20 mM sodium phosphate, 0.01% polysorbate 80.

The purification conditions of the anti-CD3 * CD19 antibody of the invention at the last chromatographic stage of the process are selected so 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, to prepare the finished dosage form, an antibody solution with a concentration of about 5 mg / ml (the 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 to store the composition and subsequent experiments.

 To obtain a lyophilized composition, a solution containing anti-CD3 * CD19 antibodies of the invention in a finished buffer (150 mM sodium chloride, 1.8% mannitol, 20 mm sodium phosphate, 0.01% polysorbate 80) was lyophilized under standard conditions including stage of freezing and vacuum dehydration. In this case, sodium chloride and mannitol were used as crystallizing components, the presence of which allows the formation of 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 for injection was used as a solvent for reconstituting the lyophilized composition. To restore the drug, 0.5 ml of the solvent was added to the vial containing the lyophilized antibody composition at room temperature and gently mixed by shaking until it was completely dissolved. Bacteriostatic water for injection can also serve as a solvent, dissolution in which allows the drug to be reused within 7 days.

Example 3. The study of biological activity

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

 Antibody MT-103 (blinatumomab) was used as a comparison drug. After incubation with the target molecules in successive 10-fold dilutions from 100 nM to 0.1 nM, a competing antibody (HD37 or OCTZ) was added to the cells at a concentration of 6 nM. Cells were washed with pH 7.0 sodium phosphate buffer and stained with anti-Human-FITS antibodies. Stained cells were analyzed by FACS Calibur.

 The binding results are shown in Table 1 and in FIG. four

Table 1

 The table shows that the GNR-047 antibody binds to both CD3 and CD 19, and in both cases it is more effective than the MTUZ comparison drug. Such low, in comparison with the comparison drug, IC50 values can be explained by the presence of 2 valencies for each specificity (CD3 and CD 19

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

 In view of the fact that the GNR-047 preparation showed improved pharmacokinetic characteristics during intravenous administration compared with MTYuZ, it was advisable to determine the pharmacokinetic parameters with more convenient subcutaneous administration, in which GNR-047 will be prolonged to be released from the depot into the bloodstream.

 For the experiment, rats of the Sprague Dawley line were used (Pushchino Kennel of Laboratory Animals, branch of the Institute of Bioorganic Chemistry named after Academicians MM Shemyakin and Yu. A. Ovchinnikov of the Russian Academy of Sciences) weighing about 400 g, 3 rats per group.

 The test antibody GNR-047 or MT-103 was administered intravenously at a dose of 0.5 mg / kg or subcutaneously at a dose of 2.0 mg / kg to each rat. Blood samples were taken at certain intervals after injection, starting from 5 min, and the concentration of the drugs in the blood plasma was determined using enzyme-linked immunosorbent assay (ELISA).

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

table 2

 ** AUC (0-inf) in this case coincides with AUC (O-t), since at the last point of the pharmacokinetic curve the concentration of MT103 is close to zero.

 *** The parameter is calculated taking into account the dose difference.

Where,

 TT - necessary to reduce the concentration of the drug in blood plasma by 50% as a result of elimination; Sshah - the highest concentration of the drug in the blood plasma; AUQo-t) is the area bounded by the pharmacokinetic curve and the abscissa axis in the time interval from the moment of drug administration until the last blood sample is taken; AUQo-inf) - the area bounded by the pharmacokinetic curve and the abscissa axis, extrapolated in time from the moment of drug administration to infinity; / a - part of the dose of the drug (in%), which reached the systemic circulation after extravascular administration.

As a result, a significant increase in half-life (T) and exposure (AU o-mf) - the area under the pharmacokinetic curve) of the GNR-047 preparation was shown in comparison with the MT103 preparation, while maintaining the maximum peak concentration (Stax) with 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. 66).

Example 5. Comparison of the antitumor activity of GNR-047 and MT 103 in a model of a tumor xenograft of Raji cells (Raji) in NOD / SCID mice during 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, on a Raja tumor cell xenograft model with simultaneous injections of human peripheral blood mononuclear cells (MCPC) to immunodeficient NOD / SCID mice . Cells of the Raja line (human Burkitt’s lymphoma) were pre-mixed with human PBMC and inoculated subcutaneously, followed by intravenous or subcutaneous administration of the studied drugs. For this, nine experimental groups of immunodeficient NOD / SCID mice (9 animals per group) were subcutaneously injected with either human Raja line Burkitt lymphoma cells or a suspension of pre-mixed Raja line cells and human PBMC. Three animals from each group received human PBMC from one of three separate healthy donors (cohorts 1, 2, and 3). 2 hours after inoculation of the cells, the animals were injected intravenously or subcutaneously with either NFB (sodium phosphate buffer solution) or the studied preparations GNR-047 or MT 103 in various doses. The injection volume was 200 μl. In all groups, tumor volume was evaluated over 45 days (days 3 through 48).

 Injected solutions were prepared by diluting the GNR-047 solution (composition) 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). The experimental groups are described in table 3.

Table 3

Исследуемый

Researched

 Cells * Inoculated

 Group drug (injection

 mouse by sc injection (d 0)

 1 time / day)

37 - 39 Cells of the Raji line + MCPC

 40 - 42 (Donor 1) GNR-047

 5 43 - 45 Cells of the Raji line + MCPC 1

 (Donor 2) mcg / animal,

 Cells of the Raji line + MCPC intravenously

 (Donor 3)

 46 - 48 Cells of the Raji line + MCPC

 49 - 51 (Donor 1) GNR-047

6 52 - 54 Cells of the Raji line + MCPC 0.1

 (Donor 2) mcg / animal,

 Cells of the Raji line + MCPC intravenously

 (Donor 3)

 55 - 57 Cells of the Raji line + MCPC

 58 - 60 (Donor 1) GNR-047 61 - 63 Cells of the Raji line + MCPC 4

7

 (Donor 2) mcg / animal,

 Cells of the Raji line + MCPC intravenously

 (Donor 3)

 64 - 66 Cells of the Raji line + MCPC

 67 - 69 (Donor 1) GNR-047 70 - 72 Cells of the Raji line + MCPC

 8 1.7

 (Donor 2) mcg / animal,

 Raji cells + PBMC subcutaneously

 (Donor 3)

 73 - 75 Cells of the Raji line + MCPC

 76 - 78 (Donor 1) GNR-047 79 - 81 Cells of the Raji line + MCPC

 9 0.17

 (Donor 2) mcg / animal,

 Raji cells + PBMC subcutaneously

 (Donor 3)

* 2.5 * 10 ⁶ cells of the Raji line, 1 * 10 ⁷ MCPC.

The following indicators were determined in experimental animals: survival, body weight, tumor volume, tumor mass.

Body weight was determined on day 0 and was 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 diameter of the tumor with a barbell. Based on the diameters, the tumor volume was calculated using the following formula:

Volume = (small diameter) ² x large diameter x 0.5 In control group 1 receiving NFB (only Raji cells), 11% of the animals survived until the study was completed. Survival in animals of group 2 treated with cells of the Raji line and human PBMC did not change. In both groups, mortality was due solely to euthanasia for ethical reasons (due to the large volume of the tumor).

 The introduction of GNR-047 and MT 103 increased survival in groups 3-9, which is reflected in table 4, and with a higher dose, the studied drug was more effective than the comparison drug:

 Table 4

Animal survival

Criteria for removing experimental animals from the study: tumor volume> 10% of body weight; tumor ulceration; 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 NPF. Moreover, in all the studied groups treated with GNR-047 or MT 103, 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 that allow us to estimate the size of the tumor during intravenous and subcutaneous administration (for groups 4, 8, and 9) are shown in Fig. 7.

Table 5 Study Day Group

 3 6 8 10 13 15 17

 1 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 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

 Study day

 Group

 20 22 24 27 29 31

 1,387.0 561.5 781.5 1,046.5 1,214.8 1,559.2

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 for the treatment of B-cell diseases or depletion of B-cells, 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, glomerulonephritis, multiple sclerosis and myasthenia gravis. The present invention can also be used in the pharmaceutical industry for the preparation of pharmaceutical compositions, formulations and kits containing an antibody of the invention for the treatment of the above diseases.

FREE TEXT OF THE LIST OF SEQUENCES SEQ ID N ° 1 - the variable domain of the light chain anti-POP antibodies

SEQ ID 3 ° 2 - the variable domain of the heavy chain anti-POP antibodies, SEQ ID jb 3 - the variable domain of the light chain of anti-C019 antibodies,

SEQ ID N ° 4 - the variable domain of the heavy chain of anti-C019 antibodies,

SEQ ID BEFORE? 5 - constant domain of CH2 SNZ IG2,

 SEQ ID Jfe 6 linker L1,

 SEQ ID jVs 7 - linker L2,

 SEQ ID jVe 8 - linker L3,

 SEQ ID jV ° 9 - hinge region H,

 SEQ ID -4 ° 10 - antibody GNR-047.

Claims

CLAIM 1. A bispecific antibody that specifically binds to CD3 and CD 19, comprising two disulfide linked chains, each of which consists of the following domains: 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 CD 19, respectively, LI, L2, L3 linker sequences, H is the hinge region of IgG immunoglobulin,  CH2-CH3 (IgG) is a constant region of the Fc domain of IgG immunoglobulin.  2. The antibody according to claim 1, characterized in that said constant region of the IgG immunoglobulin Fc domain is a constant region of the IgG immunoglobulin Fc domain.  3. The antibody according to claim 2, characterized in that the constant region of the Fc domain of IgG2 immunoglobulin contains the sequence shown in SEQ ID NO: 5. 4. The antibody according to claim 1, characterized in that the linker sequences LI, L2, L3 have a length of 5-15 amino acids.  5. The antibody according to claim 4, characterized in that the linker sequences LI, L2, L3 have sequences essentially corresponding to the sequences shown in SEQ ID NO: 6, 7, 8, respectively.  6. The antibody according to claim 1, characterized in that the hinge region of the IgG immunoglobulin essentially corresponds to the sequence shown in SEQ ID NO: 9.  7. The antibody according to claims 1 to 6, characterized in that it has an amino acid sequence essentially corresponding to the sequence represented by B SEQ ID NO: 10.  8. The antibody according to claim 1, characterized in that it has a half-life in blood, increased compared with the half-life of a pancreaticomab, measured under essentially the same conditions.  9. The antibody according to claim 1, characterized in that it has a half-life in blood of more than 40 hours.  10. A nucleic acid encoding an antibody according to any one of claims 1 to 9.  1 1. The vector containing the nucleic acid of claim 10.  12. The vector according to claim P, characterized in that it is an expression plasmid pRA1451, characterized by the following features: - consists of 1,1308 bp, - has a molecular weight of 6.99 MDa,  - contains the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 10,  - provides resistance of mammalian cells transfected with the indicated 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 pA - signal polyadenylation of bovine growth hormone,  - sv40 enh- enhancer of the SV40 virus,  - 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:  Accl (6490 bp), Agel (460 bp), Apal (995 bp), BmgBI (3270 bp), Bsml (7269 bp), EcoRV (6457 p. o.), Hindlll (1590 bp), Notl (11146 bp), Pfol (1 1021 bp), Pmll (5699 bp), Spel (4622 bp), Swal (6466 bp), Tthl 111 (10585 bp). 13. A cell containing the nucleic acid of claim 10 or the vector of claim 11.  14. The cell according to item 13, characterized in that it is a eukaryotic cell.  15. The cell according to 14, characterized in that it is a mammalian cell.  16. A cage according to claim 15, characterized in that it is a cage of a Chinese hamster. 17. A method of producing an antibody according to any one of claims 1 to 9, comprising at least culturing a cell according to any one of claims 13-16 under conditions ensuring expression of the antibody according to any one of claims 1 to 9, and the selection of the target product.  18. The method according to 17, characterized in that the cell is a cell SNO.  19. The method of purification of an antibody according to any one of claims 1 to 9, comprising at least one chromatography step, carried out under conditions ensuring the preparation of an antibody according to any one of claims 1 to 9, essentially free from cell impurities the owner. 20. The method according to claim 19, characterized in that it further includes a centrifugation step, preferably performed before the chromatography step. 21. The method according to claim 20, characterized in that it further includes another step of chromatography. 22. The method according to item 21, wherein the first stage of chromatography is performed using a sorbent based on protein A, and the second stage of chromatography is a stage of cation exchange chromatography.  23. The use of antibodies according to claims 1 to 9 for the treatment of b-cell disease, depletion of b-cells or slow the development of the pathological condition associated with b-cell disorders.  24. The use according to claim 23, characterized in that said B-cell disease, depletion of B-cells or a slowdown in 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 / 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, glomerulonephritis, multiple sclerosis and myasthenia gravis.  25. The application of item 23, wherein the antibody is administered to the patient subcutaneously. 26. The application of item 23, wherein the antibody is administered to the patient intravenously. 27. The use according to claim 23, wherein the antibody is administered to the patient in a therapeutically effective amount.  28. A pharmaceutical composition comprising a therapeutically effective amount of an antibody according to any one of claims 1 to 9 and a pharmaceutically acceptable carrier. 29. The pharmaceutical composition according to p. 28, characterized in that it is intended for b-cell disease, depletion of b-cells or slow the development of a pathological condition associated with b-cell disorders. 30. The pharmaceutical composition according to clause 29, wherein the specified b-cell disease, depletion of b-cells or slow the development of the 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 / 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, glomerulonephritis, multiple sclerosis and myasthenia gravis.  31. The pharmaceutical composition according to any one of paragraphs 28-30, characterized in that it further comprises at least one of the following sodium chloride, mannitol, sodium phosphate, polysorbate 80.  32. A method of treating a B-cell disease, depletion of B-cells or slowing the development of a pathological condition associated with B-cell disorders, comprising administering to a patient in need thereof a therapeutically effective amount of an antibody according to claims 1 to 9. 33. The method according to p, characterized in that said 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 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, glomerulonephritis, multiple sclerosis and myasthenia gravis.