Combinations of Anti-CD19 antibodies and bruton's tyrosine kinase inhibitors and uses thereof

Abstract

To provide pharmaceutical combinations of anti-CD19 antibodies and Bruton's tyrosine kinase (BTK) for treating non-Hodgkin lymphoma, chronic lymphocytic leukemia and / or acute lymphoblastic leukemia.SOLUTION: Disclosed is a synergistic combination of a CD19-specific antibody and a Bruton's tyrosine kinase (BTK) inhibitor, where the antibody comprises an HCDR1 region of sequence SYVMH (SEQ ID NO:1), an HCDR2 region of sequence NPYNDG (SEQ ID NO:2), an HCDR3 region of sequence GTYYYGTRVFDY (SEQ ID NO:3), an LCDR1 region of sequence RSSKSLQNVNGNTYLY (SEQ ID NO:4), an LCDR2 region of sequence RMSNLNS (SEQ ID NO:5), and an LCDR3 region of sequence MQHLEYPIT (SEQ ID NO:6), where the combination is used for treating non-Hodgkin lymphoma, chronic lymphocytic leukemia and / or acute lymphoblastic leukemia.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present disclosure relates to pharmaceutical combinations of anti-CD19 antibodies and Bruton's tyrosine kinase (BTK) inhibitors for the treatment of non-Hodgkin's lymphoma, chronic lymphocytic leukemia and / or acute lymphoblastic leukemia. [Background technology]

[0002] B cells are lymphocytes that play a major role in humoral immune responses. B cells are produced in the bone marrow of most mammals and represent 5-15% of the circulating lymphocyte pool. The main function of B cells is to make antibodies against various antigens, making them an essential component of the adaptive immune system.

[0003] Due to their critical role in regulating the immune system, dysregulation of B cells is associated with a variety of disorders such as lymphomas and leukemias, including non-Hodgkin's lymphoma (NHL), chronic lymphocytic leukemia (CLL) and acute lymphoblastic leukemia (ALL).

[0004] NHL is a heterogeneous malignant tumor arising from lymphocytes. The incidence in the United States (US) is estimated at 65,000 cases per year, with approximately 20,000 deaths (American Cancer Society, 2006; and SEER Cancer Statistics Review). The disease can occur at any age, with onset usually beginning in adults over 40 years of age, and incidence increasing with age. NHL is characterized by the clonal proliferation of lymphocytes that accumulate in lymph nodes, blood, bone marrow, and spleen, although any major organ may be involved. The current classification system used by pathologists and clinicians is the World Health Organization Classification of Tumours, which groups NHL into precursor and mature B-cell or mature T-cell neoplasms. PDQ currently classifies NHL as indolent or aggressive for inclusion in clinical trials. The low-grade NHL group consists mainly of follicular subtype, small lymphocytic lymphoma, MALT (mucosa-associated lymphoid tissue) and marginal zone, with low-grade encompassing approximately 50% of newly diagnosed B-cell NHL patients. High-grade NHL includes patients with histologically diagnosed diffuse large B-cell (DLBL, DLBCL or DLCL) (40% of all newly diagnosed patients have diffuse large cell), Burkitt cell and mantle cell. The clinical course of NHL is highly variable. The major determinant of clinical course is the histological subtype. Most low-grade NHLs are considered incurable diseases. Patients respond initially to either chemotherapy or antibody therapy, but the majority relapse. Studies to date have not demonstrated improved survival with early intervention. In asymptomatic patients, "watch and wait" is acceptable until the patient becomes symptomatic or the rate of disease progression appears to be accelerating. The disease may transform to more aggressive histology over time. Median survival is 8-10 years, and patients with indolent disease often undergo three or more lines of therapy during the course of their disease. Initial treatment for symptomatic indolent NHL patients has historically been combination chemotherapy.The most commonly used drugs include cyclophosphamide, vincristine, and prednisone (CVP); or cyclophosphamide, adriamycin, vincristine, and prednisone (CHOP). Approximately 70%-80% of patients respond to these initial chemotherapy regimens, with periods of remission lasting approximately 2-3 years. The majority of patients eventually relapse. The discovery and clinical use of the anti-CD20 antibody, rituximab, has significantly improved response and survival rates. The current standard of care for most patients is rituximab plus CHOP (R-CHOP) or rituximab plus CVP (R-CVP). Interferon has been approved for initial treatment of NHL in combination with alkylating agents, but its use is limited in the United States. Rituximab therapy has been shown to be effective in several types of NHL and is currently approved as first-line treatment for both low-grade (follicular lymphoma) and high-grade NHL (diffuse large B-cell lymphoma). However, anti-CD20 monoclonal antibodies (mAbs) have significant limitations, including primary resistance (50% response in relapsed low-grade patients), acquired resistance (50% response rate upon retreatment), rare complete responses (2% complete response rate in the relapsed population), and continued relapse patterns. Finally, many B cells do not express CD20, and therefore many B-cell disorders cannot be treated with anti-CD20 antibody therapy.

[0005] In addition to NHL, there are several types of leukemia that result from dysregulation of B cells. Chronic lymphocytic leukemia (also called "chronic lymphocytic leukemia" or "CLL") is a type of adult leukemia caused by abnormal accumulation of B lymphocytes. In CLL, malignant lymphocytes may appear normal and mature, but are unable to fight infections efficiently. CLL is the most common form of leukemia in adults. Men are twice as likely to develop CLL than women. However, a significant risk factor is age. More than 75% of new cases are diagnosed in patients over 50 years of age. More than 10,000 cases are diagnosed each year, with almost 5,000 deaths annually (American Cancer Society, 2006; and SEER Cancer Statistics Review). CLL is an incurable disease, but it progresses slowly in most cases. Many people with CLL lead normal, active lives for many years. Early CLL is generally not treated due to its slow onset, as early intervention is not believed to improve survival time or quality of life. Instead, the condition is monitored over time. Initial CLL treatment varies depending on the exact diagnosis and progression of the disease. There are dozens of drugs used in CLL therapy. Combination chemotherapy regimens, such as FCR (Fludarabine, Cyclophosphamide, and Rituximab), and BR (Ibrutinib and Rituximab), are effective for both newly diagnosed and relapsed CLL. Allogeneic bone marrow (stem cell) transplantation is rarely used as a first-line treatment for CLL due to its risks.

[0006] Another type of leukemia is acute lymphoblastic leukemia (ALL), also called acute lymphocytic leukemia. ALL is characterized by the overproduction and continuous proliferation of malignant immature white blood cells (also called lymphoblasts) in the bone marrow. "Acute" refers to the undifferentiated, immature state of the circulating lymphocytes ("blasts") and the rapid progression of the disease, with a life expectancy of only a few weeks to a few months if left untreated. ALL is most common in childhood, with a peak incidence between 4 and 5 years of age. Children aged 12 to 16 years are more likely to die from ALL than children of other ages. Currently, at least 80% of childhood ALL is considered curable. Fewer than 4,000 cases are diagnosed each year, with nearly 1,500 deaths annually (American Cancer Society, 2006; and SEER Cancer Statistics Review).

[0007] The human CD19 molecule is a structurally distinct cell surface receptor expressed on the surface of human B cells, including, but not limited to, pre-B cells, early developmental B cells (i.e., immature B cells), mature B cells that undergo terminal differentiation into plasma cells, and malignant B cells. CD19 is expressed on most pre-B acute lymphoblastic leukemias (ALL), non-Hodgkin's lymphomas, B-cell chronic lymphocytic leukemia (CLL), prolymphocytic leukemia, hairy cell leukemia, conventional acute lymphocytic leukemia, and some null cell acute lymphoblastic leukemias (Nadler et al, J. Immunol., 131:244-250 (1983), Loken et al, Blood, 70:1316-1324 (1987), Uckun et al, Blood, 71:13-29 (1988), Anderson et al, 1984. Blood, 63:1424-1433 (1984), Scheuermann, Leuk. Lymphoma, 18:385-397 (1995)). Expression of CD19 on plasma cells further suggests that CD19 can be expressed on differentiated B cell tumors such as multiple myeloma, plasmacytoma, and Waldenstrom's tumor (Grossbard et al., Br. J. Haematol, 102:509-15 (1998); Treon et al, Semin. Oncol, 30:248-52 (2003)).

[0008] Thus, the CD19 antigen is an immunotherapeutic target in the treatment of non-Hodgkin's lymphoma (including each of the subtypes described herein), chronic lymphocytic leukemia, and / or acute lymphoblastic leukemia.

[0009] Several CD19 therapies have been demonstrated. T cells expressing anti-CD19 chimeric antigen receptors (CARs) containing both CD3-zeta and 4-BB costimulatory domains were administered to three patients with advanced CLL. Kalos et al., T cells with Chimeric Antigen Receptors Have Potent Antitumor Effects and Can Establish Memory in Patients with Advanced Leukemia, Science Translational Medicine, vol. 3, no. 95 (10 August 2011), which is incorporated by reference in its entirety. Sadelain et al., The promise and potential pitfalls of chimeric antigen receptors, Current Opinion in Immunology, Elsevier, vol. 21, no. 2, 2 April 2009, which is incorporated by reference in its entirety, also describe anti-CD19 chimeric antigen receptors (CARs). However, neither Kalos et al. nor Sadelain et al. describe antibodies specific for CD19 in combination with Bruton's tyrosine kinase (BTK) inhibitors as exemplified herein.

[0010] The use of CD19 antibodies in non-specific B cell lymphoma is discussed in WO2007076950 (US2007154473), both of which are incorporated by reference in their entireties, with a cursory mention of ibrutinib in a long list of possible combination partners, but does not teach the antibodies exemplified herein, nor does it suggest the synergistic effects of the combination in the treatment of non-Hodgkin's lymphoma, chronic lymphocytic leukemia and / or acute lymphoblastic leukemia as exemplified herein.

[0011] The use of CD19 antibodies in CLL, NHL and ALL is described in Scheuermann et al., CD19 Antigen in Leukemia and Lymphoma Diagnosis and Immunotherapy, Leukemia and Lymphoma, Vol. 18, 385-397 (1995), which is incorporated by reference in its entirety, but does not suggest the combinations exemplified herein.

[0012] Additional antibodies specific for CD19 are described in WO 2005012493 (U.S. Patent No. 7,109,304), WO 2010053716 (U.S. Patent Application No. 12 / 266,999) (Immunomedics); WO 2007002223 (U.S. Patent No. 8,097,703) (Medarex); WO 2007002223 (U.S. Patent No. 8,097,703) (Medarex); No. 2008022152 (U.S. Patent Application No. 12 / 377,251) and WO 2008150494 (Xencor), WO 2008031056 (U.S. Patent Application No. 11 / 852,106) (Medimmune); WO 2007076950 (U.S. Patent Application No. 11 / 648,505) (Merck Patent GmbH); WO 2009 / 052431 (U.S. Ser. No. 12 / 253,895) (Seattle Genetics); and WO 2010095031 (U.S. Ser. No. 12 / 710,442) (Glenmark Pharmaceuticals), WO 2012010562 and WO 2012010561 (International Drug Development), WO 2011147834 (Roche Glycart), and WO 2012 / 156455 (Sanofi).

[0013] Combinations of antibodies specific for CD19 and other drugs are described in WO 2010151341 (U.S. Ser. No. 13 / 377,514) (The Feinstein Institute); U.S. Pat. No. 5,686,072 (University of Texas), as well as WO 2002022212 (Int. Appl. PCT / U.S. Ser. No. 01 / 29026) (IDEC Pharmaceuticals), WO 2013 / 024097 (U.S. Ser. No. 14 / 126,928) (MorphoSys AG) and WO 2013 / 024095 (U.S. Ser. No. 14 / 127,217) (MorphoSys AG), which are incorporated by reference in their entireties.

[0014] Several Bruton's tyrosine kinase inhibitors are available on the market. Ibrutinib, also known as PCI-32765 and sold under the trade name Imbruvica, is an anticancer drug that targets B-cell malignancies. Ibrutinib is described in U.S. Patent Nos. 7,514,444; 8,008,309; 8,697,711; 8,735,403; 8,957,079; and 8,754,090, all of which are incorporated by reference in their entirety.

[0015] Ibrutinib has been tested in combination with rituximab (an anti-CD20 antibody). Burger et al., Lancet Oncol., 2014 September, 15(19):1090-1099. Ibrutinib potentially inhibits the ADCC activity of certain CD20 antibodies in vitro. Duong et al., mAbs, Jan / Feb 2015, 192-198, and Kohrt et al., Blood, 2014, 123:1957-1960.

[0016] Despite recent advances in the discovery and development of anti-cancer drugs, it is clear that many forms of cancer associated with CD19-expressing tumors still have poor prognoses. Thus, there is a need for improved methods for treating such forms of cancer. Summary of the Invention

[0017] The prior art does not suggest a synergistic effect of the combination of the exemplified antibodies and ibrutinib, either alone or in combination, in the treatment of non-Hodgkin's lymphoma, chronic lymphocytic leukemia and / or acute lymphoblastic leukemia.

[0018] In one aspect, the present disclosure relates to a synergistic combination of an antibody specific for CD19 and a Bruton's tyrosine kinase (BTK) inhibitor, which is useful for the treatment of B-cell malignancies, such as non-Hodgkin's lymphoma, chronic lymphocytic leukemia, and / or acute lymphoblastic leukemia.

[0019] In vitro models are believed to be suggestive of how a compound or combination of compounds will behave in humans.

[0020] This in vitro model of MEC-1 cells suggests how this combination may work in treating human chronic lymphocytic leukemia (CLL). This in vitro model of Ramos cells suggests how this combination may work in treating human non-Hodgkin's lymphoma (NHL). MEC-1 cells (DSMZ# ACC497) are a chronic B-cell leukemia cell line. Ramos cells (ATCC number CRL-1596) are human Burkitt's lymphoma cells.

[0021] Moreover, when compounds are combined in vitro, it is expected that the combination will only have additive effects.Surprisingly, the inventors have found that the combination of a specific antibody specific for CD19 and ibrutinib is involved in synergistic levels of specific cell killing in vitro compared to antibody and ibrutinib alone.Specifically, the inventors have found that the combination of MOR00208 and ibrutinib is involved in synergistic levels of specific cell killing in vitro in MEC-1 cells compared to antibody and ibrutinib alone.

[0022] Additionally and even more unexpectedly, the inventors have found that the combination of a particular antibody specific for CD19 and ibrutinib has certain functional properties compared to the antibody and ibrutinib alone.

[0023] In summary, the combination of the exemplified anti-CD19 antibody and ibrutinib behaved synergistically in models related to CLL. Because CLL is a B cell-related disorder and CD19 is highly expressed on B cells, the exemplified combination is likely to have the same mechanism of action and should behave synergistically in the treatment of other B cell-related disorders, such as ALL and NHL.

[0024] Therefore, the combination of the exemplary CD19-specific antibody and ibrutinib should be effective in treating non-Hodgkin's lymphoma, chronic lymphocytic leukemia and / or acute lymphoblastic leukemia in humans.The expected efficacy of the exemplary CD19-specific antibody and ibrutinib combination will be confirmed in clinical trials.

[0025] Because ibrutinib and other Bruton's tyrosine kinase (BTK) inhibitors all act by inhibiting the tyrosine-protein kinase BTK enzyme, which plays a critical role in B cell development, it is believed that due to the similar mechanisms of action of ibrutinib and other Bruton's tyrosine kinase (BTK) inhibitors, synergy should also be seen when treating humans with non-Hodgkin's lymphoma, chronic lymphocytic leukemia, and / or acute lymphoblastic leukemia with combinations of the exemplary anti-CD19 antibodies and Bruton's tyrosine kinase (BTK) inhibitors other than ibrutinib.

[0026] Because the exemplified anti-CD19 antibodies and other anti-CD19 antibodies bind to CD19, it is believed that synergy should also be seen with any anti-CD19 antibody and a Bruton's tyrosine kinase (BTK) inhibitor, such as ibrutinib, when treating humans with non-Hodgkin's lymphoma, chronic lymphocytic leukemia, and / or acute lymphoblastic leukemia.

[0027] An embodiment of the present disclosure includes a synergistic combination in which an antibody specific for CD19 comprises an HCDR1 region of the sequence SYVMH (SEQ ID NO:1), an HCDR2 region of the sequence NPYNDG (SEQ ID NO:2), an HCDR3 region of the sequence GTYYYGTRVFDY (SEQ ID NO:3), an LCDR1 region of the sequence RSSKSLQNVNGNTYLY (SEQ ID NO:4), an LCDR2 region of the sequence RMSNLNS (SEQ ID NO:5) and an LCDR3 region of the sequence MQHLEYPIT (SEQ ID NO:6) and ibrutinib. In a preferred embodiment, the combination is used to treat non-Hodgkin's lymphoma, chronic lymphocytic leukemia and / or acute lymphoblastic leukemia. [Brief description of the drawings]

[0028] [Figure 1] Figure 1 shows the cytotoxic effects of MOR00208 and ibrutinib alone and in combination on Ramos cells. Ramos cells were pretreated with 40 μM ibrutinib for 24 hours. [Diagram 2]Figure 2 shows the cytotoxic effects of MOR00208 and ibrutinib alone and in combination on MEC-1 cells. MEC-1 cells were pretreated with 30 μM ibrutinib for 24 hours. [Diagram 3] Figure 3 shows the cytotoxic effects of MOR00208 and ibrutinib alone and in combination on MEC-1 cells. MEC-1 cells were pretreated with 30 μM ibrutinib for 24 hours. [Figure 4] FIG. 4 shows the amino acid sequence of the variable domain of MOR00208. [Diagram 5] FIG. 5 shows the amino acid sequence of the Fc region of MOR00208. [Figure 6] FIG. 6 shows the cytotoxicity of MOR00208 and ibrutinib alone and in combination in the MEC-1 cell line. [Figure 7] FIG. 7 shows the cytotoxicity of MOR00208 and ibrutinib alone and in combination in the MEC-1 cell line. [Figure 8] FIG. 8 shows the cytotoxicity of MOR00208 and ibrutinib alone and in combination in the MEC-1 cell line. [Figure 9] FIG. 9 shows the Chou-Talay Combination Index curve for the combination of MOR00208 and ibrutinib in MEC-1 cell line. [Figure 10] FIG. 10 shows the Chou-Talay combination index curve for the combination of MOR00208 and ibrutinib in the MEC-1 cell line. [Figure 11] FIG. 11 shows the Chou-Talay combination index curve for the combination of MOR00208 and ibrutinib in the MEC-1 cell line. [Figure 12] FIG. 12 shows the results of Clarke et al.'s synergy calculations of the combination of MOR00208 and ibrutinib in the MEC-1 cell line. [Figure 13]FIG. 13 shows the results of Clarke et al.'s synergy calculations of the combination of MOR00208 and ibrutinib in the MEC-1 cell line. [Figure 14] FIG. 14 shows the results of Clarke et al.'s synergy calculations of the combination of MOR00208 and ibrutinib in the MEC-1 cell line. [Figure 15] FIG. 15 shows the results of Clarke et al.'s synergy calculations of the combination of MOR00208 and ibrutinib in the MEC-1 cell line. [Figure 16] FIG. 16 shows the results of Clarke et al.'s synergy calculations of the combination of MOR00208 and ibrutinib in the MEC-1 cell line. [Figure 17] FIG. 17 shows the results of Clarke et al.'s synergy calculations of the combination of MOR00208 and ibrutinib in the MEC-1 cell line. [Figure 18] FIG. 18 shows the results of Clarke et al.'s synergy calculations of the combination of MOR00208 and ibrutinib in the MEC-1 cell line. [Figure 19] FIG. 19 shows the results of Clarke et al.'s synergy calculations of the combination of MOR00208 and ibrutinib in the MEC-1 cell line. [Figure 20] FIG. 20 shows the results of Clarke et al.'s synergy calculations of the combination of MOR00208 and ibrutinib in the MEC-1 cell line. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0029] "Synergy," "cooperation," or "synergistic" means more than the expected additive effect of a combination. The "synergy," "cooperation," or "synergistic" effect of a combination is determined herein by the methods of Chou et al., Clarke et al., and / or Webb et al. See Ting-Chao Chou, Theoretical Basis, Experimental Design, and Computerized Simulation of Synergism and Antagonism in Drug Combination Studies, Pharmacol Rev 58:621-681 (2006), which is incorporated by reference in its entirety. See also Clarke et al., Issues in experimental design and endpoint analysis in the study of experimental cytotoxic agents in vivo in breast cancer and other models, Breast Cancer Research and Treatment 46:255-278 (1997), which is incorporated by reference in its entirety. See also Webb, JL (1963) Enzyme and Metabolic Inhibitors, Academic Press, New York, which is incorporated by reference in its entirety.

[0030] The term "antibody" refers to monoclonal antibodies including any isotype, such as IgG, IgM, IgA, IgD, and IgE. IgG antibodies consist of two identical heavy chains and two identical light chains linked by disulfide bonds. The heavy and light chains each contain a constant region and a variable region. Each variable region contains three segments called "complementarity determining regions" ("CDRs") or "hypervariable regions" that are primarily responsible for binding to an epitope of an antigen. They are numbered sequentially from the N-terminus and are called CDR1, CDR2, and CDR3. The more highly conserved portions of the variable regions outside the CDRs are called "framework regions." "Antibody fragment" refers to Fv, scFv, dsFv, Fab, Fab', F(ab')2 fragments, or other fragments that contain at least one variable heavy chain or variable light chain, respectively, that contains the CDRs and framework regions.

[0031] "Bruton's tyrosine kinase (BTK) inhibitors" are a class of drugs that function by inhibiting the tyrosine-protein kinase BTK enzyme, which plays a key role in B cell development. Specifically, BTK contains a PH domain that binds phosphatidylinositol (3,4,5)-triphosphate (PIP3). PIP3 binding causes BTK to phosphorylate phospholipase C, which results in the hydrolysis of PIP2, phosphatidylinositol, to generate two second messengers, inositol triphosphate (IP3) and diacylglycerol (DAG), which then enter into the regulation of the activity of downstream proteins in B cell signaling.

[0032] Bruton's tyrosine kinase (BTK) inhibitors include ibrutinib. Ibrutinib is marketed by Pharmacyclics, Inc. and Johnson & Johnson's Janssen Pharmaceutical (trade name Imbruvica, also known as PCI-32765). Ibrutinib is currently labeled for the treatment of patients with mantle cell lymphoma (MCL) who have received at least one prior therapy, chronic lymphocytic leukemia (CLL) who have received at least one prior therapy, chronic lymphocytic leukemia with 17p deletion, and Waldenstrom's macroglobulinemia. The formula of ibrutinib is 1-[(3R)-3-[4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl]-1-piperidinyl]-2-propen-1-one and has the following structure: TIFF2025016532000001.tif45170

[0033] Ibrutinib is a small molecule inhibitor of BTK. Ibrutinib forms a covalent bond with a cysteine ​​residue in the BTK active site, resulting in inhibition of BTK enzymatic activity. BTK is a signaling molecule in the B cell antigen receptor (BCR) and cytokine receptor pathways. The role of BTK in signaling through the B cell surface receptor is the activation of pathways required for B cell trafficking, chemotaxis, and adhesion. Preclinical studies show that ibrutinib inhibits malignant B cell proliferation and survival in vivo, as well as cell migration and substrate adhesion in vitro.

[0034] Another Bruton's tyrosine kinase (BTK) inhibitor is ACP-196 (Acerta Pharma BV), described in WO 2012170976 (U.S. Ser. No. 14 / 112,428), WO 2013010380 (U.S. Ser. No. 14 / 233,478) and WO 2014113932 (U.S. Ser. No. 14 / 160,587), all of which are incorporated by reference in their entirety; BGB-3111 (BeiGene, Co., Ltd.) and CC-292 Evans et al.2013 Examples include:

[0035] "VH" refers to the variable region of the immunoglobulin heavy chain of an antibody or antibody fragment. "VL" refers to the variable region of the immunoglobulin light chain of an antibody or antibody fragment.

[0036] The term "CD19" refers to the protein known as CD19, which has the following synonyms: B4, B lymphocyte antigen CD19, B lymphocyte surface antigen B4, CVID3, differentiation antigen CD19, MGC12802, and T cell surface antigen Leu-12.

[0037] Human CD19 has the following amino acid sequence: TIFF2025016532000002.tif46170

[0038] "MOR00208" is an anti-CD19 antibody. The amino acid sequences of the variable domains are set forth in FIG. 4. The amino acid sequences of the heavy and light chain Fc regions of MOR00208 are shown in FIG. 5. "MOR00208" and "XmAb 5574" are used synonymously to refer to the antibody shown in FIGS. 4 and 5. The MOR00208 antibody is described in U.S. Patent Application Serial No. 12 / 377,251, which is incorporated by reference in its entirety.

[0039] US Patent Application Serial No. 12 / 377,251 describes an antibody designated 4G7 H1.52 Hybrid S239D / I332E / 4G7 L1.155 as follows (later designated MOR00208). >4G7 H1.52 Hybrid S239D / I332E TIFF2025016532000003.tif53170>4G7 L1.155 TIFF2025016532000004.tif28170

[0040] Additional antibodies specific for CD19 are described in U.S. Pat. No. 7,109,304 (Immunomedics), which is incorporated in its entirety by reference; U.S. Pat. App. No. 11 / 917,750 (Medarex), which is incorporated in its entirety by reference; U.S. Pat. App. No. 11 / 852,106 (Medimmune), which is incorporated in its entirety by reference; U.S. Pat. App. No. 11 / 648,505 (Merck Patent GmbH), which is incorporated in its entirety by reference; U.S. Pat. No. 7,968,687 (Seattle Genetics), which is incorporated in its entirety by reference; and U.S. Pat. App. No. 12 / 710,442 (Glenmark Pharmaceuticals), which is incorporated in its entirety by reference.

[0041] "Fc region" means the constant region of an antibody, which in humans may be IgG1, 2, 3, 4 subclass or other constant region. The sequences of human Fc regions are available at IMGT, Human IGH C-REGIONs, http: / / www.imgt.org / IMGTrepertoire / Proteins / protein / human / IGH / IGHC / Hu_IGHCallgenes.html (retrieved May 16, 2011).

[0042] "RefmAb33" is an antibody whose amino acid sequence is as follows: Heavy chain containing the Fc region: TIFF2025016532000005.tif40170

[0043] Light chain containing the Fc region: TIFF2025016532000006.tif21170

[0044] RefmAb33 is specific for RSV and shares the same Fc region as MOR00208, and is therefore used as an isotype control.

[0045] "Combination" means two or more items, e.g., a compound, such as an antibody and ibrutinib.

[0046] The present disclosure further relates to combinations, medicaments and pharmaceutical compositions comprising the described combinations. The two components of the synergistic combination of the present invention, such as an antibody specific for CD19 and ibrutinib, may be administered physically or temporally together, simultaneously, separately or sequentially.

[0047] Ibrutinib is currently taken orally and administered once daily, while MOR00208 is currently administered intravenously once a week or once every two weeks.

[0048] Preferably, both drugs are administered so that both drugs can be effective in patients at the same time.For example, when MOR208 is administered once a week and ibritinib is administered once a day, the active substances of both drugs are present in patients at the same time.In one embodiment, ibrutinib is administered before and / or separately from the administration of the antibody specific for CD19, such as MOR00208.

[0049] Concurrently means that the two components are administered when both components (drugs) are available to the patient at the same time. "Synergism" means that both drugs are available to the patient at the same time.

[0050] Administered together can mean administered at the same time.

[0051] The two components may be formulated in different pharmaceutical compositions. The pharmaceutical composition includes an active agent, such as an antibody for use in human therapy. The pharmaceutical composition may include an acceptable carrier or excipient.

[0052] "Administered" or "administration" includes, but is not limited to, delivery in an injectable form, such as, for example, intravenous, intramuscular, intradermal or subcutaneous routes, or mucosal routes, delivery as a nasal spray or aerosol for inhalation, or delivery as an ingestible solution, capsule or tablet.

[0053] A "therapeutically effective amount" of a compound or combination refers to an amount sufficient to cure, alleviate, or partially prevent the clinical symptoms of a given disease or disorder and its complications. The amount that is effective for a particular therapeutic purpose will vary depending on the severity of the disease or injury, as well as the weight and general condition of the subject. It will be understood that the determination of an appropriate dosage may be accomplished by constructing a matrix of values ​​using routine experimentation and testing various points within the matrix. All of these are within the ordinary skill of a trained physician or clinical scientist.

[0054] "CDR" herein is defined by Chothia et al. or Kabat et al. See Chothia C, Lesk AM. (1987) Canonical structures for the hypervariable regions of immunoglobulins. J Mol Biol., 196(4):901-17, which is incorporated by reference in its entirety. See Kabat EA, Wu TT, Perry HM, Gottesman KS and Foeller C. (1991). Sequences of Proteins of Immunological Interest. 5th edit., NIH Publication no. 91-3242, US Dept. of Health and Human Services, Washington, DC, which is incorporated by reference in its entirety.

[0055] "Cross-competition" refers to the ability of an antibody or other binding agent to interfere with the binding of another antibody or binding agent to CD19 in a standard competitive binding assay. The ability or extent to which an antibody or other binding agent can interfere with the binding of another antibody or binding molecule to CD19, and therefore whether it can be cross-competed according to the present invention, can be determined using standard competitive binding assays. One suitable assay uses Biacore technology (e.g., by using a BIAcore 3000 instrument (Biacore, Uppsala, Sweden)), which can measure the degree of interaction using surface plasmon resonance technology. Another assay for measuring cross-competition uses an ELISA-based approach. A high-throughput process for "epitope binning" antibodies based on their cross-competition is described in WO 2003 / 48731.

[0056] The term "epitope" includes any protein determinant capable of specific binding to an antibody or otherwise interacting with a molecule. Epitope determinants generally consist of chemically active surface groupings of molecules such as amino acids or carbohydrate or sugar side chains and may have specific three-dimensional structural characteristics and specific charge characteristics. Epitopes may be "linear" or "conformational". The term "linear epitope" refers to an epitope in which all of the points of interaction between the protein and the interacting molecule (such as an antibody) occur linearly along the primary amino acid sequence of the protein (continuous). The term "conformational epitope" refers to an epitope in which discontinuous amino acids are assembled in a three-dimensional structure. In a conformational epitope, the points of interaction occur across amino acid residues that are spaced apart from one another on the protein.

[0057] "Binds to the same epitope" refers to the ability of an antibody or other binding agent to bind to the same epitope as CD19 and the exemplified antibody. The epitopes of the exemplified antibodies and other antibodies to CD19 can be determined using standard epitope mapping techniques. Epitope mapping techniques are well known in the art, including Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66 (Glenn E. Morris, Ed., 1996) Humana Press, Totowa, New Jersey. For example, linear epitopes may be determined, for example, by simultaneously synthesizing large amounts of peptides corresponding to a portion of a protein molecule on a solid support and reacting the peptides with an antibody while the peptides are still bound to the support. Such techniques are known in the art and are described, for example, in U.S. Patent No. 4,708,871; Geysen et al, (1984) Proc. Natl. Acad. Sci. USA 8:3998-4002; Geysen et al, (1985) Proc. Natl. Acad. Sci. USA 82:78-182; Geysen et al, (1986) Mol. Immunol. 23:709-715. Similarly, conformational epitopes are easily identified by determining the spatial structure of amino acids, for example, by hydrogen / deuterium exchange, X-ray crystallography and two-dimensional nuclear magnetic resonance. See, for example, Epitope Mapping Protocols, supra. Antigenic regions of proteins can also be identified using standard antigenicity and hydropathy plots, such as those calculated using the Omiga version 1.0 software program available from the Oxford Molecular Group.This computer program utilizes the Hopp / Woods method, Hopp et al, (1981) Proc. Natl. Acad. Sci USA 78:3824-3828, for determining antigenic profiles and the Kyte-Doolittle technique, Kyte et al, (1982) J. Mol. Biol. 157:105-132, for hydropathy plots.

[0058] Embodiment An aspect of the disclosure is a combination comprising an antibody specific for CD19 and a Bruton's tyrosine kinase (BTK) inhibitor for use in the treatment of non-Hodgkin's lymphoma, chronic lymphocytic leukemia and / or acute lymphoblastic leukemia. In an embodiment, the combination is synergistic.

[0059] The combination of the exemplified anti-CD19 antibody and ibrutinib behaves synergistically in the in vitro model related to CLL.Since CLL is a B cell-related disorder and CD19 is highly expressed on B cells, the exemplified combination should have the same mechanism of action and should behave synergistically in the treatment of other B cell-related disorders, such as ALL and NHL.Therefore, the exemplified combination of the CD19-specific antibody and ibrutinib should be effective in the treatment of non-Hodgkin's lymphoma, chronic lymphocytic leukemia and / or acute lymphoblastic leukemia in humans.The expected effectiveness of the exemplified combination of the CD19-specific antibody and ibrutinib will be confirmed in clinical trials.

[0060] Chronic B-cell leukemia cell line MEC-1 cells (DSMZ# ACC497) were tested. This in vitro model of MEC-1 cells suggests how this combination may work in treating chronic lymphocytic leukemia (CLL) in humans. The Chou index values ​​show a clear synergistic effect of the combination of MOR00208 and ibrutinib in specific killing of MEC-1 cells compared to MOR00208 and ibrutinib alone.

[0061] The following additional cell lines are also evaluated: Ramos cells (ATCC No. CRL-1596), which are human Burkitt's lymphoma cells. HG-3 (DSMZ#ACC765) and CII (DSMZ#ACC773) are chronic lymphocytic leukemia cell lines. Su-DHL 6 (DSMZ#ACC572), U2932 (DSMZ#ACC633) and OCI-LY7 (DSMZ#ACC688) are diffuse large B-cell lymphoma (DLBCL) cell lines. JVM-2 (ATCC®CRL-3002) is a mantle cell lymphoma cell line. BALL-1 (DSMZ#ACC742) is an acute lymphoblastic leukemia cell line.

[0062] Ramos cells in this in vitro model suggest how the combination works in treating human non-Hodgkin's lymphoma (NHL). HG-3 cells and CII cells in this in vitro model suggest how the combination works in treating human chronic lymphocytic leukemia (CLL). Su-DHL 6 cells, U2932 cells and OCI-LY7 cells in this in vitro model suggest how the combination works in treating human non-Hodgkin's lymphoma. JVM-2 cells in this in vitro model suggest how the combination works in treating human non-Hodgkin's lymphoma. BALL-1 cells in this in vitro model suggest how the combination works in treating human acute lymphoblastic leukemia.

[0063] In summary, the combination of the exemplified anti-CD19 antibodies and ibrutinib behaves synergistically in models relevant to CLL.

[0064] Thus, the combination of the exemplary CD19-specific antibodies and ibrutinib should be effective in the treatment of humans with non-Hodgkin's lymphoma, chronic lymphocytic leukemia and / or acute lymphoblastic leukemia.

[0065] Because ibrutinib and other Bruton's tyrosine kinase (BTK) inhibitors all act by inhibiting the tyrosine-protein kinase BTK enzyme, which plays a critical role in B cell development, it is believed that due to the similar mechanisms of action of ibrutinib and other Bruton's tyrosine kinase (BTK) inhibitors, synergy should also be seen when treating humans with non-Hodgkin's lymphoma, chronic lymphocytic leukemia, and / or acute lymphoblastic leukemia with combinations of the exemplary anti-CD19 antibodies and Bruton's tyrosine kinase (BTK) inhibitors other than ibrutinib.

[0066] The exemplified anti-CD19 antibodies and other anti-CD19 antibodies bind to CD19 and may be used in a variety of applications, including, for example, U.S. Patent Application Serial No. 12 / 377,251 (Xencor), WO 2005012493, WO 2010053716 (Immunomedics); WO 2007002223 (Medarex); WO 2008022152 (Xencor); WO 2008031056 (Medimmune); WO 2007 / 076950 (Merck Patent GmbH); WO 2009 / 052431 (Seattle Genetics); and WO 2010095031 (Glenmark), all of which are incorporated by reference in their entirety. It is believed that synergy should also be seen when a combination of any of the anti-CD19 antibodies described in Fischer, Inc. (Franklin, MD) and a Bruton's tyrosine kinase (BTK) inhibitors is used to treat humans with non-Hodgkin's lymphoma, chronic lymphocytic leukemia, and / or acute lymphoblastic leukemia.

[0067] In embodiments, antibodies specific for CD19 include antibodies that cross-compete with an antibody comprising an HCDR1 region of the sequence SYVMH (SEQ ID NO: 1), an HCDR2 region of the sequence NPYNDG (SEQ ID NO: 2), an HCDR3 region of the sequence GTYYYGTRVFDY (SEQ ID NO: 3), an LCDR1 region of the sequence RSSKSLQNVNGNTYLY (SEQ ID NO: 4), an LCDR2 region of the sequence RMSNLNS (SEQ ID NO: 5), and an LCDR3 region of the sequence MQHLEYPIT (SEQ ID NO: 6).

[0068] In embodiments, antibodies specific for CD19 include antibodies that bind to the same epitope as an antibody comprising an HCDR1 region of the sequence SYVMH (SEQ ID NO: 1), an HCDR2 region of the sequence NPYNDG (SEQ ID NO: 2), an HCDR3 region of the sequence GTYYYGTRVFDY (SEQ ID NO: 3), an LCDR1 region of the sequence RSSKSLQNVNGNTYLY (SEQ ID NO: 4), an LCDR2 region of the sequence RMSNLNS (SEQ ID NO: 5), and an LCDR3 region of the sequence MQHLEYPIT (SEQ ID NO: 6).

[0069] In an embodiment, the antibody specific for CD19 comprises an HCDR1 region of the sequence SYVMH (SEQ ID NO: 1), an HCDR2 region of the sequence NPYNDG (SEQ ID NO: 2), an HCDR3 region of the sequence GTYYYGTRVFDY (SEQ ID NO: 3), an LCDR1 region of the sequence RSSKSLQNVNGNTYLY (SEQ ID NO: 4), an LCDR2 region of the sequence RMSNLNS (SEQ ID NO: 5) and an LCDR3 region of the sequence MQHLEYPIT (SEQ ID NO: 6).

[0070] In embodiments, the antibody specific for CD19 has the sequence The variable heavy chain of TIFF2025016532000007.tif21170, and the sequence Contains the variable light chain of TIFF2025016532000008.tif16170.

[0071] In certain embodiments, the antibody has the sequence Contains the heavy chain constant domain of TIFF2025016532000009.tif43170.

[0072] In embodiments, the antibody specific for CD19 has the sequence Contains the light chain constant domain of TIFF2025016532000010.tif14170.

[0073] In embodiments, the Bruton's tyrosine kinase (BTK) inhibitor is ibrutinib.

[0074] In embodiments, the components of the combination, the antibody specific for CD19 and ibrutinib, are administered separately. In one embodiment, ibrutinib is administered prior to administration of the antibody specific for CD19.

[0075] In an embodiment, the components of the combination are administered when both components (agents) are effective in the patient at the same time. "Synergistic" means that both agents are effective in the patient at the same time. In an embodiment, the components of the combination are administered together physically or temporally, simultaneously, separately or sequentially. In an embodiment, the components of the combination are administered simultaneously.

[0076] In embodiments, the combination is a pharmaceutical composition. In embodiments, the composition includes an acceptable carrier. In embodiments, the combination is administered in an effective amount.

[0077] In another embodiment, a synergistic combination of an antibody specific for CD19, comprising an HCDR1 region of the sequence SYVMH (SEQ ID NO: 1), an HCDR2 region of the sequence NPYNDG (SEQ ID NO: 2), an HCDR3 region of the sequence GTYYYGTRVFDY (SEQ ID NO: 3), an LCDR1 region of the sequence RSSKSLQNVNGNTYLY (SEQ ID NO: 4), an LCDR2 region of the sequence RMSNLNS (SEQ ID NO: 5), and an LCDR3 region of the sequence MQHLEYPIT (SEQ ID NO: 6), with ibrutinib, is capable of mediating the killing of MEC-1 cells by ADCC with at least 2-fold, 3-fold, 4-fold, or 5-fold greater efficacy than ibrutinib alone in the presence of isolated human PBMCs.

[0078] Aspects of the present disclosure include a synergistic combination of an antibody specific for CD19, comprising an HCDR1 region of sequence SYVMH (SEQ ID NO:1), an HCDR2 region of sequence NPYNDG (SEQ ID NO:2), an HCDR3 region of sequence GTYYYGTRVFDY (SEQ ID NO:3), an LCDR1 region of sequence RSSKSLQNVNGNTYLY (SEQ ID NO:4), an LCDR2 region of sequence RMSNLNS (SEQ ID NO:5), and an LCDR3 region of sequence MQHLEYPIT (SEQ ID NO:6), with ibrutinib for the treatment of non-Hodgkin's lymphoma, chronic lymphocytic leukemia, and / or acute lymphoblastic leukemia. In embodiments, the non-Hodgkin's lymphoma is selected from the group consisting of follicular lymphoma, small lymphocytic lymphoma, mucosa-associated lymphoid tissue, marginal zone, diffuse large B cell, Burkitt's cell, and mantle cell.

[0079] In an embodiment, the non-Hodgkin's lymphoma is a follicular lymphoma. In an embodiment, the non-Hodgkin's lymphoma is a small lymphocytic lymphoma. In an embodiment, the non-Hodgkin's lymphoma is a mucosa-associated lymphoid tissue. In an embodiment, the non-Hodgkin's lymphoma is a marginal zone lymphoma. In an embodiment, the non-Hodgkin's lymphoma is a diffuse large B-cell lymphoma. In an embodiment, the non-Hodgkin's lymphoma is a Burkitt's lymphoma. In an embodiment, the non-Hodgkin's lymphoma is a mantle cell lymphoma.

[0080] In an embodiment, the combination is for the treatment of chronic lymphocytic leukemia.In an embodiment, the combination is for the treatment of acute lymphoblastic leukemia.

[0081] Another aspect includes a method of treating non-Hodgkin's lymphoma, chronic lymphocytic leukemia, and / or acute lymphoblastic leukemia in an individual in need of such treatment, comprising administering an antibody specific for CD19 and a Bruton's tyrosine kinase (BTK) inhibitor. In an embodiment of the method, the antibody specific for CD19 includes an HCDR1 region of sequence SYVMH (SEQ ID NO: 1), an HCDR2 region of sequence NPYNDG (SEQ ID NO: 2), an HCDR3 region of sequence GTYYYGTRVFDY (SEQ ID NO: 3), an LCDR1 region of sequence RSSKSLQNVNGNTYLY (SEQ ID NO: 4), an LCDR2 region of sequence RMSNLNS (SEQ ID NO: 5), and an LCDR3 region of sequence MQHLEYPIT (SEQ ID NO: 6). In an embodiment of the method, the antibody includes an exemplary antibody specific for CD19. In an embodiment of the method, the Bruton's tyrosine kinase (BTK) inhibitor is ibrutinib.

[0082] Another embodiment includes the use of an antibody specific for CD19, comprising an HCDR1 region of the sequence SYVMH (SEQ ID NO: 1), an HCDR2 region of the sequence NPYNDG (SEQ ID NO: 2), an HCDR3 region of the sequence GTYYYGTRVFDY (SEQ ID NO: 3), an LCDR1 region of the sequence RSSKSLQNVNGNTYLY (SEQ ID NO: 4), an LCDR2 region of the sequence RMSNLNS (SEQ ID NO: 5) and an LCDR3 region of the sequence MQHLEYPIT (SEQ ID NO: 6), in the manufacture of a medicament for the treatment of non-Hodgkin's lymphoma, chronic lymphocytic leukemia and / or acute lymphoblastic leukemia, in synergistic combination with ibrutinib. EXAMPLES

[0083] Example 1: Cytotoxicity of MEC-1 cells using MOR00208 and ibrutinib alone and in combination material Cell lines tested: MEC-1 cells (DSMZ# ACC497). The following cell lines are tested: chronic B-cell leukemia cell lines; JVM-2 (ATCC® CRL-3002) mantle cell lymphoma cell line; Ramos cells (ATCC number CRL-1596), human Burkitt's lymphoma cells; HG-3 (DSMZ#ACC765) and CII (DSMZ#ACC773) are chronic lymphocytic leukemia cell lines; Su-DHL 6 (DSMZ#ACC572), U2932 (DSMZ#ACC633) and OCI-LY7 (DSMZ#ACC688) are diffuse large B-cell lymphoma (DLBCL) cell lines; JVM-2 (ATCC® CRL-3002) is a mantle cell lymphoma cell line; and BALL-1 (DSMZ#ACC742) is an acute lymphoblastic leukemia cell line.

[0084] The culture conditions for the cell lines used were in accordance with the supplier's information.

[0085] Cell culture media: Iscove's Modified Dulbecco's Medium (IMDM), Invitrogen, Cat No.: 31980-048; RPMI1640, Invitrogen, Cat No.: 31870-074; GlutaMAX, Invitrogen, Cat No.: 35050-38 Lot No.: 1504647; FCS: Sigma Cat No.: F7524 Lot No.: 111M3396.

[0086] NK: RPMI1640 with GlutaMAXTM, Invitrogen, Cat No.: 31870-074, 10% FCS; Biocoll: Biochrome AG Cat No.: L6115 Lot No.: 0034D; MACS NK Cell Isolation Kit: Miltenyi Biotec Cat No.: 130-092-657 Lot No.: 5150130115; Ibrutinib: Selleck Chem Lot No.: S2680; FCS: Sigma Cat No.: F7524 Lot No.: 111M3396; and RefmAb33 (anti-RSV) which contains the same Fc region as MOR00208.

[0087] method The cytotoxicity of MOR00208 and ibrutinib alone and in combination was tested in MEC-1 cell line (CLL).The cytotoxicity of MOR00208 and ibrutinib alone and in combination was tested in the following target cell lines: JVM-2, Ramos, HG-3, CII, Su-DHL 6, U2932, OCI-LY7, JVM-2 and BALL-1.

[0088] Ibrutinib is a covalent inhibitor of Bruton's tyrosine kinase and is believed to prevent the proliferation of target cell lines. MOR00208 targets CD19 and is involved in target cell killing via ADCC. Target cell killing is measured using the following parameters: ibrutinib at a concentration range of 0.033-33 μM, specifically 0.3 μM, 1.0 μM and 3.0 μM; MOR00208 at a concentration range of 0.001-10 nM, specifically 0.01 nM, 0.1 nM and 10 nM, and combinations of MOR00208 and ibrutinib. RefmAb33 or NK cells alone are used as controls. In the ibrutinib and MOR00208+ibrutinib combination groups, target cells are pretreated with ibrutinib for 7 days prior to ADCC assay measurement. Target cells are counted and stained using CFSE at a final concentration of 1 μg / ml. For DMSO-treated target cells, 5 × 10 5 An effector:target (E:T) ratio of 2:1 was chosen, corresponding to a cell density of 1 × 10 / ml. The E:T ratio of inhibitor-treated cells was adjusted to include the proliferative effect on target cells caused by ibrutinib treatment. NK cells were counted and 1 × 10 6The antibody was adjusted to 0.001 nM / ml. Target cell killing assays were performed as follows: 96-well plates were used, with 100 μl of target cell suspension per well, followed by 100 μl of NK cell suspension per well, resulting in an E:T ratio of 2:1. Antibodies were diluted in medium ranging from 10 to 0.001 nM. Cells were centrifuged and the target:effector cell pellet was resuspended in 100 μl of antibody-containing medium or the corresponding control solution. Assays were incubated for 4 hours at 37°C in a CO2 incubator. After a 10-minute incubation on ice, 50 μl of DAPI solution was added to each well (final concentration 1 μg / ml) and incubated on ice for 10 minutes. Measurement of cell killing was performed using a FACS-Verse. Dead target cells were DAPI positive.

[0089] Preliminary experiments were carried out using both MEC-1 cells and RAMOS cells according to the method described above. Figures 1 to 3 show the results of the preliminary experiments.

[0090] To determine the involvement of ADCC in MEC-1 cells, a total of three experiments were performed with the combination of MOR00208 and ibrutinib. The ADCC dose-response curves for experiments 1-3 are shown in Figures 6-8.

[0091] The cell death rates (%) (raw data) for Experiments 1 to 3 are shown in Tables 1 to 9 below.

[0092] Test 1: TIFF2025016532000011.tif65170TIFF2025016532000012.tif65170TIFF2025016532000013.tif64170

[0093] Test 2: TIFF2025016532000014.tif71170TIFF2025016532000015.tif71170TIFF2025016532000016.tif71170

[0094] Test 3: TIFF2025016532000017.tif70170TIFF2025016532000018.tif70170TIFF2025016532000019.tif71170

[0095] Calculating synergy The raw data (% dead cells) are analyzed as follows: 1) background (control) is subtracted from the raw data (% dead cells) to obtain the specific killing for each treatment group; then 2) the specific killing values ​​are normalized by setting the MOR00208+ibrutinib combination to 1.

[0096] To determine the synergy of the exemplary anti-CD19 antibody and ibrutinib combinations compared to MOR00208 and ibrutinib alone, a combination index (CI) calculation is completed. Such calculations are described in Ting-Chao Chou, Theoretical Basis, Experimental Design, and Computerized Simulation of Synergism and Antagonism in Drug Combination Studies, Pharmacol Rev 58:621-681 (2006), which is incorporated by reference in its entirety, and Chou TC, Talalay P, Quantitative analysis of dose-effect relationships: the combined effects of multiple drugs or enzyme inhibitors. Adv Enzyme Regul 22:27-55 (1984), which is incorporated by reference in its entirety. The Chou-Talalay method is performed using the CI-isobol method.

[0097] Median effect equation The half-effect equation is a formula that expresses the effect of an inhibitor (such as a drug) as F a / F u =(D / D50)^m, where D is the dose and F a and F uis the proportion of systems affected or not affected by dose D (F a +F u = 1); D50 is the dose that exerts half the effect (e.g., IC50, ED50, LD50). The constant m determines the shape of the dose-response curve.

[0098] We use GraphPad Prism to perform nonlinear regression calculations to estimate the parameters m and D50.

[0099] CI-isobol method The CI-isobol method provides a quantitative assessment of synergy between drugs. The combination index (CI) is estimated from dose-effect data for single and combined drug treatments. A CI less than 1 indicates synergy; CI=1 indicates additivity; CI>1 indicates antagonism. The more significant the drug interaction (synergy or antagonism), the further away from CI value 1.

[0100] Formally, the combination index (CI) for a combination drug treatment is CI = D1 / D x1 +D2 / D x2 It is defined as follows.

[0101] where D1 and D2 are the doses of drug 1 and drug 2 in the combination, respectively; Dx1 and Dx2 are the doses of treatment with only drug 1 and drug 2 that are expected to give the same effect as the combination effect. The doses Dx1 and Dx2 need to be estimated from the dose-effect data of the single drug treatments. Essentially, a median effect equation is fitted to the data for each drug. From the median effect equation for the drug, we can estimate the dose (i.e., D) required to exert the effect (i.e., Fa, Fu). The further a point is from the line of additive effect, the greater the difference between 1 and its CI, and therefore the stronger the effect (synergistic or antagonistic).

[0102] result Chou index curves are shown in Figures 9 - 11. Data obtained from three experiments (with the same concentrations) were merged to generate one curve for each Ibrutinib concentration.

[0103] Chou index values ​​indicate a clear synergistic effect of the combination of MOR00208 and ibrutinib in specific killing of MEC-1 cells compared to MOR00208 and ibrutinib alone.

[0104] Therefore, the combination of MOR00208 and ibrutinib will also behave synergistically in the treatment of human non-Hodgkin's lymphoma (NHL), chronic lymphocytic leukemia (CLL) and acute lymphoblastic leukemia (ALL).

[0105] Additional analysis Another approach to calculate and compare the effects of single drugs when used in combination is the fractional product concept, first described in 1963 by Webb JL in “Enzymes and metabolic inhibitors”. In this analysis, it is considered that the action of several drugs can be directed to the same cell fraction as long as their actions are non-competitive, which is the case for MOR00208 and ibrutinib, and therefore the measured combined effect will be less than the theoretical sum of their individual effects. According to the fractional product concept, whenever two drugs kill 50% of the target cell fraction, the effect of the combination is only 75% (applied formula: 1-(1-0.5) x (1-0.5) = 0.75) and not the expected 100%, because only 50% of the target cells are still viable and sensitive to one of the two drugs.

[0106] Another approach for calculating and comparing the effects of single agents when used in combination is that of Clarke et al., Issues in experimental design and endpoint analysis in the study of experimental cytotoxic agents in vivo in breast cancer and other models, Breast Cancer Research and Treatment 46:255-278 (1997), which is incorporated by reference in its entirety.

[0107] The % dead cells (raw data) in Tables 1-16 were analyzed as follows: Antagonistic (AB) / C<(A / C)×(B / C) Additive (AB) / C=(A / C)×(B / C) Synergistic (AB) / C>(A / C)×(B / C) A is treatment with MOR00208 alone; B is treatment with ibrutinib alone; C is the response to control DMSO+RefMab33; AB is the combination of treatments A and B.

[0108] Experiment 1 TIFF2025016532000020.tif43170

[0109] The Clarke analysis of the data presented in Table 1 is further illustrated in FIG. TIFF2025016532000021.tif43170

[0110] The Clarke analysis of the data presented in Table 2 is further illustrated in FIG. TIFF2025016532000022.tif43170

[0111] The Clarke analysis of the data presented in Table 3 is further illustrated in FIG.

[0112] Experiment 2 TIFF2025016532000023.tif43170

[0113] The Clarke analysis of the data presented in Table 4 is further illustrated in FIG. TIFF2025016532000024.tif44170

[0114] The Clarke analysis of the data presented in Table 5 is further illustrated in FIG. TIFF2025016532000025.tif44170

[0115] The Clarke analysis of the data presented in Table 6 is further illustrated in FIG.

[0116] Experiment 3 TIFF2025016532000026.tif44170

[0117] The Clarke analysis of the data presented in Table 7 is further illustrated in FIG. TIFF2025016532000027.tif44170

[0118] The Clarke analysis of the data presented in Table 8 is further illustrated in FIG. TIFF2025016532000028.tif45170

[0119] The Clarke analysis of the data presented in Table 9 is further illustrated in FIG.

[0120] Clarke results Experiments 1-3 at each concentration show clear synergy of the combination MOR00208 + ibrutinib using the method of Clarke et al. However, the method of Clarke et al. shows synergy, despite a small number of experiments in which ibrutinib activity was lower than that of the control.

[0121] It will be understood that the present description, specific examples and data, while indicating exemplary embodiments, are presented by way of illustration and are not intended to limit the invention. Various changes and modifications in the invention will become apparent from the discussion, disclosure and data contained herein and are therefore considered a part of the invention.

Claims

1. 1. A synergistic combination comprising an antibody specific for CD19, comprising an HCDR1 region of the sequence SYVMH (SEQ ID NO:1), an HCDR2 region of the sequence NPYNDG (SEQ ID NO:2), an HCDR3 region of the sequence GTYYYGTRVFDY (SEQ ID NO:3), an LCDR1 region of the sequence RSSKSLQNVNGNTYLY (SEQ ID NO:4), an LCDR2 region of the sequence RMSNLNS (SEQ ID NO:5) and an LCDR3 region of the sequence MQHLEYPIT (SEQ ID NO:6), and a Bruton's tyrosine kinase (BTK) inhibitor, for use in the treatment of non-Hodgkin's lymphoma, chronic lymphocytic leukemia and / or acute lymphoblastic leukemia.

2. 2. The combination of claim 1, wherein the antibody has the sequence and the variable heavy chain of sequence A combination comprising a variable light chain of

3. 3. The combination according to claim 1 or 2, wherein the antibody has the sequence A combination comprising a heavy chain constant domain of

4. 4. The combination according to claim 1, wherein the antibody has the sequence A combination comprising a light chain constant domain of.

5. 5. The combination according to claim 1, wherein the antibody specific for CD19 and the Bruton's tyrosine kinase (BTK) inhibitor are administered separately.

6. 6. The combination according to any one of claims 1 to 5, wherein the Bruton's tyrosine kinase (BTK) inhibitor is administered prior to administration of the antibody specific for CD19.

7. 7. The combination according to any one of claims 1 to 6, wherein the antibody specific for CD19 and the Bruton's tyrosine kinase (BTK) inhibitor are administered simultaneously.

8. 8. The combination of claim 1, wherein the antibody specific for CD19 and the Bruton's tyrosine kinase (BTK) inhibitor are administered when both agents are effective simultaneously in the patient.

9. 9. The combination according to any one of claims 1 to 8, wherein the Bruton's tyrosine kinase (BTK) inhibitor is ibrutinib.

10. 10. The combination according to any one of claims 1 to 9, for use in the treatment of chronic lymphocytic leukemia.

11. 11. The combination according to any one of claims 1 to 10, for use in the treatment of acute lymphoblastic leukemia.

12. 12. The combination of any one of claims 1 to 11, for use in the treatment of non-Hodgkin's lymphoma, wherein the non-Hodgkin's lymphoma is selected from the group consisting of follicular lymphoma, small lymphocytic lymphoma, mucosa-associated lymphoid tissue, marginal zone, diffuse large B-cell, Burkitt's cell and mantle cell.

13. 13. The combination of claim 12, wherein the non-Hodgkin's lymphoma is follicular lymphoma.

14. 13. The combination of claim 12, wherein the non-Hodgkin's lymphoma is small lymphocytic lymphoma.

15. 13. The combination of claim 12, wherein the non-Hodgkin's lymphoma is mucosa-associated lymphoid tissue.

16. 13. The combination of claim 12, wherein the non-Hodgkin's lymphoma is diffuse large B-cell lymphoma.

17. 13. The combination of claim 12, wherein the non-Hodgkin's lymphoma is Burkitt's lymphoma.

18. 13. The combination of claim 12, wherein the non-Hodgkin's lymphoma is mantle cell lymphoma.

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