RU2815679C2 - Combination therapy based on anti-cd20 antibody and btk inhibitor - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: described is a group of inventions involving a pharmaceutical combination for treating cancer wherein CD20 is expressed and using the pharmaceutical combination for preparing a drug for treating cancer wherein CD20 is expressed. Pharmaceutical combination consists of rituximab and BTK inhibitor, which is 6-amino-9-[(3R)-1-(2-butinoyl)-3-pyrrolidinyl]-7-(4-phenoxyphenyl)-7,9-dihydro-8H-purin-8-one or a salt thereof, in a synergistically effective amount, where the amount of rituximab ranges from 0.1 to 50 mg/kg, and amount 6-amino-9-[(3R)-1-(2-butinoyl)-3-pyrrolidinyl]-7-(4-phenoxyphenyl)-7,9-dihydro-8H-purin-8-one or its salt ranges from 1 mcg/kg to 50 mg/kg.

EFFECT: invention extends the range of agents for treating cancer in which CD20 is expressed.

5 cl, 5 dwg, 7 tbl, 3 ex

Description

The present invention provides a combination therapy based on an anti-CD20 antibody and a BTK inhibitor for the treatment of cancer. BACKGROUND OF THE INVENTION Afucosylated Antibodies

The cell-mediated effector functions of monoclonal antibodies can be enhanced by engineering their oligosaccharide component according to the method described by Umana R. et al., Nature Biotechnol. 17, 1999, pp. 176-180 and in US 6602684. IgG1 antibodies, which are the antibodies most commonly used in cancer immunotherapy, are glycoproteins that have a conserved N-linked glycosylation site at position Asn297 in each CH2 domain. The two complex biantennary oligosaccharides that are attached to Asn297 are sandwiched between the CH2 domains, forming extensive contacts with the polypeptide backbone, and their presence is essential for the ability of the antibody to mediate effector functions such as antibody-mediated cell-dependent cytotoxicity (ADCC) (Lifely MR et al. ., Glycobiology, 5, 1995, pp. 813-822; Jefferis R. et al., Immunol. Rev., 163, 1998, pp. 59-76; Wright A. and Morrison SL, Trends Biotechnol., 15, 1997 , pp. 26-32).U R. et al., Nature Biotechnol. 17, 1999, pp. 176-180 and WO 1999/54342 demonstrated that overexpression in Chinese hamster ovary (CHO) cells of β(1,4)-N-acetylglucosaminyltransferase III (“GnTIII”), i.e. glycosyltransferase, which catalyzes the formation of bisectional oligosaccharides, significantly increases the ADCC activity of antibodies in vitro. Changing the composition of the N297 carbohydrate or its elimination also affects the binding of Fc to FcγR and C1q (Umana R. et al., Nature Biotechnol. 17, 1999, pp. 176-180; Davies J. et al., Biotechnol. Bioeng. 74, 2001, pp. 288-294; Mimura Y. et al., J. Biol. Chem. 276, 2001, pp. 45539-45547; Radaev S. et al., J. Biol. Chem. 276, 2001, cc. 16478-16483; Shields RL et al., J. Biol. Chem. 276, 2001, cc. 6591-6604; Shields RL et al., J. Biol. Chem. 277, 2002, cc. 26733-26740; Simmons LC et al., J. Immunol. Methods 263, 2002, pp. 133-147).

Studies have been published that discuss the activity of afucosylated and fucosylated antibodies, including antibodies to CD20 (see, for example, Iida S. et al., Clin. Cancer Res. 12, 2006, pp. 2879-2887; Natsume A. et al. , J. Immunol. Methods 306, 2005, pp. 93-103; Satoh M., et al., Expert Opin. Biol. Ther. 6, 2006, pp. 1161-1173; Kanda Y., et al., Biotechnol. Bioeng. 94, 2006, pp. 680-688; Davies J. et al., Biotechnol. Bioeng. 74, 2001, pp. 288-294).

CD20 and anti-CD20 antibodies

CD20 (also called evolutionarily conserved human B cell differentiation antigen or BP35) is a well-known hydrophobic transmembrane protein localized on pre-B and mature B cells (Valentine M.A. et al., J. Biol. Chem 264, 1989, pp. 11282-11287; and Einfeld D. A. et al., EMBO J. 7, 1988, 711-717; Tedder T. F. et al., Proc. Natl. Acad. Sci. U.S.A. 85, 1988, cc. 208-212; Stamenkovic I. et al., J. Exp. Med. 167, 1988, pp. 1975-1980; Tedder T. F. et al., J. Immunol. 142, 1989, pp. 2560-2568). In non-Hodgkin's lymphomas (NHL), CD20 is expressed on the surface of more than 90% of B cells (Anderson K.S. et al., Blood 63, 1984, pp. 1424-1433), but it is not detected on hematopoietic stem cells, pro-B -cells, healthy plasma cells or in other healthy tissues (Tedder T.F. et al., J. Immunol. 135, 1985, pp. 973-979).

There are two different types of CD20 antibodies that differ significantly in their CD20 binding mechanism and biological activity (Cragg M.S. et al., Blood 103, 2004, pp. 2738-2743; and Cragg M.S. et al., Blood, 101, 2003, pp. 1045-1052). Type I antibodies, such as rituximab (non-afucosylated antibody with 85% or more fucose content), ofatumumab, veltuzumab, ocrelizumab, have strong complement-mediated cytotoxicity.

Type II antibodies, such as tositumomab (B1), 11 B8, AT80 or humanized B-Ly1 antibodies, effectively initiate target cell death through caspase-independent apoptosis with concomitant exposure to phosphatidylserine.

BTK and BTK inhibitors

Bruton's tyrosine kinase, or the tyrosine kinase whose mutation is associated with Bruton's agammaglobulinemia (abbreviated Btk or BTK), is a member of the TEC family of kinases. BTK is associated with agammaglobulinemia, a disease associated with X-linked primary immunodeficiency (Bruton's agammaglobulinemia). The exact mechanism of its action is not known. The BTK gene encodes the BTK protein, which is critical for B cell development and maturation, such as mast cell activation via the high-affinity IgE receptor. Patients with X-linked agammaglobulinemia have normal populations of pre-B cells in the bone marrow, but these populations fail to mature and enter the bloodstream. The BTK gene is localized on the X chromosome. More than 400 mutations have been identified in the BTK gene.

BTK is activated in the reverse direction by Src-family kinases such as Blk, Lyn and Fyn, and leads to forward activation of critical survival pathways such as NF-κB and MAP kinase. BTK contains a pleckstrin homologous (PH) domain that binds phosphatidylinositol (3,4,5)-triphosphate (PI(3,4,5)P ₃ ). Binding of PI(3,4,5)P ₃ induces phosphorylation of Btk phospholipase Cγ (PLCγ), which in turn hydrolyzes PI(4,5)P2, phosphatidylinositol, to form two second messengers, inositol triphosphate (IP3) and diacylglycrein (DAG). , which then go on to modulate the activity of downstream proteins during B cell signaling. Then

Ca ²⁺ is mobilized and the NF-κB and MAP kinase pathways are activated.

An example of a BTK inhibitor known in the art is ibrutinib (PCI-32765; Advani et al., J Clin Oncol. 31(1), January 1, 2013, pp. 88-94), which is a small molecule reversible BTK inhibitor.

Summary of the invention

When creating the invention, it was found that the combination of an antibody to CD20 type I or an afucosylated antibody to CD20 type II with a BTK inhibitor had significantly increased antiproliferative effects. When creating the invention, it was unexpectedly found that the combination had an effect exceeding additive, i.e. highly synergistic effect.

One aspect of the invention is an afucosylated anti-CD20 antibody in which the fucose content is 60% or less of the total oligosaccharides (sugars) on Asn297 for the treatment of cancer in combination with a BTK inhibitor.

Another aspect of the invention is the use of an afucosylated anti-CD20 antibody, in which the fucose content is 60% or less of the total oligosaccharides (sugars) on Asn297, for the preparation of a drug for the treatment of cancer in combination with a BTK inhibitor.

Another aspect of the invention is a method of treating a patient suffering from cancer comprising administering an afucosylated anti-CD20 antibody in which the fucose content is 60% or less of the total oligosaccharides (sugars) on Asn297, in combination with a BTK inhibitor, to a patient in need of in such treatment.

According to one embodiment of the invention, the fucose content is from 40% to 60% of the total oligosaccharides (sugars) on Asn297. According to another embodiment of the invention, the fucose content is 0% of the total oligosaccharides (sugars) on Asn297.

In one embodiment, the anti-CD20 type I antibody is rituximab. In one embodiment, the afucosylated anti-CD20 antibody is an antibody of the IgG1 isotype. In another embodiment of the invention, said cancer is a cancer that expresses CD20, preferably a lymphoma or lymphocytic leukemia. In one embodiment, the afucosylated anti-CD20 antibody is a humanized B-Ly1 antibody. In another embodiment of the invention, said afucosylated anti-CD20 antibody is an anti-CD20 type P antibody.

In one embodiment of the invention, said BTK inhibitor is a compound selected from those described in WO 2011/152351 and WO 2013/081016. Said BTK inhibitor is preferably a compound of formula I or formula I-1 as defined herein. Preferably the BTK inhibitor is 6-amino-9-[(3R)-1-(2-butinoyl)-3-pyrrolidinyl]-7-(4-phenoxyphenyl)-7,9-dihydro-8H-purin-8-one or its salt.

In one embodiment of the invention, said anti-CD20 type I antibody is rituximab, said afucosylated anti-CD20 antibody is a humanized B-Ly1 antibody, and said BTK inhibitor is selected from the group consisting of compounds that are described in WO 2011/152351 and WO 2013 /081016. Said BTK inhibitor is preferably a compound of formula I or formula I-1 as defined herein. Preferably the BTK inhibitor is 6-amino-9-[(3R)-1-(2-butinoyl)-3-pyrrolidinyl]-7-(4-phenoxyphenyl)-7,9-dihydro-8H-purin-8-one or a salt thereof, and said cancer is a cancer that expresses CD20, in one embodiment, lymphoma or lymphocytic leukemia.

According to one embodiment of the invention, the binding of an afucosylated anti-CD20 antibody to CD20 has a KD value of 10 ^-8 M to 10 ^-13 M.

One embodiment of the invention is a pharmaceutical composition comprising a combination of an anti-CD20 type I antibody (in one embodiment, rituximab) or an afucosylated anti-CD20 antibody in which the fucose content is 60% or less of the total oligosaccharides (sugars) on Asn297 ( in one embodiment, an afucosed humanized B-Ly1 antibody) and a BTK inhibitor (in one embodiment, said BTK inhibitor is selected from the group consisting of compounds that are described in WO 2011/152351 and WO 2013/081016). Said BTK inhibitor is preferably a compound of formula I or formula I-1 as defined herein. Preferably the BTK inhibitor is 6-amino-9-[(3R)-1-(2-butinoyl)-3-pyrrolidinyl]-7-(4-phenoxyphenyl)-7,9-dihydro-8H-purin-8-one or its salt) intended for the treatment of cancer.

Description of drawings

The drawings show:

in fig. 1 - results of a study of antitumor activity in mice carrying subcutaneous TMD8 cells: curves showing the average tumor volumes (a) and curves showing the median tumor volumes (b).

Mice were xenografted at D0. Randomization occurred on D14 (designated day 0 in Fig. 1). Mice were administered orally (PO) vehicle alone and Compound A at 10 mg/kg twice daily (Q0.5D x 24) alone or in combination with one IV injection of GA101 at 1 and 3 mg/kg once weekly. (Q7D x 4). Mice were administered by intravenous (IV) injection of rituximab alone (3 mg/kg) or by IV injection of GA101 alone (1 and 3 mg/kg) once weekly (D14 (day 0), 21 (day 7), 28 (day 14) and D35 (day 21): Q7D × 4). Tumor volumes were monitored and recorded twice weekly;

in fig. 2 - results of a study of antitumor activity in mice carrying subcutaneous TMD8 cells: curves showing the average tumor volumes (a), and curves showing the median tumor volumes (b), in relation to mice.

Mice were xenografted at D0. Randomization occurred on D14 (400-450 mm ³ : designated as day 0 in Fig. 2). Mice were administered via the PO route with vehicle alone and Compound A at 10 mg/kg twice daily (Q0.5D x 24) alone or in combination with one IV injection of GA101/RTX at 3 mg/kg once weekly (Q7D × 4). Mice received one IV injection of rituximab (3 mg/kg) or one IV injection of GA101 (3 mg/kg) once a week (D14 (day 0), 21 (day 7), 28 (day 14), and 35 ( day 21): Q7D × 4). Tumor volumes were monitored and recorded twice weekly;

in fig. 3 results of a study of antitumor activity in mice bearing subcutaneous TMD8 cells: curves showing the average tumor volumes in relation to mice.

Mice were xenografted at D0. Randomization occurred at D14 (approximately 450 mm ³ : designated as day 0 in Fig. 3). Mice were fed a diet containing Compound A at a concentration of 0.0037% (corresponding to a dose of 6 mg/kg/day) or Compound B at a concentration of 0.012% (corresponding to a dose of 20 mg/kg/day), alone or in combination with a single IV injection RTX 3 mg/kg once weekly (Q7D × 3). Mice received one IV injection of rituximab (3 mg/kg) once a week (D14 (day 0), 21 (day 7), and 28 (day 14): Q7D × 3). Tumor volumes were monitored and recorded on day 3, day 7, day 11, day 14, day 18 and day 21.

Detailed Description of the Invention

The invention provides an anti-CD20 type I antibody or an afucosylated anti-CD20 antibody of the IgG1 or IgG3 isotype, in which the fucose content is 60% or less of the total oligosaccharides (sugars) on Asn297, for the treatment of cancer in combination with a BTK inhibitor.

The invention provides the use of an anti-CD20 type I antibody or an afucosylated anti-CD20 antibody of the IgG1 or IgG3 isotype, in which the fucose content is 60% or less of the total oligosaccharides (sugars) on Asn297, for the preparation of a drug intended for the treatment of cancer in combinations with a BTK inhibitor.

According to one embodiment of the invention, the fucose content is from 40% to 60% of the total oligosaccharides (sugars) on Asn297.

The term "antibody" refers to various forms of antibodies, including, but not limited to, complete antibodies, human antibodies, humanized antibodies and genetically engineered antibodies such as monoclonal antibodies, chimeric antibodies or recombinant antibodies, as well as fragments of these antibodies if they retain the distinctive features of the antibodies proposed in the invention. The terms “monoclonal antibody” or “monoclonal antibody composition” as used herein refer to a preparation containing antibody molecules that have the same amino acid composition. Accordingly, the term “human monoclonal antibody” refers to antibodies having the same binding specificity that have variable and constant regions derived from human germline immunoglobulin sequences. According to one embodiment of the invention, human monoclonal antibodies are produced using a hybridoma that includes a B cell derived from a transgenic non-human animal, for example, a transgenic mouse, having a genome that contains a human heavy chain transgene and a human light chain transgene fused to an immortalized cell.

The term "chimeric antibody" refers to a monoclonal antibody containing a variable region, i.e. a binding region derived from one source or species, and at least a portion of a constant region derived from another source or species, which is typically produced by recombinant DNA techniques. Most preferred are chimeric antibodies containing a murine variable region and a human constant region. Such mouse/human chimeric antibodies are the product of expressed immunoglobulin genes containing DNA segments that encode murine immunoglobulin variable regions and DNA segments that encode human immunoglobulin constant regions. Other forms of "chimeric antibodies" within the scope of the present invention are antibodies in which the class or subclass is modified or altered from the parent antibody. Such “chimeric” antibodies are also called “class switched antibodies”. Methods for producing chimeric antibodies are based on conventional recombinant DNA and gene transfection techniques, and are now well known in the art (see, for example, Morrison S.L. et al., Proc. Natl. Acad Sci. USA 81, 1984, cc 6851-6855; US 5202238 and US 5204244).

The term “humanized antibody” refers to antibodies in which the framework region or “hypervariable regions” (CDRs) are modified such that they contain the CDRs of an immunoglobulin of a different specificity compared to the parent immunoglobulin. In a preferred embodiment of the invention, a mouse CDR is transplanted into a human antibody framework, resulting in a “humanized antibody” (see, for example, Riechmann L. et al., Nature 332, 1988, pp. 323-327; and Neuberger M.S. and al., Nature 314, 1985, pp. 268-270). The most preferred CDRs correspond to regions that are represented by sequences that recognize the antigens mentioned above for chimeric and bi- or multispecific antibodies.

As used herein, the term “human antibody” is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. Human antibodies are well known in the art (van Dijk M.A. and van de Winkel J.G., Curr. Opin. Chem. Biol. 5, 2001, pp. 368-374). Based on this technology, it is possible to obtain human antibodies to a wide variety of targets. Examples of human antibodies are described in Kellermann S.A. et al., Curr Opin Biotechnol. 13, 2002, pp. 593-597.

The term "recombinant human antibody" as used herein is intended to include all human antibodies that are produced, expressed, generated, or isolated by recombination techniques, such as antibodies isolated from a host cell, such as an NS0 or CHO cell, or from an animal (eg, from a mouse) that is transgenic for human immunoglobulin genes, or antibodies expressed using a recombinant expression vector that is transfected into a host cell. Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences in converted form. Recombinant human antibodies proposed in the invention were subjected to in vivo somatic hypermutation. Thus, the amino acid sequences of the VH and VL regions of a recombinant antibody are sequences that, although derived from and related to human germline VH and VL region sequences, may not occur in the naturally occurring in vivo germline spectrum. human antibody lines.

As used herein, the term “bi- or multispecific antibody” refers to monoclonal antibodies that have specificities that bind to at least two different sites. In some embodiments, one specificity binds to CD20 and the other to any other antigen. In some embodiments, bispecific antibodies can bind to two different CD20 epitopes. Bispecific antibodies can also be used to localize cytotoxic agents in cells that express CD20. Bispecific antibodies can be produced as full-length antibodies or antibody fragments.

As used herein, the term “binds” or “specifically binds” refers to an antibody that specifically binds to an epitope of a tumor antigen as determined by an in vitro assay, preferably by a (surface) plasmon resonance assay (BIAcore, Inc.). GE-Healthcare, Uppsala, Sweden) using purified wild-type antigen. Binding affinity is defined in terms of ka (rate constant of antibody association when forming an antibody/antigen complex), k _D (dissociation constant) and K _D (k _D /ka). Binding or specific binding is considered to have occurred if the binding affinity (K _D ) is 10 ^-8 M or less, preferably 10 ^-8 M to 10 ^-13 M (in one embodiment, 10 ^-9 M to 10 ^-13 M). Thus, the afucosylated antibody of the invention specifically binds to a tumor antigen, which is characterized by a binding affinity (K _D ) of 10 ^-8 mol/l, preferably from 10 ^-8 M to 10 ^-13 M (in one embodiment inventions from 10 ^-9 M to 10 ^-13 M).

The term "nucleic acid molecule" as used herein is intended to refer to DNA molecules and RNA molecules. The nucleic acid molecule can be single-stranded or double-stranded, but preferably it is double-stranded DNA.

"Constant domains" are not directly involved in antibody-antigen binding, but they are involved in effector functions (such as ADCC, complement fixation, and CDC).

The terms “variable region” (variable light chain (VL), variable heavy chain (VH)) as used herein refer to regions of each of a pair of light and heavy chains that are directly involved in the binding of an antibody to an antigen. The human light and heavy chain variable domains have the same general structure, and each domain contains four framework regions (FRs), the sequences of which are largely conserved, connected by three “hypervariable regions” (or complementarity determining regions, CDRs). The framework regions are adapted to a β-sheet conformation, and the CDRs can form loops connecting the β-sheet structure. CDRs in each chain maintain their three-dimensional structure with framework regions and, together with CDRs from the other chain, form an antigen-binding center.

The term “hypervariable region” as used herein refers to the amino acid residues of an antibody that are responsible for binding to an antigen. The hypervariable region contains amino acid residues from the “complementarity determining regions” or “CDRs”. "Framework" or "FR" regions are regions of the variable region other than the remnants of the hypervariable region as defined herein. Thus, the light and heavy chains of an antibody contain in the direction from N- to C-terminus the following regions: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The heavy chain CDR3 is the region that makes the major contribution to antigen binding. CDR and FR regions are defined according to the standard nomenclature of Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD., 1991) and/or as regions from the “hypervariable loop”.

The term "afucosylated antibody" refers to an antibody of the IgG1 or IgG3 isotype (preferably the IgG1 isotype) with an altered glycosylation pattern in the Fc region at position Asn297, which has a reduced number of fucose residues. Glycosylation of human IgG1 or IgG3 occurs at position Asn297, it is due to the presence of a core fucosylated biantennary complex oligosaccharide at the glycosylation site, having up to 2 Gal residues at the end. These structures are designated as G0-, G1- (α1,6 or α1,3) or G2-glycan residues depending on the number of terminal Gal residues (Raju T.S., BioProcess Int. 1, 2003, pp. 44-53). The CHO type of glycosylation of the Fc regions of antibodies is described, for example, in Routier F.H., Glycoconjugate J. 14, 1997, pp. 201-207. Antibodies produced by recombinant expression in CHO host cells lacking glycosylation modification (glycomodification) typically have a level of fucosylation at Asn297 of at least 85%. Obviously, in the context of the present description, the term afucosylated antibody also includes antibodies that do not have fucose in their glycosylation pattern. It is well known that the usual position of the glycosylated residue in an antibody corresponds to asparagine at position 297 according to the EU numbering system (“Asn297”).

The "EU numbering system" or "EU index" is typically used to designate the position of a residue in the immunoglobulin heavy chain constant region (for example, the EU index is described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed. ., Public Health Service, National Institutes of Health, Bethesda, MD., 1991, publication is incorporated herein by reference in its entirety).

Thus, the afucosylated antibody of the present invention is an antibody of the IgG1 or IgG3 isotype (preferably the IgG1 isotype) in which the fucose content is 60% or less of the total oligosaccharides (sugars) on Asn297 (meaning that at least 40% or more of the oligosaccharides on Asn297 in the Fc region are afucosylated). In one embodiment of the invention, the fucose content is from 40% to 60% of the amount of oligosaccharides on Asn297 in the Fc region. In another embodiment, the fucose content is 50% or less, and in a further embodiment, the fucose content is 30% or less of the amount of oligosaccharides on Asn297 in the Fc region. According to the invention, "fraction (amount) of fucose" means the amount of the specified oligosaccharide (fucose) in the oligosaccharide (sugar) chain on Asn297 relative to the sum of all oligosaccharides (sugars) attached to Asn297 (for example, complex, hybrid and high-mannose structures) according to MALDI-TOF mass spectrometry and calculated as an average value (a detailed method for determining the amount of fucose is described, for example, in WO 2008/077546). According to yet another embodiment of the invention, the Fc region oligosaccharides are bisectional. The afucosylated antibody of the invention can be expressed in a glycomodified host cell engineered to express at least one nucleic acid encoding a polypeptide that has GnTIII activity in an amount sufficient to partially fucosylate the oligosaccharides in the Fc region. According to one embodiment of the invention, the polypeptide having GnTIII activity is a fusion polypeptide. Alternatively, the α1,6-fucosyltransferase activity of the host cell can be reduced or eliminated according to the method described in US 6,946,292 to produce glycomodified host cells. The fucosylation level of an antibody can be determined, for example, either by changing fermentation conditions (eg, fermentation duration) or by combining at least two antibodies with different fucosylation levels. These afucosylated antibodies and corresponding glycoconstruction methods (glycosylation pattern design, glycoengineering) are described in WO 2005/044859, WO 2004/065540, WO 2007/031875, Umana R. et al., Nature Biotechnol. 17, 1999, pp. 176-180, WO 99/154342, WO 2005/018572, WO 2006/116260, WO 2006/114700, WO 2005/011735, WO 2005/027966, WO 97/028267, US 2006/0134709, US 2005/0054048, US 2005/0152894, WO 2003/035835, WO 2000/061739. These antibodies, obtained using glycoengineering methods, have increased ADCC. Other glycoengineering methods that produce afucosylated antibodies according to the invention are described, for example, in Niwa R. et al., J. Immunol. Methods 306, 2005, pp. 151-160; Shinkawa T. et al., J. Biol. Chem. 278, 2003, pp. 3466-3473; WO 03/055993 or US 2005/0249722.

Thus, one aspect of the invention is an anti-CD20 type I antibody or an afucosylated anti-CD20 antibody of the IgG1 or IgG3 isotype (preferably the IgG1 isotype) that specifically binds to CD20 in which the fucose content is 60% or less of the total oligosaccharides ( sugars) on Asn297, for the treatment of cancer in combination with a BTK inhibitor. Another aspect of the invention is the use of an afucosylated anti-CD20 antibody of the IgG1 or IgG3 isotype (preferably the IgG1 isotype) specifically binding to CD20, in which the fucose content is 60% or less of the total oligosaccharides (sugars) on Asn297, for the preparation of a medicament , intended for the treatment of cancer in combination with a BTK inhibitor. According to one embodiment of the invention, the fucose content is between 60% and 20% of the total oligosaccharides (sugars) on Asn297. According to one embodiment of the invention, the fucose content is from 60% to 40% of the total oligosaccharides (sugars) on Asn297. According to one embodiment of the invention, the fucose content is 0% of the total oligosaccharides (sugars) on Asn297.

CD20 (also called B-lymphocyte antigen CD20, B-lymphocyte surface antigen B1, Leu-16, BP35, BM5 and LF5; the sequence is presented in the SwissProt database under number P11836) is a hydrophobic transmembrane protein with a molecular weight of approximately 35 kDa, localized on pre-B and mature B lymphocytes (Valentine M.A. et al., J. Biol. Chem. 264, 1989, pp. 11282-11287; Tedder T.F. et al., Proc. Natl. Acad. Sci. U.S.A. 85, 1988, pp. 208-212; Stamenkovic I. et al., J. Exp. Med. 167, 1988, pp. 1975-1980; Einfeld D. A. et al., EMBO J. 7, 1988, pp. 711 -717; Tedder T.F. et al., J. Immunol. 142, 1989, cc. 2560-2568). The corresponding human gene is a four-transmembrane domain member of subfamily A 1, also known as MS4A1. This gene encodes a member of the 4A transmembrane gene family. Members of this new protein family share common structural features and similar intron/exon junctions (splicing boundaries) and have unique expression patterns for hematopoietic cells and nonlymphoid tissues. This gene encodes a B cell surface molecule that plays a role in the development and differentiation of B cells into plasma cells. This family member is located on 11q12 in a cluster of family members. Alternative splicing of this gene results in the formation of two transcriptional variants that encode the same protein.

The terms “CD20” and “CD20 antigen” are used interchangeably as used herein and include any variants, isoforms and species homologues of human CD20 that are naturally expressed by cells or expressed on cells transfected with the CD20 gene. Binding of the antibody of the invention to the CD20 antigen mediates the destruction of cells expressing CD20 (eg, a tumor cell) by inactivating CD20. Destruction of CD20-expressing cells can occur through one or more of the following mechanisms: induction of cell death/apoptosis, ADCC, and CDC.

Synonyms for CD20 in the art include B cell antigen CD20, B cell surface antigen B1, Leu-16, BP35, BM5, and LF5.

The term “anti-CD20 antibody” as used herein refers to an antibody that specifically binds to the CD20 antigen. Depending on the binding characteristics and biological activity of the CD20 antibody against the CD20 antigen, two types of CD20 antibodies (type I and type II CD20 antibodies) can be distinguished according to Cragg M.S. et al., Blood 103, 2004, pp. 2738-2743; and Cragg M.S. et al., Blood 101, 2003, pp. 1045-1052 (see table 1).

Examples of anti-CD20 type II antibodies are, for example, the humanized antibody B-Ly1 of the IgG1 isotype (a chimeric humanized antibody of the IgG1 isotype described in WO 2005/044859), 11 B8 IgG1 (described in WO 2004/035607) and AT80 IgG1. As a rule, antibodies to CD20 type II of the IgG1 isotype have characteristic CDC properties. Anti-CD20 type II antibodies have a reduced CDC (if they are of the IgG1 isotype) compared to type I antibodies of the IgG1 isotype.

Examples of anti-CD20 type I antibodies are, for example, rituximab, HI47 IgG3 (ECACC, hybridoma), 2C6 IgG1 (described in WO 2005/103081), 2F2 IgG1 (described in WO 2004/035607 and WO 2005/103081) and 2H7 IgG1 ( described in WO 2004/056312).

In one embodiment, the afucosylated anti-CD20 antibody of the invention is an anti-CD20 type II antibody, and in another embodiment, it is an afucosylated humanized B-Ly1 antibody.

The afucosylated anti-CD20 antibodies of the invention have increased antibody-mediated cell-dependent cytotoxicity (ADCC) compared to anti-CD20 antibodies that do not have reduced fucose content.

“Afucosylated anti-CD20 antibody with increased antibody-mediated cell-dependent cytotoxicity (ADCC)” refers to an afucosylated anti-CD20 antibody, as defined herein, having increased ADCC, as determined by any suitable method known in the art. One acceptable in vitro ADCC assay method is that:

1) the assay uses target cells that are known to express a target antigen recognized by the antigen-binding region of the antibody;

2) human peripheral blood mononuclear cells (PBMCs) isolated from the blood of a randomly selected healthy donor are used as effector cells in the analysis;

3) the analysis is carried out according to the following protocol:

I) PBMCs are isolated using standard density gradient centrifugation processes and suspended at 5 x 10 ⁶ cells/ml in RPMI cell culture medium;

ii) Grow target cells using standard tissue culture techniques, collect cells at exponential growth phase that are greater than 90% viability, wash in RPMI cell culture medium, label with ⁵¹ Cr (100 μCi), wash twice in cell culture medium and resuspended in cell culture medium at a density of 10 ⁵ cells/ml;

III) transfection is carried out using 100 μl of the above final target cell suspension for each well of a 96-well microtiter plate;

IV) carry out a serial dilution of the antibody from 4000 to 0.04 ng/ml in a cell culture medium and add 50 μl of the resulting solutions of the antibody to the target cells in a 96-well microtiter plate, evaluate the antibody in triplicate at different concentrations, covering the entire the range of concentrations indicated above;

V) to control maximum release (MR), add 50 μl of a 2% (VN) aqueous solution of nonionic surfactant (Nonidet, Sigma, St. Louis) to 3 additional wells in the plate containing labeled target cells. instead of an antibody solution (point IV, above);

VI) to control spontaneous release (SR), add 50 μl of RPMI cell culture medium instead of the antibody solution to 3 additional wells in the plate containing labeled target cells (point IV above);

vii) then the 96-well microtiter plate is centrifuged at 50 × g for 1 min and incubated for 1 h at 4°C;

VIII) add 50 μl of the PBMC suspension (step I above) to each well to ensure an effector cell:target cell ratio of 25:1, and the plates are placed in an incubator in an atmosphere containing 5% CO ₂ for 4 hours at 37° WITH;

ix) collect the cell-free supernatant from each well and quantify the experimentally released radioactivity (ER) using a gamma counter;

X) calculate the percentage of specific lysis for each antibody concentration using the formula (ER-MR)/(MR-SR) × 100, where ER represents the average radioactivity (see point IX above) determined for the specified antibody concentration, MR represents is the average radioactivity (see paragraph IX above) determined for MR controls (see paragraph V above), and SR is the average radioactivity (see paragraph IX above) determined for SR controls (see paragraph VI, above);

4) consider that there is an “increased ADCC level” if there is either an increase in the maximum percentage of specific lysis found in the above range of antibody concentrations, and/or a decrease in the antibody concentration required to achieve half of the maximum percentage of specific lysis found in the above range of antibody concentrations. The increase in ADCC level is determined relative to the ADCC level measured using the assay described above, mediated by the same antibody, produced using the same type of host cells, using the same standard purification, formulation and storage methods that are well known to those skilled in the art. , but which is not produced using host cells engineered to overexpress GnTIII.

Said “increased ADCC” can be obtained by modifying said antibodies by glycoengineering techniques, which means enhancing the naturally occurring cell-mediated effector functions of monoclonal antibodies by engineering their oligosaccharide component according to the method described in Umana, R., et al. Nature Biotechnol. 17, 1999, pp. 176-180 and in US 6602684.

The term “complement-dependent cytotoxicity (CDC)” refers to the lysis of human target tumor cells by the antibody of the invention in the presence of complement. CDC is preferably assessed by treating a preparation of CD20 expressing cells with the anti-CD20 antibody of the invention in the presence of complement. CDC is considered to be present if the antibody, when used at a concentration of 100 nM, induces lysis (cell death) of 20% or more tumor cells within 4 hours. The assay is preferably performed using ⁵¹ Cr or Eu labeled tumor cells and assessing the release of ⁵¹ Cr or Eu. As a control, target tumor cells are incubated with complement without antibody.

Rituximab (an example of an anti-CD20 type I antibody) is a genetically engineered chimeric human/mouse human gamma-1 constant region-containing monoclonal antibody to the human CD20 antigen.

Rituximab (an example of an anti-CD20 type I antibody) is a genetically engineered chimeric human/mouse human gamma-1 constant region-containing monoclonal antibody to the human CD20 antigen. This chimeric antibody contains human gamma-1 constant regions and is identified as "C2 B8" in US 5,736,137 (Anderson K.C. et al.), issued April 17, 1998 to IDEC Pharmaceuticals Corporation.

Rituximab is approved for the treatment of patients with relapsed or refractory low-grade or follicular, CD20-positive, B-cell non-Hodgkin lymphoma. In vitro studies of the mechanism of action have demonstrated that rituximab has human complement-dependent cytotoxicity (CDC) (Reff M.E. et al., Blood 83(2), 1994, pp. 435-445). In addition, it has significant activity in antibody-mediated cell-dependent cytotoxicity (ADCC) assays. Rituximab is non-fucosylated.

The term “humanized B-Ly1 antibody” refers to the humanized B-Ly1 antibody described in WO 2005/044859 and WO 2007/031875, which was prepared from the mouse anti-CD20 monoclonal antibody B-Ly1 (mouse heavy chain variable region (VH): SEQ ID NO: 1; murine light chain variable region (VL): SEQ ID NO: 2 - see Poppema S. and Visser L., Biotest Bulletin 3, 1987, pp. 131-139) by chimerization using the human constant region from IgG1 and subsequent humanization (see WO 2005/044859 and WO 2007/031875). These "humanized B-Ly1 antibodies" are described in detail in WO 2005/044859 and WO 2007/031875.

According to one embodiment of the invention, the “humanized B-Ly1 antibody” has a heavy chain variable region (VH) selected from SEQ ID NO: 3 to SEQ ID NO: 20 (B-HH2 - B-HH9 and B-HL8 -B- HL17, which are described in WO 2005/044859 and WO 2007/031875). In one specific embodiment, such a variable domain is selected from the group consisting of SEQ ID NOs: 3, 4, 7, 9, 11, 13 and 15 (B-HH2, B-HH3, B-HH6, B-HH8, B-HL8, B-HL11 and B-HL13, which are described in WO 2005/044859 and WO 2007/031875). In one particular embodiment, the “humanized B-Ly1 antibody” has a lung variable region (VL) as set forth in SEQ ID NO: 20 (B-KV1 in WO 2005/044859 and WO 2007/031875). In one specific embodiment of the invention, the “humanized B-Ly1 antibody” has a heavy chain variable region (VH) as set forth in SEQ ID NO: 7 (B-HH6 in WO 2005/044859 and WO 2007/031875) and has a variable the light chain region (VL), which is presented in SEQ ID NO: 20 (B-KV1 in WO 2005/044859 and WO 2007/031875). In addition, the humanized B-Ly1 antibody is preferably an antibody of the IgG1 isotype. These afucosylated humanized B-Ly1 antibodies of the invention are glycoengineered (GE) in the Fc region according to the methods described in WO 2005/044859, WO 2004/065540, WO 2007/031875, Umana R. et al. , Nature Biotechnol. 17, 1999, pp. 176-180 and WO 99/154342. In one embodiment, the afucosylated glycoengineered humanized B-Ly1 antibody is B-HH6-B-KV1 GE. In one embodiment, the anti-CD20 antibody is obinutuzumab (recommended international nonproprietary name (INN), WHO Drug Information, vol. 26, no. 4, 2012, p. 453). As used herein, obinutuzumab is synonymous with GA101. Trade name - GAZYVA. The WHO Drug Information document replaces all previous versions (e.g. Vol. 25, No. 1, 2011, pp. 75-76), and its previously known name is afutuzumab (recommended by INN, WHO Drug Information , v. 23, no. 2, 2009, p. 176; v. 22, no. 2, 2008, p. 124).

BTK as used herein means "Bruton's tyrosine kinase" or "Bruton's agammaglobulinemia mutated tyrosine kinase" (abbreviated Btk or BTK), which is a member of the TEC family of kinases. The BTK gene encodes the BTK protein, which is critical for B cell development and maturation, such as mast cell activation via the high-affinity IgE receptor. The BTK gene is localized on the X chromosome. More than 400 mutations have been identified in the BTK gene.

The term "BTK inhibitor" according to the invention refers to agents that interfere with the manifestation of BTK kinase activity, which is characterized by an IC ₅₀ value of from 0.001 μM to about 2 μM, in one embodiment of the invention from 0.002 μM to about 2 μM. In one embodiment of the invention, BTK inhibitors are antibodies, antisense oligonucleotides, peptides.

In another embodiment of the invention, BTK inhibitors are small molecule compounds with a molecular weight (MW) of less than 1500 Daltons (Da).

In one embodiment of the invention, said small molecule BTK inhibitors are compounds described in WO 2011/152351 and WO 2013/081016.

In one embodiment, the small molecule BTK inhibitor of the invention is characterized by the general formula (I)

In the formula, L stands for (1) -O-, (2) -S-, (3) -SO-, (4) -SO ₂ - (5) -NH-, (6) -C(O)- (7 ) -CH ₂ -O- (8) -O-CH ₂ - (9) -CH ₂ - or (10) -CH(OH)-;

R ¹ represents (1) a halogen atom, (2) a C ₁ -C ₄ alkyl group, (3) a C ₁ -C ₄ alkoxy group, (4) a C ₁ -C ₄ haloalkyl group, or (5) a C ₁ -C _{4 g} haloalkoxy group;

ring 1 denotes a 4-7 membered cyclic group which may be substituted by 1-5 substituents independently selected from the group consisting of (1) halogen atoms, (2) C ₁ -C ₄ alkyl groups, (3) C ₁ -C ₄ alkoxy groups, (4) nitrile, (5) C ₁ -C ₄ haloalkyl groups and (6) C ₁ -C ₄ haloalkoxy groups, and when two or more substituents are present on ring 1, then these substituents can form a 4-7 membered cyclic group in combination with the atoms in ring 1 to which these substituents are bonded;

ring 2 denotes a 4-7 membered saturated heterocycle, which can be substituted with 1-3 -KR ² groups;

K denotes (1) bond, (2) C ₁ -C ₄ alkylene, (3) -C(O)-, (4) -C(O)-CH ₂ -, (5) -CH ₂ -C(O )-, (6) -C(O)O- or (7) -SO ₂ - (in which the bond indicated on the left is connected to ring 2);

R ² denotes (1) C ₁ -C ₄ alkyl, (2) C ₂ -C ₄ alkenyl or (3) C ₂ -C ₄ alkynyl group, each of which may be substituted by 1-5 substituents, each of which is independently from each other selected from the group consisting of (1) NR ³ R ⁴ , (2) halogen atoms, (3) CONR ⁵ R ⁶ , (4) CO ₂ R ⁷ and (5) OR ⁸ ;

R ³ and R ⁴ each independently represent (1) a hydrogen atom or (2) a C ₁ -C ₄ alkyl group, which may be substituted by OR ⁹ or CONR ¹⁰ R ¹¹ ;

R ³ and R ⁴ together with the nitrogen atom to which they are connected can form a 4-7 membered nitrogen-containing saturated heterocycle, which can be replaced by an oxo group or a hydroxyl group;

R ⁵ and R ⁶ each independently represent (1) a hydrogen atom, (2) a C ₁ -C ₄ alkyl group, or (3) a phenyl group;

R ⁷ represents (1) a hydrogen atom or (2) a C ₁ -C ₄ alkyl group;

R ⁸ represents (1) a hydrogen atom, (2) a C ₁ -C ₄ alkyl group, (3) a phenyl group, or (4) a benzotriazolyl group;

R ⁹ represents (1) a hydrogen atom or (2) a C ₁ -C ₄ alkyl group; R ¹⁰ and R ¹¹ each independently represent (1) a hydrogen atom or (2) a C ₁ -C ₄ alkyl group;

n denotes an integer from 0 to 4; m is an integer from 0 to 2 and, when n is 2 or more, R ¹ ' may be the same or different from each other, an optical isomer of the compound or a mixture thereof, a salt, solvate, N-oxide or prodrug thereof;

[2] a compound as described in paragraph [1], in which R ² denotes a C ₂ -C ₄ alkenyl group or a C ₂ -C ₄ alkynyl group, each of which may be substituted by 1-5 substituents, each of which is independently from each other selected from the group consisting of (1) NR ³ R ⁴ , (2) halogen atoms, (3) CONR ⁵ R ⁶ , (4) CO ₂ R ⁷ and (5) OR ⁸ ;

[3] a compound as described in paragraph [1], wherein ring 1 is a benzene, cyclohexane or pyridine ring, each of which may be substituted with 1 to 5 substituents, each of which is independently selected from the group consisting of of (1) halogen atoms, (2) C ₁ -C ₄ alkyl groups, (3) C ₁ -C ₄ alkoxy groups, (4) nitrile and (5) CF ₃ ;

[4] a compound as described in paragraph [1], in which ring 2 is a 4-7 membered nitrogen-containing saturated heterocycle, which may be substituted by 1-3 KR ² groups;

[5] a compound as described in [4], wherein the 4-7 membered nitrogen-containing saturated heterocycle is an azetidine, pyrrolidine or piperidine ring;

[6] a compound falling under the same as described in paragraph [1], represented by the general formula (I-1)

In the formula, ring 1-1 is a benzene, cyclohexane or pyridine ring, each of which may be substituted with 1-5 substituents, each independently selected from the group consisting of (1) halogen atoms, (2) C ₁ -C ₄ alkyl groups, (3) C ₁ -C ₄ alkoxy groups, (4) nitrile and (5) CF ₃ , and ring 2-1 is a 4-7 membered nitrogen-containing saturated heterocycle that can be substituted with 1-3 groups -KR ² , where other characters have the meanings indicated above;

[7] a compound as described in paragraph [6], in which R ² denotes a C ₂ -C ₄ alkenyl group or a C ₂ -C ₄ alkynyl group, each of which may be substituted by 1-5 substituents, each of which is independently from each other selected from the group consisting of (1) NR ³ R ⁴ , (2) halogen atoms, (3) CONR ⁵ R ⁶ , (4) CO ₂ R ⁷ and (5) OR ⁸ ;

[8] a compound as described in paragraph [1], which is (1) 9-(1-acryloyl-3-azetidyl)-6-amino-7-(4-phenoxyphenyl)-7,9-dihydro- 8H-purin-8-one, (2) 6-amino-9-{(3R)-1-[(2E)-4-(dimethylamino)-2-butenoyl]-3-pyrrolidinyl}-7-(4- phenoxyphenyl)-7,9-dihydro-8H-purin-8-one, (3) 9-[(1-acryloyl-4-piperidinyl)methyl]-6-amino-7-(4-phenoxyphenyl)-7,9 -dihydro-8H-purin-8-one, (4) 6-amino-9-[(3R)-1-(2-butinoyl)-3-pyrrolidinyl]-7-(4-phenoxyphenyl)-7,9- dihydro-8H-purin-8-one, (5) 6-amino-9-{(3S)-1-[(2E)-4-(dimethylamino)-2-butenoyl]-3-pyrrolidinyl}-7-( 4-phenoxyphenyl)-7,9-dihydro-8H-purin-8-one, (6) 6-amino-7-[4-(3-chlorophenoxy)phenyl]-9-{(3R)-1-[( 2E)-4-(dimethylamino)-2-butenoyl]-3-pyrrolidinyl}-7,9-dihydro-8H-purin-8-one, (7) 6-amino-9-[1-(2-butinoyl) -3-pyrrolidinyl]-7-(4-phenoxyphenyl)-7,9-dihydro-8H-purin-8-one or (8) 6-amino-9-{1-[(2E)-4-(dimethylamino) -2-butenoyl]-3-pyrrolidinyl}-7-(4-phenoxyphenyl)-7,9-dihydro-8H-purin-8-one, or its isomer or mixtures thereof.

Preferably, the BTK inhibitor of the invention is 6-amino-9-[(3R)-1-(2-butinoyl)-3-pyrrolidinyl]-7-(4-phenoxyphenyl)-7,9-dihydro-8H- purine-8-one or its salt.

The preparation of the BTK inhibitor proposed in the invention is carried out according to the method described in WO 2011/152351.

The term "salt" refers to salts of compounds in the form of a pharmaceutically acceptable salt. Examples of these salts may be salts with alkali metals (potassium, sodium, etc.), salts of alkaline earth metals (calcium, magnesium, etc.), ammonium salt, salts with pharmaceutically acceptable organic amines (tetramethylammonium, triethylamine, methylamine, dimethylamine, cyclopentylamine, benzylamine, phenethylamine, piperidine, monoethanolamine, diethanolamine, tris(hydroxymethyl)aminomethane, lysine, arginine, N-methyl-D-glucamine, etc.), and acid addition salts (inorganic acid salts (hydrochloride, hydrobromide, hydroiodide, sulfate, phosphate, nitrate, etc.) and salts of organic acids (acetate, trifluoroacetate, lactate, tartrate, oxalate, fumarate, maleate, benzoate, citrate, methanesulfonate, ethanesulfonate, benzenesulfonate, toluenesulfonate, isethionate, glucuronate, gluconate, etc.)).

The term "IC ₅₀ " refers to the concentration of a particular compound required to inhibit 50% of a particular measured activity. The IC ₅₀ of BTK inhibitors that inhibit BTK kinase activity can be measured, among other things, according to the method described below.

In vitro activity assay to determine the IC ₅₀ of the BTK inhibitor of the invention:

Btk enzyme inhibitory activity was measured based on the protocol provided by the manufacturer using Btk (Invitrogen Corporation) and the Z'-LYTE™ -Tyr1-peptide kinase activity assay kit (Invitrogen Corporation) which contained the following reagents: Tur- 1-peptide, Thy-1-phosphopeptide, 5x kinase buffer, ATP, developing reagent B, developing buffer and stop reagent. 5 μl/well of BTK inhibitor solution diluted with dimethyl sulfoxide (DMSO), or DMSO alone, and 10 μl/well of substrate/enzyme mixture solution were added to a 96-well assay plate and reacted for 20 min at 30°C. A substrate/enzyme mixture solution was prepared by diluting with kinase buffer (DL-dithiothreitol (DTT, 2.7 mM), 1.33× kinase buffer) to obtain a final Tyr-1 peptide concentration of 4 μM and a final Btk concentration of 5 nM. Then 5 μl/well of adenosine triphosphate (ATP, final concentration 36 μM) was added and the reaction was carried out for 1 hour at 30°C. After completion of the reaction, 10 μl of developing solution, which was prepared by diluting developing reagent B 128 times using developing buffer, was added, and the reaction was carried out for another 1 hour at 30°C. The enzymatic reaction was then stopped by adding 10 μL of stop solution. The fluorescence intensity at 445 nm and 520 nm in each well was measured using a Fusion universal microplate analyzer (PerkinElmer Inc.), such as a fluorescence plate reader. The percentage of phosphorylation was determined using the ratio of emission at 445 nm (coumarin emission) and emission at 520 nm (fluorescein emission), according to the protocol supplied with the kit.

The percentage inhibition (%) by BTK inhibitor was calculated using the following equation:

Percentage (%) of phosphorylation inhibition =1 - {(AC - ACh)/(AC - AB)} × 100, where ACh denotes % phosphorylation when adding BTK inhibitor, AB denotes % phosphorylation without adding ATP (blank control), AC denotes % phosphorylation when adding only DMSO (control).

The 50% inhibition value (IC ₅₀ value) for the BTK inhibitor was determined from the inhibition curve based on the % inhibition at each concentration of the BTK inhibitor.

The oligosaccharide component can have a significant impact on properties relevant to the effectiveness of the therapeutic glycoprotein, including its physical stability, resistance to proteases, interactions with the immune system, pharmacokinetic parameters and specific biological activities. These properties may depend not only on the presence or absence of oligosaccharides, but also on their specific structures. Certain generalizations can be made regarding the relationship between oligosaccharide structure and glycoprotein function. For example, some oligosaccharide structures mediate rapid clearance of the glycoprotein from the bloodstream through interactions with specific carbohydrate-binding proteins, while others can be bound by antibodies and trigger unwanted immune responses (Jenkins N. et al., Nature Biotechnol. 14, 1996, pp. 975-981 ).

Mammalian cells are the preferred host for the production of therapeutic glycoproteins due to their ability to glycosylate proteins into the most suitable form for use in humans (Cumming D.A. et al., Glycobiology 1, 1991, pp. 115-130; Jenkins N. et al., Nature Biotechnol 14, 1996, pp. 975-981). Bacteria very rarely glycosylate proteins, and like other types of common hosts such as cells of yeast, filamentous fungi, insects and plants, provide glycosylation patterns associated with rapid clearance from the bloodstream, unwanted immune interactions and, in some specific cases, reduced biological activity. Among mammalian cells, Chinese hamster ovary (CHO) cells have found the most widespread use over the past two decades. In addition to providing acceptable glycosylation patterns, these cells enable the sustainable generation of genetically stable, high-yielding clonal cell lines. They can be cultured to high densities in simple bioreactors using serum-free media and can be used to develop safe and reproducible bioprocesses. Other commonly used animal cells are baby hamster kidney (BHK) cells, murine myeloma NS0 and SP2/0 cells. In recent years, the possibility of their production in transgenic animals has also been studied (Jenkins N. et al., Nature Biotechnol. 14, 1996, pp. 975-981).

All antibodies contain carbohydrate structures in conserved positions in the heavy chain constant regions, with each isotype characterized by a different organization of N-linked carbohydrate structures that have different effects on protein assembly, secretion and functional activity (Wright A. and Monison S.L., Trends Biotech. 15, 1997, pp. 26-32). The structure of the attached N-linked carbohydrate varies greatly depending on the degree of processing and can include high-mannose, multi-branched, and biantennary complex oligosaccharides (Wright A. and Morrison S.L., Trends Biotech. 15, 1997, pp. 26-32) . Typically, heterogeneous processing of core oligosaccharide structures attached at a specific glycosylation site occurs, resulting in even monoclonal antibodies existing in multiple glycoforms. It has also been found that there are large differences in the glycosylation of antibodies between cell lines, and even when a given cell line is grown under different culture conditions, there are small differences (Lifely M.R. et al., Glycobiology 5(8), 1995, pp. 813- 822).

One way to achieve significant increases in efficiency while maintaining a simple manufacturing process and avoiding significant unwanted side effects is to enhance the natural cell-mediated effector functions of monoclonal antibodies by engineering the oligosaccharide moiety according to the method described in Umana, R. et al., Nature Biotechnol., 17. 1999, pp. 176-180 and US 6,602,684. IgG1 antibodies, which are the antibodies most commonly used in cancer immunotherapy, are glycoproteins that have a conserved N-linked glycosylation site at position Asn297 in each CH2 domain. The two complex biantennary oligosaccharides that are attached to Asn297 are sandwiched between the CH2 domains, forming extensive contacts with the polypeptide backbone, and their presence is essential for the ability of the antibody to mediate effector functions such as antibody-mediated cell-dependent cytotoxicity (ADCC) (Lifely M.R. et al. ., Glycobiology, 5, 1995, pp. 813-822; Jefferis R. et al., Immunol. Rev., 163, 1998, pp. 59-76; Wright A. and Morrison S. L., Trends Biotechnol., 15, 1997 , pp. 26-32).

Previously, overexpression of β(1,4)-N-acetylglucosaminyltransferase III (“GnTIII”), a glycosyltransferase that catalyzes the formation of bisectional oligosaccharides, in Chinese hamster ovary (CHO) cells was found to significantly enhance the in vitro ADCC activity of an anti-neuroblastoma chimeric monoclonal antibody ( chCE7) produced by engineered CHO cells (see Umafia R. et al., Nature Biotechnol., 17, 1999, pp. 176-180; and WO 1999/54342, the entire contents of which are incorporated herein by reference). The chCE7 antibody belongs to a large class of unconjugated monoclonal antibodies that have high affinity and specificity for tumors, but are too low for clinical use when produced in standard commercial cell lines that lack the GnTIII enzyme (Umana R. et al ., Nature Biotechnol., 17, 1999, pp. 176-180). This study was the first to demonstrate that significant increases in ADCC activity can be achieved by engineering antibody-producing cells expressing GnTIII, which also results in an increase in the relative abundance of constant region (Fc)-associated bisectional oligosaccharides, including bisectional non-fucosylated oligosaccharides, compared to with levels characteristic of naturally occurring antibodies.

The term “cancer” as used herein refers to cancers such as lymphomas, lymphocytic leukemias, lung cancer, non-small cell lung cancer (NSCL), bronchoalveolar cell lung cancer, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, cutaneous or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, anal cancer, stomach cancer, gastric cancer, colon cancer, breast cancer, fallopian tube carcinoma, endometrial carcinoma, cervical carcinoma, vaginal carcinoma, vulvar carcinoma, disease Hodgkin's cancer, esophageal cancer, small intestinal cancer, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, bladder cancer, kidney or ureter cancer, renal cell carcinoma, renal pelvic carcinoma, mesothelioma, hepatocellular carcinoma, biliary cancer, central nervous system (CNS) neoplasms, spinal tumor, brainstem glioma, glioblastoma multiforme, astrocytomas, schwannomas, ependymomas, medulloblastomas, meningiomas, squamous cell carcinomas, adenoma pituitary gland, including resistant variants of any of the above cancers or a combination of one or more of the above cancers. According to one embodiment of the invention, the term cancer refers to cancer in which CD20 is expressed.

The term “CD20 antigen expression” is intended to refer to a significant level of CD20 antigen expression in a cell, preferably on the cell surface of a T or B cell, more preferably a B cell, of a tumor or cancer, respectively, preferably a tumor that is not solid tumors. Patients who have “CD20 expressing cancers” can be identified using standard assays known in the art. For example, CD20 antigen expression can be assessed by immunohistochemistry (IHC), FACS, or by PCR detection of the corresponding mRNA.

The term “CD20-expressing cancer” as used herein refers to all cancers in which the cancer cells express the CD20 antigen. In the context of the present description, cancers in which CD20 expression occurs are preferably lymphomas (preferably B-cell non-Hodgkin lymphomas (NHL)) and lymphocytic leukemias. These lymphomas and lymphocytic leukemias include, for example, a) follicular lymphomas, b) small cell nuclear lymphomas/Burkitt lymphoma (including endemic Burkitt lymphoma, sporadic Burkitt lymphoma and non-Burkitt lymphoma), c) marginal zone lymphomas (including extranodal B-cell lymphoma) marginal zone lymphoma (mucosal lymphoid tissue lymphoma, MALT), nodal B-cell marginal zone lymphoma and splenic marginal zone lymphoma), d) mantle cell lymphoma (MCL), e) large cell lymphoma (including B-cell diffuse large cell lymphoma (DLCL), diffuse mixed cell lymphoma, immunoblastic lymphoma, primary mediastinal B-cell lymphoma, angiocentric lymphoma-pulmonary B-cell lymphoma), f) hairy cell leukemia, g) lymphocytic lymphoma, Waldenström's macroglobulinemia, h) acute lymphoblastic leukemia (ALL ), chronic lymphocytic leukemia (CLL)/small cell lymphocytic leukemia (SLL), B-cell prolymphocytic leukemia, i) plasma cell neoplasms, plasma cell myelomas, multiple myeloma, plasmacytoma, j) Hodgkin's disease.

In one embodiment, the cancers that express CD20 are B-cell non-Hodgkin's lymphomas (NHL). In another embodiment, the cancers that express CD20 are mantle cell lymphoma (MCL), acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), B-cell diffuse large cell lymphoma (DLCL), Burkitt's lymphoma, hair cell leukemia, follicular lymphoma, multiple myeloma, marginal zone lymphoma, post-transplant lymphoproliferative disorder (PTLD), HIV-associated lymphoma, Waldenström macroglobulinemia, or primary CNS lymphoma.

The term "treatment", "treatment" or its equivalent, as applied to cancer, for example, refers to a procedure or course of treatment that is designed to reduce or eliminate a certain number of cancer cells in a patient's body or to alleviate the symptoms of cancer. A “treatment” for cancer or other proliferative disorder does not necessarily imply that the cancer cells or other disorder should actually be eliminated, that the number of cells or the level of the disorder should actually be reduced, or that other symptoms of the cancer or other disorder should actually be alleviated. Often a cancer treatment method can be carried out even with a small probability of success, but if, taking into account the patient's medical history and the assessed prospects for surgery, it is believed that it may nevertheless have an overall beneficial effect.

The terms "combination", "co-administration" or "co-use" refers to the use of said anti-CD20 type I antibody or said afucosylated anti-CD20 antibody and said BTK inhibitor in two different formulations (or in a single formulation). Co-administration can be simultaneous or sequential in any order, and there is preferably a period of time during which two active substances (or all active substances) simultaneously exert their biological effects. Said anti-CD20 type I antibody or said afucosylated anti-CD20 antibody and said BTK inhibitor are administered together either simultaneously or sequentially (e.g., intravenously (i.v.) by continuous infusion (one for the anti-CD20 antibody and then one for said BTK inhibitor; or e.g. the anti-CD20 antibody is administered intravenously (i.v.) by continuous infusion, and the BTK inhibitor is administered orally.) When both therapeutic agents are co-administered sequentially, the administered dose is administered either on the same day in two different administrations, or one of the agents is administered on day 1, and the second co-administered agent is administered on days 2 to 7, preferably days 2 to 4. Thus, “sequentially” means within 7 days after dosing of the first component (anti-CD20 antibody or BTK inhibitor), preferably on within 4 days after dosing of the first component; and the term “concurrently” means at the same time. The term “co-administration" with respect to maintenance doses of the specified anti-CD20 type I antibody or afucosylated anti-CD20 antibody and the specified BTK inhibitor means that maintenance doses can be co-administer simultaneously if the treatment cycle of both drugs is suitable, for example every week. Or the BTK inhibitor is administered, for example, every first to third day, and the specified afucosylated antibody is administered every week. Or maintenance doses are administered together sequentially, either on the same day or within several days.

Obviously, the antibodies are administered to the patient in a "therapeutically effective amount" (or simply "effective amount"), which is the amount of the corresponding compound or combination that produces the biological or medical response of the tissue, system, animal or person expected by the researcher, veterinarian, treating doctor or other clinician.

The amount used for co-administration of said anti-CD20 type I antibody or afucosylated anti-CD20 antibody and said BTK inhibitor and the time of co-administration will depend on the type (species, sex, age, weight, etc.) and condition of the patient being treated and the severity of the disease or condition being treated. The specified anti-CD20 type I antibody or afucosylated anti-CD20 antibody and the specified BTK inhibitor can be used for co-administration to a patient simultaneously or in a series of treatments, for example, on the same day or the next day.

If administration is intravenous, the duration of the initial infusion of said anti-CD20 type I antibody or afucosylated anti-CD20 antibody and said BTK inhibitor may be longer than the duration of subsequent infusions, for example, about 90 minutes for the initial infusion and about 30 minutes for subsequent infusions ( if the initial infusion was well tolerated).

Depending on the type and severity of the disease, the initial possible dose when both drugs are coadministered to a patient may be from about 1 mcg/kg to 50 mg/kg (e.g., 0.1 to 20 mg/kg) for the indicated anti-CD20 type I antibody or afucosylated anti-CD20 antibody; and from 1 μg/kg to 50 mg/kg (eg, 0.1-20 mg/kg) for said BTK inhibitor. In one embodiment, the preferred dose of said afucosylated anti-CD20 antibody (preferably the afucosylated humanized B-Ly1 antibody, more preferably obinutuzumab) is from about 0.05 mg/kg to about 30 mg/kg. Thus, one or more may be used for co-administration to a patient. several of the following doses: approximately 0.5, 2.0, 4.0, 10, or 30 mg/kg (or any combination thereof). According to one embodiment of the invention, the dose of said BTK inhibitor (preferably 6-amino-9-[(3R)-1-(2-butinoyl)-3-pyrrolidinyl]-7-(4-phenoxyphenyl)-7,9-dihydro- 8H-purin-8-one or salts thereof) should be from about 0.05 to about 30 mg/kg. Thus, one or more of the following doses may be used for coadministration to a patient: about 0.5, 2.0, 4.0, 10, or 30 mg/kg (or any combination thereof).

Depending on the type (species, sex, age, weight, etc.) and condition of the patient and on the type I CD20 antibody, preferably rituximab, or the type of afucosylated anti-CD20 antibody, the dose and schedule of administration of said afucosylated antibody, preferably an afucosylated humanized antibody B-Ly1, more preferably obinutuzumab, the dose and schedule of administration of said anti-CD20 type I antibody or said afucosylated anti-CD20 antibody may be different from that used for said BTK inhibitor. For example, an anti-CD20 type I antibody or said afucosylated anti-CD20 antibody can be administered, for example, every 1-3 weeks, and said BTK inhibitor can be administered daily or every 2-10 days. An initial higher loading dose may also be used, followed by one or more lower doses.

In one embodiment, the preferred dose of an anti-CD20 type I antibody (preferably rituximab) may be from about 100 to about 1000 mg/m ² per day 1, 8, 15, 22, 29, 36, 43, 50, 57 when administered 8 week dosing cycle.

In one embodiment, a preferred dose of said afucosylated anti-CD20 antibody (preferably an afucosylated humanized B-Ly1 antibody, more preferably obinutuzumab) may be 800 to 1600 mg (in one embodiment, 800 to 1200 mg) per day 1.8 15 over a 3-6 week dosing cycle, and then the dose may range from 400 to 1200 (in one embodiment, 800 to 1200 mg) per day 1 over up to nine 3-4 week dosing cycles.

In one embodiment, the dose of said BTK inhibitor selected from the group consisting of: (1) 9-(1-acryloyl-3-azetidyl)-6-amino-7-(4-phenoxyphenyl)-7,9-dihydro -8H-purin-8-one, (2) 6-amino-9-{(3R)-1-[(2E)-4-(dimethylamino)-2-butenoyl]-3-pyrrolidinyl}-7-(4 -phenoxyphenyl)-7,9-dihydro-8H-purin-8-one, (3) 9-[(1-acryloyl-4-piperidinyl)methyl]-6-amino-7-(4-phenoxyphenyl)-7, 9-dihydro-8H-purin-8-one, (4) 6-amino-9-[(3R)-1-(2-butinoyl)-3-pyrrolidinyl]-7-(4-phenoxyphenyl)-7,9 -dihydro-8H-purin-8-one, (5) 6-amino-9-{(3S)-1-[(2E)-4-(dimethylamino)-2-butenoyl]-3-pyrrolidinyl}-7- (4-phenoxyphenyl)-7,9-dihydro-8H-purin-8-one, (6) 6-amino-7-[4-(3-chlorophenoxy)phenyl]-9-{(3R)-1-[ (2E)-4-(dimethylamino)-2-butenoyl]-3-pyrrolidinyl}-7,9-dihydro-8H-purin-8-one, (7) 6-amino-9-[1-(2-butinoyl )-3-pyrrolidinyl]-7-(4-phenoxyphenyl)-7,9-dihydro-8H-purin-8-one and (8) 6-amino-9-{1-[(2E)-4-(dimethylamino )-2-butenoyl]-3-pyrrolidinyl}-7-(4-phenoxyphenyl)-7,9-dihydro-8H-purin-8-one, as follows. The dose of said BTK inhibitor, selected from the group of optical isomers of said compounds (1)-(8) and mixtures thereof, is as follows. Dose of BTK inhibitor 6-amino-9-[(3R)-1-(2-butinoyl)-3-pyrrolidinyl]-7-(4-phenoxyphenyl)-7,9-dihydro-8H-purin-8-one or its salts corresponds to those indicated below. The specified dose proposed in the invention is from 10 to 70 mg/kg, preferably from 20 to 55 mg/kg, which is administered daily or every other day by oral administration.

The recommended dose may vary depending on the additional coadministration of the chemotherapy agent and depending on the type of chemotherapy agent.

In one embodiment, the invention can be used to prevent or reduce metastases or their further spread in a patient suffering from cancer, preferably cancer in which CD20 is expressed. This invention can be used to prolong the life expectancy of the specified patient, prolong the life expectancy without disease progression in the specified patient, prolong the duration of response, which leads to a statistically significant and clinically noticeable improvement in the condition of the treated patient as measured by life expectancy, life expectancy without disease progression, level of response or duration of response. In a preferred embodiment, the invention can be used to improve response rates in a group of patients.

As used herein, additional other cytotoxic, chemotherapeutic, or anticancer agents, or compounds, or ionizing radiation that enhance the effectiveness of said agents (eg, cytokines) may be used for the treatment of cancer, in combination with an anti-CD20 type I antibody or an afucosylated anti-CD20 antibody and said BTK inhibitor for the treatment of cancer. These molecules must be present in combination in amounts effective for the intended purposes. According to one embodiment of the invention, a combination treatment using said anti-CD20 type I antibody, preferably rituximab, or said afucosylated anti-CD20 antibody, preferably an afucosylated humanized B-Ly1 antibody, more preferably obinutuzumab, and said BTK inhibitor is administered without said additional cytotoxic, chemotherapeutic or anticancer agents or compounds that enhance the effectiveness of said additional agents.

Said additional agents are, for example, alkylating agents or agents having alkylating activity, such as cyclophosphamide (CTX; e.g. Cytoxan®), chlorambucil (CHL; e.g. leukeran®), cisplatin (CisP; e.g. platinol®), busulfan (eg, myleran®), melphalan, carmustine (BCNU), streptozotocin, triethylenemelamine (TEM), mitomycin C, etc.; antimetabolites such as methotrexate (MTX), etoposide (VP16; e.g. vepesid®), 6-mercaptopurine (6MP), 6-thioguanine (6TG), cytarabine (Ara-C), 5-fluorouracil (5-FU), capecitabine (eg Xeloda®), dacarbazine (DTIC), etc.; antibiotics such as actinomycin D, doxorubicin (DXR; eg adriamycin®), daunorubicin (daunomycin), bleomycin, mithramycin and the like; alkaloids such as vinca alkaloids such as vincristine (VCR), vinblastine and the like; and other antineoplastic agents such as paclitaxel (eg taxol®) and paclitaxel derivatives, cytostatic agents, glucocorticoids such as dexamethasone (DEX; eg decadron®) and corticosteroids such as prednisone, nucleoside enzyme inhibitors such as hydroxyurea, amino acid depleters enzymes such as asparaginase, leucovorin and other folic acid derivatives, and similar other antitumor agents. The following agents may also be used as additional agents: arniphostin (eg, ethyol®), dactinomycin, mechlorethamine (nitrogen analogue of mustard gas), streptozocin, cyclophosphamide, lomustine (CCNU), doxorubicin lipo (eg, doxil®), gemcitabine (eg, gemzar®), daunorubicin lipo (e.g. daunoxome®), procarbazine, mitomycin, docetaxel (e.g. taxotere®), aldesleukin, carboplatin, oxaliplatin, cladribine, camptothecin, CPT 11 (irinotecan), 10-hydroxy-7-ethylcamptothecin (SN38 ), floxuridine, fludarabine, ifosfamide, idarubicin, mesna, interferon beta, interferon alpha, mitoxantrone, topotecan, leuprolide, megestrol, melphalan, mercaptopurine, plicamycin, mitotane, pegaspargase, pentostatin, pipobroman, plicamycin, tamoxifen, teniposide, testolactone, thioguanine, thiotepa, uracil analogue of mustard gas, vinorelbine, chlorambucil. In one embodiment of the invention, a combination therapy based on an afucosylated anti-CD20 antibody and said BTK inhibitor is used without said additional agents.

The use of cytotoxic and anticancer agents described above, as well as antiproliferative target-specific anticancer drugs such as protein kinase inhibitors, in chemotherapeutic regimens is generally well characterized in the field of cancer treatment, and is provided for in the context of the present description and similar provisions regarding monitoring should be taken into account when using them tolerance and effectiveness and control of routes of administration and doses with certain adjustments. For example, actual doses of cytotoxic agents may be modified depending on the response of the patient's cultured cells determined using histoculture-based methods. As a rule, doses should be lower compared to the amount of agent in which it is used in the absence of other additional agents.

Typical doses of an effective cytotoxic agent may be within the manufacturer's recommended ranges and, based on in vitro responses or responses obtained in animal models, the concentration or amount may be reduced by up to approximately one order of magnitude. Thus, the actual dose should depend on the recommendations of the treating physician, the patient's condition, and the effectiveness of the therapeutic modality as determined by the in vitro sensitivity of primary malignant cell cultures or histoculture tissue samples, or the responses found in appropriate animal models.

In the context of the present invention, in addition to the combination treatment of cancer in which CD20 expression occurs, based on an anti-CD20 type I antibody or an afucosylated anti-CD20 antibody and said BTK inhibitor, ionizing radiation and/or radiopharmaceuticals can be used in an effective amount (dose). The radiation source may be internal or external to the patient being treated. When the source is external to the patient, the therapy is called external beam radiation therapy (EBRT). When the radiation source is internal to the patient, the treatment is referred to as brachytherapy (BT). Radioactive atoms that can be used in accordance with the present invention can be selected from the group including, but not limited to, radium, cesium-137, iridium-192, americium-241, gold-198, cobalt-57, copper-67, technetium-99, iodine-123, iodine-131 and indium-111. It is also possible to label the antibody with appropriate radioactive isotopes. In one embodiment, a combination therapy of an anti-CD20 type I antibody or an afucosylated anti-CD20 antibody and said BTK inhibitor is administered without ionizing radiation.

Radiation therapy is the standard treatment for controlling unresectable or inoperable tumors and/or tumor metastases. Improved results were obtained when radiation therapy was combined with chemotherapy. Radiation therapy is based on the principle that a high dose of radiation delivered to the target area can lead to the death of reproductive cells in both the tumor and healthy tissue. The radiation regimen is typically defined in terms of absorbed radiation dose (Gy), time, and fractionation, and must be carefully selected by the oncologist. The amount of radiation a patient receives should depend on various circumstances, but the two most important are the location of the tumor relative to other critical structures or organs in the body and the extent of the tumor's spread. A typical course of treatment for a patient undergoing radiation therapy is a treatment regimen over a period of 1 to 6 weeks, with a total dose of 10 to 80 Gy administered to the patient in a single daily fractionated dose of 1.8 to 2 ,0 Gy, 5 days a week. In a preferred embodiment of the present invention, synergy occurs when tumors in human patients are subjected to the combination therapy of the invention and radiation. In other words, the inhibition of tumor growth by the agents included in the combination of the invention is enhanced when combined with radiation, optionally in combination with additional chemotherapeutic or anticancer agents. Parameters for adjuvant radiotherapy are described, for example, in WO 99/60023.

The anti-CD20 type I antibody or afucosylated anti-CD20 antibody is administered to a patient by known methods using intravenous bolus or continuous infusion over a period of time, via the intramuscular, intraperitoneal, spinal, subcutaneous, intra-articular, intrasynovial or intrathecal route. In one embodiment of the invention, the antibody is administered intravenously or subcutaneously.

The BTK inhibitor is administered to a patient by known methods using intravenous bolus or continuous infusion over a period of time, via the oral, intramuscular, intraperitoneal, spinal, subcutaneous, intra-articular, intrasynovial or intrathecal route. In one embodiment, the BTK inhibitor is administered intravenously or orally.

As used herein, the term "pharmaceutically acceptable carrier" includes any and all materials suitable for pharmaceutical administration, including solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonicizing and absorption retarding agents, and other materials and compounds that are suitable for pharmaceutical administration. Unless they are incompatible with the active substance, any conventional media or agents can be used in the compositions of the invention. The compositions can also include additional active ingredients.

Pharmaceutical compositions:

Pharmaceutical compositions can be prepared by treating the anti-CD20 antibody and/or BTK inhibitor of the invention with pharmaceutically acceptable inorganic or organic carriers. As carriers, for example, for tablets, coated tablets, dragees and hard gelatin capsules, lactose, corn starch or derivatives thereof, talc, stearic acids or salts thereof, and the like can be used. Suitable carriers for soft gelatin capsules are, for example, vegetable oils, waxes, fats, semi-solid and liquid polyols and the like. However, depending on the nature of the active substance, carriers are generally not required for soft gelatin capsules. Suitable carriers for the preparation of solutions and syrups include, for example, water, polyols, glycerin, vegetable oil, and the like. Acceptable carriers for suppositories include, for example, naturally occurring or hydrogenated oils, waxes, fats, semi-solid or liquid polyols and the like.

In addition, the pharmaceutical compositions may include preservatives, solubilizers, stabilizers, wetting agents, emulsifiers, sweeteners, colorants, flavoring agents, osmotic salts, buffers, masking agents, or antioxidants. They may also contain other therapeutically effective substances.

One embodiment of the invention is a composition comprising either said anti-CD20 type I antibody (preferably rituximab) or said afucosylated anti-CD20 antibody with a fucose content of 60% or less (preferably an afucosylated humanized B-Ly1 antibody, more preferably obinutuzumab), and and said BTK inhibitor, for use in the treatment of cancer, in particular cancer that expresses CD20 (preferably lymphoma or lymphocytic leukemia, more preferably B-cell non-Hodgkin lymphoma (NHL)), mantle cell lymphoma (MCL), acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), B-cell diffuse large cell lymphoma (DLCL), Burkitt's lymphoma, hairy cell leukemia, follicular lymphoma, multiple myeloma, marginal zone lymphoma, post-transplant lymphoproliferative disorder (PTLD), HIV-associated lymphoma , Waldenström macroglobulinemia or primary CNS lymphoma).

Said pharmaceutical composition may also contain one or more pharmaceutically acceptable carriers.

The present invention also relates to a pharmaceutical composition, which is used in particular in cancer, containing (I) as a first agent an anti-CD20 type I antibody (preferably rituximab) or an afucosylated anti-CD20 antibody with a fucose content of 60% or less (preferably an afucosylated humanized antibody B-Ly1), in an effective amount; and (II) a BTK inhibitor in an effective amount. Said composition optionally contains pharmaceutically acceptable carriers and/or excipients.

Pharmaceutical compositions containing only anti-CD20 type I antibody or afucosylated anti-CD20 antibody used in the present invention are prepared for storage by mixing the antibody having the required purity, optionally in combination with pharmaceutically acceptable carriers, excipients or stabilizers (Remington's Pharmaceutical Sciences, edited by Osol A.E., 16th ed., 1980), in the form of lyophilized formulations or aqueous solutions. Suitable carriers, excipients or stabilizers are non-toxic to recipients at the dosages and concentrations used and include buffers such as phosphate, citrate and other organic acid buffers; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkylparabens such as methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and meta-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates, including glucose, mannose or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions such as sodium; metal complexes (eg Zn-protein complexes); and/or nonionic surfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG).

Pharmaceutical compositions of BTK inhibitors may be similar to those described for anti-CD20 type I antibody or afucosylated anti-CD20 antibody.

Pharmaceutical compositions of a small molecule BTK inhibitor include compositions suitable for oral, nasal, topical (including buccal and sublingual), rectal, vaginal and/or parenteral administration. Conveniently, the compositions are unit dosages of the drug and can be prepared by methods well known in the art of pharmacy. The amount of active ingredient that can be combined with the carrier to form a unit dosage form may vary depending on the host being treated as well as the particular route of administration. The amount of active ingredient that can be combined with the carrier to form a unit dosage form will generally be the amount of BTK inhibitor that provides a therapeutic effect. Typically, when not 100%, the amount will be from about 1% to about 99% of the active ingredient, preferably from about 5% to about 70%, most preferably from about 10% to about 30%. Methods for preparing these compositions include the step of contacting the BTK inhibitor with a carrier and optionally one or more auxiliary ingredients. Typically, BTK inhibitor pharmaceutical compositions are prepared by uniformly and intimately contacting the BTK inhibitor with a liquid carrier or finely divided solid carriers, or both, and then, if necessary, shaping the product. Pharmaceutical compositions suitable for oral administration may take the form of capsules, cachets, sachets, pills, tablets, lozenges (prepared using a flavoring base, typically sucrose, gum arabic or tragacanth), powders, granules, or in the form of a solution or suspension in an aqueous or non-aqueous liquid, or in the form of an oil-in-water or water-in-oil liquid emulsion, or in the form of an elixir or syrup or lozenge (prepared using an inert base such as gelatin and glycerin, or sucrose and gum arabic), and/or mouthwash, etc., each form containing as an active ingredient a BTK inhibitor in a predetermined amount. The BTK inhibitor can be administered as a bolus, electuary or paste.

In one additional embodiment of the invention, an anti-CD20 type I antibody or an afucosylated anti-CD20 antibody and a BTK inhibitor are formulated as two different pharmaceutical compositions.

The active substances can be included in microcapsules obtained, for example, by coacervation or interfacial polymerization, for example, hydroxymethylcellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules, respectively, in colloidal systems for the administration of drugs (for example, liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or in a macroemulsion. These methods are described in Remington's Pharmaceutical Sciences, ed. Osol A.E., 16th ed., 1980.

Slow-release preparations can be prepared. Suitable examples of sustained release formulations are semi-permeable matrices of solid hydrophobic polymers containing the antibody, said matrices being in the form of articles of a specific configuration, such as films or microcapsules. Examples of sustained release matrices are polyesters, hydrogels (e.g. poly(2-hydroxyethyl methacrylate) or poly(vinyl alcohol)), polylactides (US 3773919), copolymers of L-glutamic acid and gamma-ethyl-L-glutamate, indegradable ethylene vinyl acetate, degradable lactic acid-glycolic acid copolymers such as LUPRON DEPOT™ (injectable microspheres consisting of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(-)-3-hydroxybutyric acid.

Formulations intended for administration in vivo must be sterile. For this purpose, filtration through sterile filter membranes can be used.

One embodiment of the invention is a pharmaceutical composition comprising a combination of an anti-CD20 type I antibody or a humanized B-Ly1 antibody that is afucosylated with a fucose content of 60% or less of the total oligosaccharides (sugars) on Asn297, and 6-amino- 9-[(3R)-1-(2-butinoyl)-3-pyrrolidinyl]-7-(4-phenoxyphenyl)-7,9-dihydro-8H-purin-8-one or its salts, intended for the treatment of cancer.

The present invention also relates to a method of treating cancer, comprising administering to a patient in need of such treatment (I) as a first agent, an anti-CD20 type I antibody or an afucosylated anti-CD20 antibody having a fucose content of 60% or less ( preferably an afucosylated humanized antibody B-Ly1), in an effective amount, and (ii) as a second agent, a BTK inhibitor in an effective amount.

In one embodiment, fucose is present in an amount of 40% to 60%.

Preferably, the cancer is a cancer that expresses CD20.

Preferably, the cancer in which CD20 expression occurs is a lymphoma or lymphocytic leukemia.

Preferably, the anti-CD20 type I antibody is rituximab.

Preferably, said afucosylated anti-CD20 antibody is an anti-CD20 type II antibody.

Preferably, said antibody is a humanized B-Ly1 antibody as provided herein.

Preferably, said antibody is obinutuzumab.

Preferably, said BTK inhibitor is selected from the group consisting of: (1) 9-(1-acryloyl-3-azetidyl)-6-amino-7-(4-phenoxyphenyl)-7,9-dihydro-8H-purine-8- she, (2) 6-amino-9-{(3R)-1-[(2E)-4-(dimethylamino)-2-butenoyl]-3-pyrrolidinyl}-7-(4-phenoxyphenyl)-7.9 -dihydro-8H-purin-8-one, (3) 9-[(1-acryloyl-4-piperidinyl)methyl]-6-amino-7-(4-phenoxyphenyl)-7,9-dihydro-8H-purine -8-one, (4) 6-amino-9-[(3R)-1-(2-butinoyl)-3-pyrrolidinyl]-7-(4-phenoxyphenyl)-7,9-dihydro-8H-purine- 8-one, (5) 6-amino-9-{(3S)-1-[(2E)-4-(dimethylamino)-2-butenoyl]-3-pyrrolidinyl}-7-(4-phenoxyphenyl)-7 ,9-dihydro-8H-purin-8-one, (6) 6-amino-7-[4-(3-chlorophenoxy)phenyl]-9-{(3R)-1-[(2E)-4-( dimethylamino)-2-butenoyl]-3-pyrrolidinyl}-7,9-dihydro-8H-purin-8-one, (7) 6-amino-9-[1-(2-butinoyl)-3-pyrrolidinyl]- 7-(4-phenoxyphenyl)-7,9-dihydro-8H-purin-8-one and (8) 6-amino-9-{1-[(2E)-4-(dimethylamino)-2-butenoyl]- 3-pyrrolidinyl}-7-(4-phenoxyphenyl)-7,9-dihydro-8H-purin-8-one. Preferably, said BTK inhibitor is also selected from the group of optical isomers of said compounds (1) to (8) and mixtures thereof.

Preferably, said BTK inhibitor is 6-amino-9-[(3R)-1-(2-butinoyl)-3-pyrrolidinyl]-7-(4-phenoxyphenyl)-7,9-dihydro-8H-purine-8- he or his salt.

Preferably, said salt is a hydrochloride.

Preferably, said anti-CD20 type I antibody is rituximab or said afucosylated anti-CD20 antibody is a humanized B-Ly1 antibody and said BTK inhibitor is selected from the group consisting of: (1) 9-(1-acryloyl-3-azetidyl)-6 -amino-7-(4-phenoxyphenyl)-7,9-dihydro-8H-purin-8-one, (2) 6-amino-9-{(3R)-1-[(2E)-4-(dimethylamino )-2-butenoyl]-3-pyrrolidinyl}-7-(4-phenoxyphenyl)-7,9-dihydro-8H-purin-8-one, (3) 9-[(1-acryloyl-4-piperidinyl)methyl ]-6-amino-7-(4-phenoxyphenyl)-7,9-dihydro-8H-purin-8-one, (4) 6-amino-9-[(3R)-1-(2-butinoyl)- 3-pyrrolidinyl]-7-(4-phenoxyphenyl)-7,9-dihydro-8H-purin-8-one, (5) 6-amino-9-{(3S)-1-[(2E)-4- (dimethylamino)-2-butenoyl]-3-pyrrolidinyl}-7-(4-phenoxyphenyl)-7,9-dihydro-8H-purin-8-one, (6) 6-amino-7-[4-(3 -chlorophenoxy)phenyl]-9-{(3R)-1-[(2E)-4-(dimethylamino)-2-butenoyl]-3-pyrrolidinyl}-7,9-dihydro-8H-purin-8-one, (7) 6-amino-9-[1-(2-butynoyl)-3-pyrrolidinyl]-7-(4-phenoxyphenyl)-7,9-dihydro-8H-purin-8-one and (8) 6- amino-9-{1-[(2E)-4-(dimethylamino)-2-butenoyl]-3-pyrrolidinyl}-7-(4-phenoxyphenyl)-7,9-dihydro-8H-purin-8-one. Preferably, said BTK inhibitor is also selected from the group of optical isomers of said compounds (1) to (8) and mixtures thereof. Preferably, said BTK inhibitor is 6-amino-9-[(3R)-1-(2-butinoyl)-3-pyrrolidinyl]-7-(4-phenoxyphenyl)-7,9-dihydro-8H-purine-8- it or a salt thereof, and said cancer is a cancer that expresses CD20, preferably lymphoma or lymphocytic leukemia.

As used herein, the term “patient” preferably refers to a person who is in need of treatment with an anti-CD20 type I antibody or an afucosylated anti-CD20 antibody (eg, a patient suffering from a cancer in which CD20 is expressed), for any purpose, and most preferably to a person who is in need of said treatment for cancer or a precancerous condition or lesion. However, the term “patient” can also refer to non-human animals, preferably mammals, among others, such as dogs, cats, horses, cows, pigs, sheep and non-human primates.

The present invention also provides an anti-CD20 type I antibody or an afucosylated anti-CD20 antibody with a fucose content of 60% or less and a BTK inhibitor for use in the treatment of cancer.

Preferably, said anti-CD20 type I antibody is rituximab.

Preferably, said afucosylated anti-CD20 antibody is a humanized B-Ly1 antibody.

Preferably, said afucosylated humanized B-Ly1 antibody is obinutuzumab.

Preferably, said BTK inhibitor is selected from the group consisting of: (1) 9-(1-acryloyl-3-azetidyl)-6-amino-7-(4-phenoxyphenyl)-7,9-dihydro-8H-purine-8- she, (2) 6-amino-9-{(3R)-1-[(2E)-4-(dimethylamino)-2-butenoyl]-3-pyrrolidinyl}-7-(4-phenoxyphenyl)-7.9 -dihydro-8H-purin-8-one, (3) 9-[(1-acryloyl-4-piperidinyl)methyl]-6-amino-7-(4-phenoxyphenyl)-7,9-dihydro-8H-purine -8-one, (4) 6-amino-9-[(3R)-1-(2-butinoyl)-3-pyrrolidinyl]-7-(4-phenoxyphenyl)-7,9-dihydro-8H-purine- 8-one, (5) 6-amino-9-{(3S)-1-[(2E)-4-(dimethylamino)-2-butenoyl]-3-pyrrolidinyl}-7-(4-phenoxyphenyl)-7 ,9-dihydro-8H-purin-8-one, (6) 6-amino-7-[4-(3-chlorophenoxy)phenyl]-9-{(3R)-1-[(2E)-4-( dimethylamino)-2-butenoyl]-3-pyrrolidinyl}-7,9-dihydro-8H-purin-8-one, (7) 6-amino-9-[1-(2-butinoyl)-3-pyrrolidinyl]- 7-(4-phenoxyphenyl)-7,9-dihydro-8H-purin-8-one and (8) 6-amino-9-{1-[(2E)-4-(dimethylamino)-2-butenoyl]- 3-pyrrolidinyl}-7-(4-phenoxyphenyl)-7,9-dihydro-8H-purin-8-one. Preferably, said BTK inhibitor is also selected from the group of optical isomers of said compounds (1) to (8) and mixtures thereof.

Preferably, said BTK inhibitor is 6-amino-9-[(3R)-1-(2-butinoyl)-3-pyrrolidinyl]-7-(4-phenoxyphenyl)-7,9-dihydro-8H-purine-8- he or his salt. The specified connection is also presented in the form of the following structure:

Preferably, said salt is a hydrochloride.

Preferably, said anti-CD20 type I antibody is rituximab or said afucosylated anti-CD20 antibody is a humanized B-Ly1 antibody, more preferably obinutuzumab, and said BTK inhibitor is selected from the group consisting of: (1) 9-(1-acryloyl-3 -azetidyl)-6-amino-7-(4-phenoxyphenyl)-7,9-dihydro-8H-purin-8-one, (2) 6-amino-9-{(3R)-1-[(2E) -4-(dimethylamino)-2-butenoyl]-3-pyrrolidinyl}-7-(4-phenoxyphenyl)-7,9-dihydro-8H-purin-8-one, (3) 9-[(1-acryloyl- 4-piperidinyl)methyl]-6-amino-7-(4-phenoxyphenyl)-7,9-dihydro-8H-purin-8-one, (4) 6-amino-9-[(3R)-1-( 2-butinoyl)-3-pyrrolidinyl]-7-(4-phenoxyphenyl)-7,9-dihydro-8H-purin-8-one, (5) 6-amino-9-{(3S)-1-[( 2E)-4-(dimethylamino)-2-butenoyl]-3-pyrrolidinyl}-7-(4-phenoxyphenyl)-7,9-dihydro-8H-purin-8-one, (6) 6-amino-7- [4-(3-chlorophenoxy)phenyl]-9-{(3R)-1-[(2E)-4-(dimethylamino)-2-butenoyl]-3-pyrrolidinyl}-7,9-dihydro-8H-purine -8-one, (7) 6-amino-9-[1-(2-butinoyl)-3-pyrrolidinyl]-7-(4-phenoxyphenyl)-7,9-dihydro-8H-purin-8-one and (8) 6-amino-9-{1-[(2E)-4-(dimethylamino)-2-butenoyl]-3-pyrrolidinyl}-7-(4-phenoxyphenyl)-7,9-dihydro-8H-purine -8-she. Preferably, said BTK inhibitor is also selected from the group of optical isomers of said compounds (1) to (8) and mixtures thereof. Preferably, said BTK inhibitor is 6-amino-9-[(3R)-1-(2-butinoyl)-3-pyrrolidinyl]-7-(4-phenoxyphenyl)-7,9-dihydro-8H-purine-8- it or a salt thereof, and said cancer is a cancer that expresses CD20, preferably lymphoma or lymphocytic leukemia.

The following examples, sequence listing and drawings are presented for the purpose of explaining the present invention, the true scope of which is set forth in the appended claims. It is obvious that modifications can be made to the presented procedures without departing from the spirit of the invention.

Description of the sequence list:

SEQ ID NO: 1 is the amino acid sequence of the heavy chain variable region (VH) of the mouse anti-CD20 monoclonal antibody B-Ly1.

SEQ ID NO: 2 is the amino acid sequence of the light chain variable region (VL) of the mouse anti-CD20 monoclonal antibody B-Ly1.

SEQ ID NO: 3-19 - amino acid sequences of the heavy chain variable region (VH) of humanized antibodies B-Ly1 (B-HH2 - B-HH9, B-HL8 and B-HL10 - B-HL17).

SEQ ID NO: 20 - amino acid sequences of the light chain variable region (VL) of the humanized antibody B-Ly1 B-KV1.

Experimental procedures

Example 1:

Study of the antitumor efficacy of a BTK inhibitor in combination with an anti-CD20 antibody in SCID mice with subcutaneous xenografts of the TMD8 cell line (ABC-DLBCL)

1. Materials and methods

1.1. Substances tested

Test substance, 6-amino-9-[(3R)-1-(2-butinoyl)-3-pyrrolidinyl]-7-(4-phenoxyphenyl)-7,9-dihydro-8H-purin-8-one hydrochloride ( referred to below in the context of this description as “compound A”), were stored in sealed containers at ambient temperature, protected from light.

GA101 (obinutuzumab, marketed as Gazyva® or Gazyvaro®), 500 mg, rituximab (RTX, marketed as Rituxan® and MabThera®), 200 mg, obtained from Roche Glycart AG and stored at 4 °C.

1.2. Filler for substances

Compound A was weighed and suspended in a solution of 0.5% (w/v) 400 cP methylcellulose solution (Wako Pure Chemical Industries, Ltd., referred to herein below as "0.5% MS"). using a mortar and pestle to obtain a suspension with a concentration of 1 mg/ml. The suspension was used within 7 days after preparation. The suspension was confirmed to be stable and homogeneous for 7 days when stored in a refrigerator, protected from light, and for an additional 24 hours at ambient temperature when exposed to indoor lighting.

Stock solutions of both GA101 (25 mg/ml) and rituximab (10 mg/ml) were maintained at 4°C. Before administration to mice, stock solutions were diluted with 0.9% NaCl to obtain concentrations of 0.3, 1, and 3 mg/ml, which were used in the experiment for assessing doses, and 0.1 and 0.3 mg/ml, which were used for studying combinations .

1.3. Route of drug administration

Compound A was administered to mice orally (PO) twice daily by oral lavage using a disposable oral feeding needle (Fuchigami Kikai Co.) at a dose of 10 ml/kg. The GA101/rituximab mixture was injected intravenously (IV) at a dose of 10 ml/kg into the caudal vein of mice. For all treatments, the volumes used for administration were adapted to the body weight of each mouse measured at the time point closest to the time of administration. The group that received b.i.d. Compound A and the Compound A vehicle and 0.5% MS group, respectively, were treated in the morning and afternoon at intervals of 8 hours or more.

2. Animals

We used female mice of the C.B-17/1cr-SCID/SCIDJcl line (Clea Japan Inc., which were 6 weeks old at the beginning of the experiments; hereinafter in the context of this description they are designated as “Scid mice”). Mice were housed in metal cages at 3-5 mice per cage and allowed to acclimate for approximately 1 week before inclusion in the experiment. While in the applicants' animal facility, the animals had free access to solid food CRF-1 (Oriental Yeast Co., Ltd.) and tap water (from an automatic dispenser). The excreta were treated using an automatic cleaning system. The general condition of the animals was examined upon receipt and at the end of the acclimatization period. The experiment included animals that did not have problems with their general condition. Animals were kept in rooms with controlled temperature: 24 ± 2°C, humidity: 55 ± 15%, ventilation: 15 ± 5 cycles/h with 100% fresh outside air, lighting conditions: 12 h dark/light cycles with fluorescent lamps. (light: from 08:00 to 20:00).

3. Cancer cell lines and culture conditions

The TMD8 line was created from cells from a patient with B-cell diffuse large cell lymphoma (Tohda S. et al., Leuk Res., 30, 2006, pp. 1385-1390). RPMI 1640 medium (Invitrogen Corporation, hereinafter referred to as "RPMI") containing 10 vol.% inactivated FBS and 1 vol.% penicillin-streptomycin liquid (Invitrogen Corporation) was prepared and used as the medium. . The medium was stored at 5°C (acceptable range 1°C to 9°C). 150-mm dishes containing medium were inoculated with the cell suspension at a rate of 4 × 10 ⁶ to 2.0 × 10 ⁷ cells per dish. The plates were incubated at 37°C, 5% CO ₂ , 95% air. Cell numbers were counted using a hemocytometer and cell viability was at least 90% on the day of cell injection.

4. Cell inoculation

On day 0, cell debris was suspended in RPMI, pre-chilled on ice, to obtain a cell suspension containing 2×10 ⁸ cells/ml. The cell suspension in RPMI and an equal volume of BD Matrigel GFR (BD Biosciences) were combined to obtain a cell suspension containing 1 x 10 ⁸ cells/ml. It was used as a cell suspension for implantation. Then, 0.1 ml of the cell suspension was injected subcutaneously into the right flank of the mice under pentobarbital anesthesia using a 25-gauge needle (Terumo Corporation).

5. Processing scheme

When the average size of the tumors reached 100-200 mm ³ and 300-400 mm ³ , mice were randomly divided into groups of 8-10 individuals based on individual tumor sizes and treatment began. The mean tumor volume in each group did not differ from the tumor volume in the other groups (analysis of mean deviations).

6. Monitoring the condition of animals bearing xenografts

After implantation of TMD8, the general condition of all animals was recorded every day, measuring the volume of tumors.

7. Measuring tumor diameters

The long axis and short axis of the tumors were measured using an electronic caliper (Mitutoyo Corporation) every 2 or 4 days after TMD8 implantation until the day of final assessment.

8. Endpoints and assessment methods

The primary endpoint was tumor volume on the day of final assessment. In addition, the degree of tumor growth inhibition (TGI, %) in the drug-treated groups compared with the vehicle-treated group was calculated. Change over time in tumor volume and body weight from day 0 were used as secondary endpoints. Tumor volume was calculated using the following formula.

Tumor volume = long axis of tumor x (short axis of tumor) ² × 0.5. Individual tumor volumes were calculated to the first decimal place.

Finally, the mice were sacrificed and necropsy was performed when the tumors reached a maximum of 3000 mm ³ .

9. Form of presentation and data processing

9.1. Data presentation form

Tumor volume and body weight in each group were expressed as mean ± square error (S.E.). TGI(%) in treated groups was calculated to the first decimal place.

9.2. Statistical analysis

Microsoft Office Excel 2007 SP-1 was used to calculate means and square errors and included in tables and graphs. To evaluate statistical criteria, we used the EXSUS system, version 7.7.1 (CAC Corporation), based on SAS 9.2 TS2M3 (SAS Institute Japan). Student's t test was used to compare tumor volumes between GA101 monotherapy and combination therapy with GA101 and Compound A. All statistical tests were 2-sided and the significance level was 5%.

10. Results

Description of experimental groups.

Figure 1 shows the results of an experiment to evaluate the antitumor activity of Compound A in combination with GA101 in a TMD8 xenotransplantation model. At D14, when the average volume of tumors reached 400-450 mm ³ , mice were randomly distributed into 7 groups of 10 mice each depending on the volume of tumors. As shown in FIG. 1, GA101 monotherapy had antitumor effects. However, in the groups receiving combination therapy, the effect was more pronounced compared with the corresponding monotherapy. Statistically significant differences were found on days 14, 17, 21 and 24. At D35 (day 21), 4 mice in the vehicle-treated group were sacrificed because their tumor volumes exceeded 3000 mm ³ . All mice were sacrificed at D38 (day 24).

In this experiment, it was found that the combination of GA101-compound A significantly exceeded the activity of the indicated monotherapy (statistical analysis is presented below).

When creating the invention, a regression of tumor volumes was unexpectedly found in some individuals b D 14 and 21 in the groups treated with GA101 at a dose of 3 mg/kg in combination with compound A at a dose of 10 mg/kg, while in the group that was subjected to monotherapy , complete remission of tumors was not detected. These results clearly demonstrate that combination therapy may have superior efficacy in an advanced tumor model.

Example 2:

The experiment described in Example 2 was carried out according to the method described in Example 1, except as specified for the “substance excipient” and “processing scheme”.

1. 1. Filler for substances

Compound A was weighed and suspended in a solution of 0.5% (w/v) 400 cP methylcellulose solution (Wako Pure Chemical Industries, Ltd., referred to herein below as "0.5% MS"). using a mortar and pestle to obtain a suspension with a concentration of 1 mg/ml (10 mg/kg). The suspension was used within 7 days after preparation. The suspension was confirmed to be stable and homogeneous for 7 days when stored in a refrigerator, protected from light, and for an additional 24 hours at ambient temperature when exposed to indoor lighting.

Stock solutions of both GA101 (25 mg/ml) and rituximab (10 mg/ml) were maintained at 4°C. Before administration to mice, the stock solutions were diluted with 0.9% NaCl to obtain the concentration of 0.3 mg/ml (3 mg/kg) that was used in this experiment.

1.2. Processing scheme

When the average size of the tumors reached 400-450 mm ³ , mice were randomly divided into 6 groups based on individual tumor sizes and treatment began. The mean tumor volume in each group differed from the tumor volume in the other groups (analysis of mean deviations).

2. Results

Description of experimental groups.

In fig. 2 demonstrates the results of an experiment to evaluate the antitumor activity of compound A in combination with GA101/RTX in a model obtained by TMD8 xenotransplantation. At D14 (day 0), when the average volume of tumors reached 400-450 mm ³ , mice were randomly divided into 6 groups of 10 mice each, depending on the volume of tumors. As shown in FIG. 2, each monotherapy had an antitumor effect. Statistically significant differences were found after day 4 for GA101 and RTX and after day 11 for Compound A. Additionally, treatment with Compound A in combination with GA101 or RTX at a suboptimal dose of 3 mg/kg once weekly resulted in significant growth inhibition tumors in a model obtained using a TMD8 xenograft. Statistically significant differences were also found between the monotherapy and combination therapy groups using Compound A, indicating that the combination of Compound A with GA101 or RTX, when used at a suboptimal dose, had significantly higher activity than the corresponding Ab or Compound A at their use as monotherapy. At D35 (day 21), 3 mice from the vehicle group were sacrificed because their tumor volumes exceeded 3000 mm ³ . All mice were sacrificed on D39 (day 25).

Tumor growth inhibition (TGI) data on days 0, 4, 7, 11, 14, 18, and 21 in each group are presented below. GA101-based monotherapy had superior antitumor activity compared to RTX-based monotherapy in terms of tumor growth inhibition (TGI, 77.9% vs. 54.3% at day 21).

Tumor growth inhibition (TGI).

Tumor diameters were measured and tumor volumes were calculated every 3 or 4 days after group evaluation. Tumor growth inhibition (TGI) data in the drug-treated group compared with the volume of tumors in the vehicle-treated group on each day measured was calculated and expressed to the first decimal place.

Example 3:

The experiment described in Example 3 was carried out according to the method described in Example 1, except for the specified “test substance”, “route of administration” and “processing scheme”.

1.1 Test substance

The reference substance, ibrutinib (hereinafter referred to as “compound B”), was stored in sealed containers at ambient temperature, protected from light.

1.2 Route of administration of drugs

Mice were fed a commercial diet (CRF1, Oriental Yeast Co., Ltd.) supplemented with either 0.0037 wt. % (included in pellets) of compound A, or 0.012 wt. % (included in pellets) of compound B, respectively. The dose of compound A or compound B corresponded to a daily intake of 6 or 20 mg/kg body weight. These calculations were based on the fact that the average daily feed intake ranged from 160 to 180 mg/g body weight. 1.3 Processing scheme

Two groups (vehicle and RTX): mice were kept on regular chow (CRF1). Drug groups: Mice were fed the same diet containing Compound A or Compound B after randomization. Rituximab was administered intravenously at a dose of 3 mg/kg once a week.

2. Results

Description of experimental groups.

In fig. Figure 3 demonstrates the results of an experiment to evaluate the antitumor activity of compound A or compound B in combination with RTX in a model obtained by xenotransplantation of TMD8. At D14 (day 0), when the average tumor volume reached 450 mm ³ , mice were randomly assigned to 6 groups of 9 mice each depending on tumor volume. As shown in FIG. 3, each monotherapy had an antitumor effect. Statistically significant differences were found after day 7 for RTX and Compound A and after day 11 for Compound B. No statistically significant differences were found between either. Thus, the antitumor activity of compound A (0.0037%) turned out to be comparable to the activity of compound B (0.012%). Importantly, Compound A in combination with RTX was more effective than the corresponding monotherapies in the TMD8 xenograft model. Statistically significant differences were also found between monotherapy and combination therapy. However, no statistically significant differences were found when Compound B was used with RTX and the corresponding monotherapy, indicating that Compound A in combination with RTX has a synergistic effect.

Tumor growth inhibition (TGI) data on days 0, 3, 7, 11, 14, 18 and 21 in each group are presented below. Compound A monotherapy had superior antitumor activity compared to RTX monotherapy in terms of tumor growth inhibition (TGI, 91.1% vs. 64.2% at day 21, p<0.001).

Tumor diameters were measured and tumor volumes were calculated every 3 or 4 days after group evaluation. Tumor growth inhibition (TGI) data in the drug-treated group compared with the volume of tumors in the vehicle-treated group on each day measured was calculated and expressed to the first decimal place.

Conclusion

Based on the results reported in Example 1, Example 2 and Example 3, it is apparent that the use of a combination of Compound A with GA101 or RTX may be an effective treatment for ABC-DLBCL. Compound B has been shown to antagonize RTX-dependent NK cell-mediated cytotoxicity (see Kohrt NE et al., Blood, vol. 123, 2014, pp. 1957-1960). However, it should be noted that compound A in combination with RTX or GA101 is more effective than compound B in combination with RTX in this model obtained using DLBCL xenotransplantation, which suggests the possibility of using this combination (compound A + RTX or GA101 ) in clinical practice.

The following sequences indicated in the present description, as well as in the attached sequence listing, are part of the present invention:

Sequences

SEQ ID NO: 1

Amino acid sequence of the variable heavy chain (VH) region of the mouse anti-CD20 monoclonal antibody B-Ly1

Gly Pro Glu Leu Val Lys Pro Gly Ala Ser Val Lys Ile Ser Cys Lys

Ala Ser Gly Tyr Ala Phe Ser Tyr Ser Trp Met Asn Trp Val Lys Leu

Arg Pro Gly Gln Gly Leu Glu Trp ILe Gly Arg Ile Phe Pro Gly Asp

Gly Asp Thr Asp Tyr Asn Gly Lys Phe Lys Gly Lys Ala Thr Leu Thr

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

Ser Val Asp Ser Ala Val Tyr Leu Cys Ala Arg Asn Val Phe Asp Gly

Tyr Trp Leu Val Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ala

SEQ ID NO: 2

Amino acid sequence of the variable light chain (VL) region of the mouse anti-CD20 monoclonal antibody B-Ly1

Asn Pro Val Thr Leu Gly Thr Ser Ala Ser Ile Ser Cys Arg Ser Ser

Lys Ser Leu Leu His Ser Asn Gly Ile Thr Tyr Leu Tyr Trp Tyr Leu

Gln Lys Pro Gly Gln Ser Pro Gln Leu Leu Ile Tyr Gln Met Ser Asn

Leu Val Ser Gly Val Pro Asp Arg Phe Ser Ser Ser Gly Ser Gly Thr

Asp Phe Thr Leu Arg Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val

Tyr Tyr Cys Ala Gln Asn Leu Glu Leu Pro Tyr Thr Phe Gly Gly Gly

Thr Lys Leu Glu Ile Lys Arg

SEQ ID NO: 3

Amino acid sequence of the heavy chain variable region (VH) of the humanized antibody B-Ly1 (B-HH2)

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Tyr Ser

Trp Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

Gly Arg Ile Phe Pro Gly Asp Gly Asp Thr Asp Tyr Asn Gly Lys Phe

Lys Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

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

Ala Arg Asn Val Phe Asp Gly Tyr Trp Leu Val Tyr Trp Gly Gln Gly

Thr Leu Val Thr Val Ser Ser

SEQ ID NP: 4

Amino acid sequence of the heavy chain variable region (VH) of the humanized antibody B-Ly1 (B-HH3)

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Tyr Ser

Trp Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

Gly Arg Ile Phe Pro Gly Asp Gly Asp Thr Asp Tyr Asn Gly Lys Phe

Lys Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

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

Ala Arg Asn Val Phe Asp Gly Tyr Trp Leu Val Tyr Trp Gly Gln Gly

Thr Leu Val Thr Val Ser Ser

SEQ ID NP: 5

Amino acid sequence of the heavy chain variable region (VH) of the humanized antibody B-Ly1 (B-HH4)

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

Ser Val Lys Val Ser Cys Lys Val Ser Gly Tyr Ala Phe Ser Tyr Ser

Trp Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

Gly Arg Il Phe Pro Gly Asp Gly Asp Thr Asp Tyr Asn Gly Lys Phe

Lys Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

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

Ala Arg Asn Val Phe Asp Gly Tyr Trp Leu Val Tyr Trp Gly Gln Gly

Thr Leu Val Thr Val Ser Ser

SEQ ID NO: 6

Amino acid sequence of the heavy chain variable region (VH) of the humanized antibody B-Ly1 (B-HH5)

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Tyr Ser

Trp Met Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

Gly Arg Ile Phe Pro Gly Asp Gly Asp Thr Asp Tyr Asn Gly Lys Phe

Lys Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

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

Ala Arg Asn Val Phe Asp Gly Tyr Trp Leu Val Tyr Trp Gly Gln Gly

Thr Leu Val Thr Val Ser Ser

SEQ ID NO: 7

Amino acid sequence of the heavy chain variable region (VH) of the humanized antibody B-Ly1 (B-HH6)

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Tyr Ser

Trp Ile Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

Gly Arg Ile Phe Pro Gly Asp Gly Asp Thr Asp Tyr Asn Gly Lys Phe

Lys Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

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

Ala Arg Asn Val Phe Asp Gly Tyr Trp Leu Val Tyr Trp Gly Gln Gly

Thr Leu Val Thr Val Ser Ser

SEQ ID NO: 8

Amino acid sequence of the heavy chain variable region (VH) of the humanized antibody B-Ly1 (B-HH7)

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Tyr Ser

Trp Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

Gly Arg Ile Phe Pro Gly Asp Gly Asp Thr Asp Tyr Asn Gly Lys Phe

Lys Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

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

Ala Arg Asn Val Phe Asp Gly Tyr Trp Leu Val Tyr Trp Gly Gln Gly

Thr Leu Val Thr Val Ser Ser

SEQ ID NO: 9

Amino acid sequence of the heavy chain variable region (VH) of the humanized antibody B-Ly1 (B-HH8)

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Tyr Ser

Trp Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

Gly Arg Ile Phe Pro Gly Asp Gly Asp Thr Asp Tyr Asn Gly Lys Phe

Lys Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

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

Ala Arg Asn Val Phe Asp Gly Tyr Trp Leu Val Tyr Trp Gly Gln Gly

Thr Leu Val Thr Val Ser Ser

SEQ ID NO: 10

Amino acid sequence of the heavy chain variable region (VH) of the humanized antibody B-Ly1 (B-HH9)

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Tyr Ser

Trp Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

Gly Arg Ile Phe Pro Gly Asp Gly Asp Thr Asp Tyr Asn Gly Lys Phe

Lys Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

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

Ala Arg Asn Val Phe Asp Gly Tyr Trp Leu Val Tyr Trp Gly Gln Gly

Thr Leu Val Thr Val Ser Ser

SEQ ID NP: 11

Amino acid sequence of the heavy chain variable region (VH) of the humanized antibody B-Ly1 (B-HL8)

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Tyr Ser

Trp Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

Gly Arg Ile Phe Pro Gly Asp Gly Asp Thr Asp Tyr Asn Gly Lys Phe

Lys Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

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

Ala Arg Asn Val Phe Asp Gly Tyr Trp Leu Val Tyr Trp Gly Gln Gly

Thr Leu Val Thr Val Ser Ser

SEQ ID NP: 12

Amino acid sequence of the heavy chain variable region (VH) of the humanized antibody B-Ly1 (B-HL10)

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ala Phe Ser Tyr Ser

Trp Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

Gly Arg Ile Phe Pro Gly Asp Gly Asp Thr Asp Tyr Asn Gly Lys Phe

Lys Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

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

Ala Arg Asn Val Phe Asp Gly Tyr Trp Leu Val Tyr Trp Gly Gln Gly

Thr Leu Val Thr Val Ser Ser

SEQ ID NO: 13

Amino acid sequence of the variable heavy chain (VH) region of the humanized antibody B-Ly1 (B-HL11)

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Tyr Ser

Trp Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

Gly Arg Ile Phe Pro Gly Asp Gly Asp Thr Asp Tyr Asn Gly Lys Phe

Lys Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

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

Ala Arg Asn Val Phe Asp Gly Tyr Trp Leu Val Tyr Trp Gly Gln Gly

Thr Leu Val Thr Val Ser Ser

SEQ ID NO: 14

Amino acid sequence of the variable heavy chain (VH) region of the humanized antibody B-Ly1 (B-HL12)

Glu Val Gln Leu Val Glu Ser Gly Ala Gly Leu Val Lys Pro Gly Gly

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Tyr Ser

Trp Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Met

Gly Arg Ile Phe Pro Gly Asp Gly Asp Thr Asp Tyr Asn Gly Lys Phe

Lys Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

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

Ala Arg Asn Val Phe Asp Gly Tyr Trp Leu Val Tyr Trp Gly Gln Gly

Thr Leu Val Thr Val Ser Ser

SEQ ID NO: 15

Amino acid sequence of the variable heavy chain (VH) region of the humanized antibody B-Ly1 (B-HL13)

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Lys Pro Gly Gly

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Tyr Ser

Trp Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Met

Gly Arg Ile Phe Pro Gly Asp Gly Asp Thr Asp Tyr Asn Gly Lys Phe

Lys Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

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

Ala Arg Asn Val Phe Asp Gly Tyr Trp Leu Val Tyr Trp Gly Gln Gly

Thr Leu Val Thr Val Ser Ser

SEQ ID NO: 16

Amino acid sequence of the heavy chain variable region (VH) of the humanized antibody B-Ly1 (B-HL14)

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Lys Lys Pro Gly Gly

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Tyr Ser

Trp Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Met

Gly Arg Ile Phe Pro Gly Asp Gly Asp Thr Asp Tyr Asn Gly Lys Phe

Lys Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

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

Ala Arg Asn Val Phe Asp Gly Tyr Trp Leu Val Tyr Trp Gly Gln Gly

Thr Leu Val Thr Val Ser Ser

SEQ ID NO: 17

Amino acid sequence of the variable heavy chain (VH) region of the humanized antibody B-Ly1 (B-HL15)

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Ser

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Tyr Ser

Trp Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Met

Gly Arg Ile Phe Pro Gly Asp Gly Asp Thr Asp Tyr Asn Gly Lys Phe

Lys Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

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

Ala Arg Asn Val Phe Asp Gly Tyr Trp Leu Val Tyr Trp Gly Gln Gly

Thr Leu Val Thr Val Ser Ser

SEQ ID NO: 18

Amino acid sequence of the heavy chain variable region (VH) of the humanized antibody B-Ly1 (B-HL16)

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

Ser Leu Arg Val Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Tyr Ser

Trp Met Asn Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Met

Gly Arg Ile Phe Pro Gly Asp Gly Asp Thr Asp Tyr Asn Gly Lys Phe

Lys Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

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

Ala Arg Asn Val Phe Asp Gly Tyr Trp Leu Val Tyr Trp Gly Gln Gly

Thr Leu Val Thr Val Ser Ser

SEQ ID NP: 19

Amino acid sequence of the heavy chain variable region (VH) of the humanized antibody B-Ly1 (B-HL17)

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Tyr Ser

Trp Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Met

Gly Arg Ile Phe Pro Gly Asp Gly Asp Thr Asp Tyr Asn Gly Lys Phe

Lys Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

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

Ala Arg Asn Val Phe Asp Gly Tyr Trp Leu Val Tyr Trp Gly Gln Gly

Thr Leu Val Thr Val Ser Ser

SEQ ID NO: 20

Amino acid sequence of the variable light chain (VL) region of the humanized antibody B-Ly1 B-KV1

Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Thr Pro Gly

Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Lys Ser Leu Leu His Ser

Asn Gly Ile Thr Tyr Leu Tyr Trp Tyr Leu Gln Lys Pro Gly Gin Ser

Pro Gln Leu Leu Ile Tyr Gln Met Ser Asn Leu Val Ser Gly Val Pro

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ala Gln Asn

Leu Glu Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

Arg Thr Val

Claims (5)

1. Pharmaceutical combination for the treatment of cancer in which CD20 expression occurs, consisting of rituximab and a BTK inhibitor, which is 6-amino-9-[(3R)-1-(2-butynoyl)-3-pyrrolidinyl]-7-( 4-phenoxyphenyl)-7,9-dihydro-8H-purin-8-one or salts thereof, in a synergistically effective amount, wherein the amount of rituximab is from 0.1 to 50 mg/kg and the amount of 6-amino-9-[ (3R)-1-(2-butinoyl)-3-pyrrolidinyl]-7-(4-phenoxyphenyl)-7,9-dihydro-8H-purin-8-one or its salt is from 1 μg/kg to 50 mg /kg. 2. The pharmaceutical combination according to claim 1, characterized in that said cancer in which CD20 is expressed is a lymphoma or lymphocytic leukemia. 3. Use of the pharmaceutical combination according to claim 1 for the preparation of a medicinal product intended for the treatment of cancer in which CD20 expression occurs. 4. Use according to claim 3, characterized in that the cancer in which CD20 is expressed is a lymphoma or lymphocytic leukemia. 5. Use according to claim 3, characterized in that one or more additional other cytotoxic, chemotherapeutic, or anticancer agents or compounds, or ionizing radiation are used, which increases the effect of these agents.

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