Use of anti-CCR7 antibodies in combination therapy with BTK inhibitors and / or BCL2 inhibitors to treat hematological malignancies

Abstract

The present invention provides novel uses and methods comprising antibodies or antigen-binding fragments thereof that bind to the CCR7 receptor for use as novel combination therapies with BTK inhibitors and / or Bcl-2 inhibitors in the treatment of hyperproliferative hematological malignancies, preferably B-cell lymphomas such as CLL. The combinations can be used as first-line or in naive patients who have not previously been treated with BTK inhibitors and / or Bcl-2 inhibitors, or in patients with BTK inhibitor and / or Bcl-2 inhibitor-resistant / relapsed disease. The antibodies and antigen-binding fragments can selectively eliminate CCR7-expressing malignant cells ex vivo or in vitro and can impair / block the migration of these tumor cells toward CCR7 ligands. These effects are unrelated to previous or current treatment with BTK inhibitors and / or Bcl-2 inhibitors. Similarly, the efficacy of the antibodies is not affected by patients with relapsed / resistant disease. The use of said antibodies as monotherapy or in combination with BTK inhibitors and / or Bcl-2 inhibitors to eliminate, kill, and impair / block migration and activation of CCR7-expressing tumor cells is disclosed, thus providing an alternative therapy for treating hyperproliferative hematological cancers.

Description

[Technical field]

[0001] The present invention relates generally to the fields of medicine and pharmacy, and more particularly to the field of oncology. [Background technology]

[0002] Effective treatment of hyperproliferative disorders is a continuing goal in the oncology field. Generally, cancer is characterized by the proliferation of malignant cells with the potential for unrestrained growth, local expansion, and systemic metastasis as a result of deregulation of normal processes that control cell division, differentiation, and apoptotic cell death, which may include abnormalities in signal transduction pathways.

[0003] Bruton's tyrosine kinase (BTK), a member of the Tec family of cytoplasmic tyrosine kinases, is intimately involved in multiple signaling pathways that control the survival, activation, proliferation, and differentiation of B-lineage lymphoid cells. BTK is an upstream activator of multiple anti-apoptotic signaling molecules and networks, including the signal transducer and activator of transcription 5 (STAT5) protein, the phosphatidylinositol (PI) 3-kinase / AKT / mammalian target of rapamycin (mTOR) pathway, and nuclear factor kappa B (NF-κB). In addition, BTK associates with the death receptor Fas via its kinase and pleckstrin homology (PH) domains and prevents the interaction of Fas with Fas-associated death domain protein (FADD), which is essential for Fas-induced recruitment and activation of caspase-8 / FLICE during apoptotic signaling. This dysfunction by BTK prevents the assembly of the pro-apoptotic death-inducing signaling complex (DISC) following Fas ligation.

[0004] BTK is abundantly expressed in malignant cells from patients with B-cell precursor (BCP) acute lymphoblastic leukemia (ALL, the most common form of cancer in children and adolescents), chronic lymphocytic leukemia (CLL), and non-Hodgkin's lymphoma (NHL). Thus, BTK has emerged as an important molecular target for the treatment of B-lineage leukemias and lymphomas.

[0005] There are several BTK inhibitors in the clinic. The first molecule approved is ibrutinib (1-(3-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)prop-2-en-1-one, disclosed in WO 2008 / 039218. Ibrutinib is currently used to treat B-cell cancers such as mantle cell lymphoma, chronic lymphocytic leukemia, and Waldenstrom's macroglobulinemia. Ibrutinib is used as a first-line treatment in patients with chronic lymphocytic leukemia (CLL) who require treatment and who are newly diagnosed, and may also be used in relapsed CLL. Ibrutinib is further used to treat Waldenstrom's macroglobulinemia, and as a second-line treatment for mantle cell lymphoma (MCL), marginal zone lymphoma, and chronic graft-versus-host disease. Recently, acalabrutinib was approved by the FDA for the treatment of mantle cell lymphoma (www.fda.gov / news-events / press-announcements / fda-approves-new-treatment-adults-mantle-cell-lymphoma).

[0006] Both primary (natural) and secondary (acquired) resistance to ibrutinib has been reported in various lymphomas, including CLL and MCL (Kaur, 2017, Ann Hematol. doi:10.1007 / s00277-017-2973-2). Accordingly, various patent publications have proposed the use of ibrutinib as part of a combination therapy with other treatment modalities (see, e.g., U.S. Patent Application Publication No. 2017239351, WO 2017 / 023815(A1), U.S. Patent Application Publication No. 2018 / 0153892(A1), U.S. Patent Application Publication No. 2017360796, U.S. Patent Application Publication No. 2015105409, U.S. Patent Application Publication No. 2017354655, U.S. Patent Application Publication No. 2017224819, and U.S. Patent Application Publication No. 2016 / 0303130).

[0007] B-cell lymphoma 2 (Bcl-2) is an anti-apoptotic protein localized to the outer membrane of mitochondria, where it plays a key role in promoting cell survival and inhibiting the action of pro-apoptotic proteins. Bcl-2 protein belongs to the Bcl-2 family, a family composed of two groups of highly conserved proteins: anti-apoptotic and pro-apoptotic proteins. A homeostatic balance between these two groups of proteins is required for cells to control cell death and cell survival (Scheffold et al., Recent Results Cancer Res. 2018;212:215-242. doi:10.1007 / 978-3-319-91439-8_11; Moia et al., Expert Rev Hematol. 2018 May;11(5):391-402. doi:10.1080 / 17474086.2018.1456332).

[0008] In several hematological malignancies, upregulation of Bcl-2 protein is well documented. For example, in CLL patients, increased levels of Bcl-2 protein may be the result of epigenetic dysregulation of the Bcl-2 gene promoter or (in most cases) a deletion at locus 13q14. This deleted region contains two Bcl-2 repressors, namely microRNA 15a and 16-1, which bind to the mRNA of the Bcl-2 gene and inhibit the translation of the Bcl-2 protein (Scheffold et al., 2018, supra; Moia et al., 2018, supra).

[0009] In the last few years, new therapies targeting the anti-apoptotic Bcl-2 protein have been developed. Venetoclax (ABT-199) was approved by the FDA as a second-line treatment for CLL with 17p deletion (Deeks, 2016, Drugs.doi:10.1007 / s40265-016-0596-x). Despite the encouraging results obtained in CLL patients treated with venetoclax (Moia et al., 2018, supra), some studies have reported the eventual development of venetoclax resistance. Zhao et al. (Cancer Cell. 2019;35(5):752-766.e9.doi:10.1016 / j.ccell.2019.04.005) found that during venetoclax treatment, some populations of cells lost a region of chromosome 18 containing the Bcl-2 gene, which contributed to the survival of these cell populations. Tahir et al. (BMC Cancer. 2017;17(1):399.doi:10.1186 / s12885-017-3383-5) and Chiron et al. (Oncotarget. 2015;6(11):8750-9) independently identified another mechanism of venetoclax resistance in several cell lines based on upregulation of antiapoptotic MCL-1 and BCL-XL proteins, and downregulation of antiapoptotic BAX, BIM, and NOXA proteins. In addition, in cases of MCL, venetoclax resistance was achieved via an increase in the apoptotic threshold.Finally, Chiron et al. (2015; supra) reported that CD40-CD40L interactions between pathological cells and stroma could also circumvent venetoclax activity by potent activation of both classical and alternative NF-kB pathways, which mediate BCL-XL upregulation.

[0010] Human CC motif receptor 7 (hereafter referred to as "CCR7") is a seven-transmembrane domain G protein-coupled receptor (GPCR) that was initially found to be lymphocyte-selectively expressed upon EBV infection (Birkenbach et al., 1993, J. Virol. 67:2209-2220). CCR7 selectively binds two chemokines, designated CCL19 and CCL21. In homeostasis and inflammation, CCR7 is expressed on naive T and B lymphocytes, central memory T cells (TCM), some subsets of natural killer cells (NK cells), semi-mature and mature DCs, and plasmacytoid DCs (Forster R et al., Cell 1999;99:23-33; Comerford I et al., Cytokine Growth Factor Rev. 2013 June;24(3):269-83). In these leukocyte subsets, CCR7 regulates migration, organization, and activation.

[0011] Alfonso-Perez et al. (J Leukoc Biol. 2006 June;79(6):1157-65) disclose that anti-human CCR7 antibodies mediated potent complement-dependent cytotoxicity (CDC) against CLL cells while sparing normal T lymphocytes from the same patient, and that anti-human CCR7 antibodies blocked in vitro migration of CLL cells in response to physiological ligands of CCR7. Thus, WO 2007 / 003426 discloses the use of anti-human CCR7 antibodies to treat tumors expressing the CCR7 receptor, including hematological tumors such as CLL and MCL.

[0012] Patrussi et al. (Cancer Res. 2015 Oct. 1;75(19):4153-63) disclosed that ibrutinib treatment of CLL cells resulted in significant downregulation of surface CCR7, and based on this disclosure, combining an anti-CCR7 antibody with ibrutinib would not be expected to improve the therapeutic efficacy of the anti-CCR7 antibody.

[0013] In addition, De Rooij et al. (Blood, 2012;119(11):2590-4) reported that ibrutinib treatment of primary CLL cells impaired integrin-mediated adhesion and / or migration mediated by CCR7 activation. Thus, it is believed that ibrutinib may inhibit homing of CLL and B-cell lymphoma cells to secondary lymphoid organs (SLOs) where its ligand is produced. However, combining an antibody targeting CCR7 with ibrutinib would not be expected to improve therapeutic inhibition of malignant cell migration to SLOs. Furthermore, antibody-triggered target cell killing is less likely to occur due to loss of CCR7 on the target cell surface.

[0014] In addition to BTK inhibitors, B-cell malignancies (and CLL in particular) can be treated with inhibitors of the B-cell lymphoma 2 (Bcl-2) protein, an oncogenic protein known to inhibit apoptosis (Gentile et al., Expert Opin Investig Drugs. 2017;26(11):1307-1316). In cancer, CCR7 has long been thought to play a role in protecting T cells from apoptosis (Kim et al., Clin Cancer Res. 2005;11(21):7901-10). Furthermore, in the study by Kim et al., evaluation by multicolor flow cytometry showed a significant correlation between higher levels of Bcl-2 and higher expression of CCR7 in T cells, and activation of CCR7 caused phosphorylation of P3K / Akt and a subsequent increase in Bcl-2 expression. It is therefore believed that treating patients with Bcl-2 inhibitors may reduce the CCR7-induced anti-apoptotic effect and thus prevent the combination of these compounds with anti-CCR7 antibodies. Summary of the Invention [Problem to be solved by the invention]

[0015] It is therefore an object of the present invention to provide pharmaceutical and therapeutic approaches for preventing and treating hematological malignancies, particularly B-cell malignancies such as CLL, which overcome the disadvantages of prior art approaches. In particular, it is an object of the present invention to improve survival rates of such B-cell malignancies. [Means for solving the problem]

[0016] In a first aspect, the present invention relates to an anti-CCR7 antibody for use in the treatment of a hyperproliferative hematological disorder, the disorder being at least one of a) a disorder treated with at least one of a Bruton's tyrosine kinase (BTK) inhibitor and a B-cell lymphoma 2 (Bcl-2) inhibitor, b) a disorder that has relapsed after treatment with at least one of a BTK inhibitor and a Bcl-2 inhibitor, and c) a disorder that is resistant to treatment with at least one of a BTK inhibitor and a Bcl-2 inhibitor. In the treatment, the anti-CCR7 antibody can be administered simultaneously, separately, or sequentially with at least one of a BTK inhibitor and a Bcl-2 inhibitor. The hyperproliferative hematological disorder to be treated according to the present invention is preferably a disorder in which the hyperproliferative cells are cells of the B-cell lineage, more preferably the disorder is a B-cell hematological malignancy, most preferably a lymphoma or leukemia. Preferably, the hematological malignancy treated according to the present invention is a hematological malignancy selected from the group consisting of acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), acute monocytic leukemia (AMoL), chronic lymphocytic leukemia (CLL), high-risk CLL, small lymphocytic lymphoma (SLL), high-risk SLL, multiple myeloma (MM), non-Hodgkin's lymphoma (NHL), mantle cell lymphoma (MCL), follicular lymphoma (FL), Waldenstrom's macroglobulinemia (WM), diffuse large B-cell lymphoma (DLBCL), marginal zone lymphoma (MZL), Burkitt's lymphoma (BL), hairy cell leukemia (HCL), Richter's transformation, and T-cell prolymphocytic leukemia (T-PLL).

[0017] Anti-CCR7 antibodies for use in treatment according to the present invention preferably have an IC50 of 100 nM or less with respect to inhibiting at least one of CCR7-dependent intracellular signaling and CCR7 receptor internalization by at least one CCR7 ligand selected from CCL19 and CCL21. 50 The anti-CCR7 antibody further preferably inhibits CCR7-dependent intracellular signaling without substantial agonistic effects. Preferred anti-CCR7 antibodies for use in treatment according to the present invention have a K d K for the N-terminal extracellular domain of human CCR7 is up to 20-fold higher than d and the reference anti-CCR7 antibody is a murine anti-CCR7 antibody, the amino acid sequence of the heavy chain variable domain of which is SEQ ID NO: 1 and the amino acid sequence of the light chain variable domain of which is SEQ ID NO: 2. The anti-CCR7 antibody is preferably a chimeric, humanized or human antibody. A preferred chimeric, humanized or human anti-CCR7 antibody for use in the treatment according to the invention is an antibody having the HVR of the anti-human CCR7 antibody, the amino acid sequence of the heavy chain variable domain of which is SEQ ID NO: 1 and the amino acid sequence of the light chain variable domain of which is SEQ ID NO: 2.

[0018] Preferred BTK inhibitors for use in treatment according to the present invention are ibrutinib, zanbrutinib, or acalabrutinib, and preferred Bcl-2 inhibitors for use in treatment according to the present invention are venetoclax or navitoclax.

[0019] The hyperproliferative hematological disorders treated according to the present invention are preferably disorders in treatment-naive patients, more preferably, the hyperproliferative hematological disorders are disorders in patients who are treatment-naive with at least one of a BTK inhibitor, a Bcl-2 inhibitor, and an anti-CCR7 antibody.

[0020] In one embodiment, the hyperproliferative hematological disorder treated according to the invention is a hyperproliferative hematological disorder that is resistant to and / or has relapsed after treatment with a chemotherapeutic agent other than a BTK inhibitor, a Bcl-2 inhibitor, and an anti-CCR7 antibody, preferably one or more of fludarabine, cyclophosphamide, idelalisib, an anti-CD20 antibody, preferably rituximab, obinituzumab, ocrelizumab, veltuzumab, or ofatumumab, or an anti-CD52 antibody, preferably alemtuzumab.

[0021] In another embodiment, the hyperproliferative hematological disorder treated according to the present invention is a hyperproliferative hematological disorder that is resistant to and / or has relapsed following treatment with at least one of a BTK inhibitor and a Bcl-2 inhibitor. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0022] definition Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Those skilled in the art will recognize many methods and materials similar or equivalent to those described herein that could be used in the practice of the present invention. Indeed, the present invention is in no way limited to the methods and materials described.

[0023] For purposes of the present invention, the following terms are defined hereinafter.

[0024] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. For example, a method for administering a drug or an agent includes administration of multiple molecules (e.g., 10, 100, 1000, 10,000, 100,000, or more than 1 million molecules) and multiple drugs or agents.

[0025] As used herein, the term "and / or" indicates that one or more of the stated instances may occur alone or in combination with at least one of the stated instances up to all of the stated instances.

[0026] As used herein, a particular value followed by "at least" means a value greater than or equal to the particular value. For example, "at least 2" should be understood to be the same as "2 or greater," i.e., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, ... etc.

[0027] As used herein, "cancer" and "cancerous" refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. Cancer is also called a malignant neoplasm.

[0028] As used herein, "in combination with" is intended to refer to all forms of administration that provide a first drug together with an additional (second, third) drug. The drugs can be administered simultaneously, separately, or sequentially and in any order, unless otherwise specified. Drugs administered in combination have coordinated biological activity to the subject to which they are delivered.

[0029] As used herein, "simultaneous" administration refers to the administration of two or more drugs at the same time, not necessarily via the same route of administration or in the form of one combined formulation. For example, one drug may be administered orally and the other intravenously during a patient visit. Separately includes administration of drugs in separate forms and / or at separate times, again not necessarily via the same route of administration. Sequentially refers to administration of a first drug followed immediately by administration of a second drug, or a predetermined time apart from administration of the first drug.

[0030] As used herein, the word "comprise" and its conjugations are used in an open-ended sense and mean that the items following the word are included but not to the exclusion of items not specifically mentioned. This word also encompasses the more restrictive "consisting of."

[0031] As used herein, "compositions," "products," or "combinations" useful in the methods of the disclosure include those suitable for a variety of routes of administration, including, but not limited to, intravenous, subcutaneous, intradermal, subdermal, intranodal, intratumoral, intramuscular, intraperitoneal, oral, nasal, topical (including buccal and sublingual), rectal, vaginal, aerosol, and / or parenteral or mucosal application. Compositions, formulations, and products of the disclosure or invention typically include a drug (alone or in combination) and one or more suitable pharma- ceutically acceptable excipients.

[0032] As used herein, "effective amount" refers to the amount of drug required to ameliorate the symptoms of a disease compared to an untreated patient. The effective amount of the active agent(s) used to practice the present invention for the therapeutic treatment of cancer will vary depending on the method of administration, the age, weight, and general health of the subject. Ultimately, the attending physician or veterinarian will determine the appropriate amount and administration schedule. Such an amount is referred to as an "effective" amount. Thus, in the context of this disclosure, in the context of administering a drug, "effective against" a disease or condition indicates that administration in a clinically relevant manner will result in at least a statistically significant proportion of patients with a beneficial effect, such as amelioration of symptoms, cure, reduction of at least one disease sign or symptom, prolongation of life, improvement in quality of life, or other effect generally recognized as good by physicians familiar with treating a particular type of disease or condition.

[0033] The term "antibody" is used in the broadest sense and specifically includes, for example, antagonists, neutralizing antibodies, single anti-CCR7 monoclonal antibodies, including full-length or complete monoclonal antibodies, anti-CCR7 antibody compositions with polyepitopic specificity, polyclonal antibodies, multivalent antibodies, single-chain anti-CCR7 antibodies, and fragments of anti-CCR7 antibodies, including Fab, Fab', F(ab')2, and Fv fragments, diabodies, single domain antibodies (sdAbs) (see below), so long as they exhibit the desired biological and / or immunological activity. The term "immunoglobulin" (Ig) is used interchangeably with antibody herein. Antibodies may be human and / or humanized antibodies.

[0034] The term "anti-CCR7 antibody" or "antibody that binds to CCR7" refers to an antibody capable of binding to CCR7 with sufficient affinity to be useful as a diagnostic and / or therapeutic agent in targeting CCR7. Preferably, the extent of binding of the anti-CCR7 antibody to unrelated non-CCR7 proteins is less than about 10% of the binding of the antibody to CCR7, as measured, for example, by radioimmunoassay (RIA) or ELISA. In certain embodiments, an antibody that binds to CCR7 has a dissociation constant (K d In certain embodiments, the anti-CCR7 antibody binds to an epitope of CCR7 that is conserved among CCR7 from different species. It is further understood herein that the term "anti-CCR7 antibody" as used herein includes fragments of antibodies that bind to CCR7.

[0035] An "isolated antibody" is one that has been identified and separated and / or recovered from a component of its natural environment.

[0036] The basic four-chain antibody unit is a heterotetrameric glycoprotein composed of two identical light (L) chains and two identical heavy (H) chains (IgM antibodies consist of five basic heterotetrameric units and an additional polypeptide called the J chain, thus containing 10 antigen-binding sites, and secretory IgA antibodies can polymerize to form multivalent assemblies containing two to five basic four-chain units and a J chain). For IgG, the four-chain unit is generally about 150,000 daltons. Each L chain is linked to a H chain by one covalent disulfide bond, and the two H chains are linked to each other by one or more disulfide bonds depending on the H chain isotype. Each H and L chain also has regularly spaced intrachain disulfide bridges. Each H chain contains a variable domain (V H ), followed by three constant domains for each of the α and γ chains (C H ), and four C for μ and ε isotypes. H Each L chain has a variable domain (V L ), followed by a constant domain (C L ) V L is V H and C L is the first constant domain of the heavy chain (C H 1). Certain amino acid residues are believed to form an interface between the light-chain variable domain and the heavy-chain variable domain. H and V L Together, the pairing of these two amino acids forms a single antigen-binding site. Regarding the structure and properties of different classes of antibodies, see, e.g., Basic and Clinical Immunology, 8th ed., Daniel P. Stites, Abba I. Terr, and Tristram G. Parslow (eds.), Appleton & Lange, Norwalk, CT, 1994, Chapter 6, p. 71.

[0037] The L chains from any vertebrate species can be assigned to one of two clearly distinct types, called kappa and lambda, based on the amino acid sequence of the constant domain. The heavy chain constant domain (C HDepending on the amino acid sequence of the immunoglobulins, they can be assigned to different classes or isotypes. There are five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, with heavy chains designated α, δ, ε, γ, and μ, respectively. The γ and α classes are H They are further divided into subclasses based on relatively minor differences in sequence and function; for example, humans express the following subclasses: IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2.

[0038] The "variable region" or "variable domain" of an antibody refers to the amino-terminal domain of either the heavy or light chain of an antibody. The variable domain of the heavy chain is designated "V H The variable domain of the light chain is sometimes called the "V L These domains are generally the most variable parts of the antibody and contain the antigen-binding sites.

[0039] The term "variable" refers to the fact that certain segments of the variable domains differ widely in sequence among antibodies. The V domains mediate antigen binding and define the specificity of a particular antibody for a particular antigen. However, variability is not evenly distributed across the 110 amino acid span of the variable domains. Instead, the V regions consist of relatively invariant stretches of 15-33 amino acids called framework regions (FRs) separated by shorter highly variable regions called "hypervariable regions" (HVRs) of 7-25 amino acids in length. Native heavy and light chain variable domains each contain four FRs connected by three hypervariable regions, mostly in a β-sheet configuration, which form loops that connect and in some cases form part of the β-sheet structure. The hypervariable regions in each chain are held together in close proximity by the FRs and, together with the hypervariable regions from the other chain, contribute to the formation of the antigen-binding site of antibodies (see Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD. (1991)). The constant domains are not directly involved in binding the antibody to an antigen, but are involved in various effector functions, such as antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and antibody-dependent cellular phagocytosis (ADCP).

[0040] A "complete" antibody is one that comprises the antigen binding site, the C and at least the heavy chain constant domain, i.e., H 1. C H 2, and C H 3. The constant domains may be native sequence constant domains (e.g., human native sequence constant domains) or amino acid sequence variants thereof. Preferably, the intact antibody has one or more effector functions.

[0041] A "naked antibody" for purposes herein is an antibody that is not conjugated to a cytotoxic moiety or radiolabel.

[0042] An "antibody fragment" comprises a portion of an intact antibody, preferably the antigen-binding or variable region of the intact antibody. Examples of antibody fragments include Fab, Fab', F(ab') 2 and Fv fragments; diabodies; linear antibodies (see U.S. Pat. No. 5,641,870, Example 2; Zapata et al., Protein Eng. 8(10):1057-1062

[1995] ); single-chain antibody molecules; and multispecific antibodies formed from antibody fragments. In one embodiment, an antibody fragment contains the antigen-binding site of an intact antibody and thus retains the ability to bind antigen.

[0043] The Fc fragment contains the carboxy-terminal portions of both H chains held together by disulfides. The effector functions of an antibody are determined by sequences in the Fc region, which is also the portion recognized by Fc receptors (FcR) found on certain types of cells.

[0044] The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific and directed against a single antigenic site, in contrast to polyclonal antibody preparations which include different antibodies, each directed against a different determinant (epitope). Monoclonal antibodies are advantageous in that they may be synthesized uncontaminated by other antibodies. The modifier "monoclonal" should not be construed to require production of the antibody by any particular method. For example, monoclonal antibodies useful in the present invention may be prepared by the hybridoma method first described in Kohler et al., Nature, 256:495 (1975), or may be made using recombinant DNA methods in bacterial, eukaryotic, or plant cells (see, e.g., U.S. Pat. No. 4,816,567). The "monoclonal antibodies" may also be isolated from phage antibody libraries using the techniques described in, for example, Clackson et al., Nature, 352:624-628 (1991) and Marks et al., J. Mol. Biol., 222:581-597 (1991).

[0045] Monoclonal antibodies herein include "chimeric" antibodies in which a portion of the heavy and / or light chain is identical or homologous to corresponding sequences in antibodies from a particular species or belonging to a particular antibody class or subclass, and the remainder of the chain(s) is identical or homologous to corresponding sequences in antibodies from another species or belonging to another antibody class or subclass, and fragments of such antibodies, so long as they exhibit the desired biological activity (see U.S. Pat. No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)). Chimeric antibodies of interest herein include "primatized" antibodies that contain variable domain antigen-binding sequences derived from a non-human primate (e.g., Old World Monkey, Ape, etc.) and human constant region sequences.

[0046] "Humanized" forms of non-human (e.g. rodent) antibodies are chimeric antibodies that contain minimal sequence derived from the non-human antibody. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit, or non-human primate having the desired specificity, affinity, and capacity of the antibody. In some cases, some framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are not found in the recipient antibody or the donor antibody. These modifications are made to further improve antibody performance. In general, humanized antibodies may contain typically two variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence. The humanized antibody may optionally also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992). See also the following review articles and references cited therein: Vaswani and Hamilton, Ann. Allergy, Asthma and Immunol., 1:105-115 (1998); Harris, Biochem. Soc. Transactions, 23:1035-1038 (1995); Hurle and Gross, Curr. Op. Biotech., 5:428-433 (1994).

[0047] The term "hypervariable region," "HVR," as used herein, refers to the regions of an antibody variable domain that are hypervariable in sequence and / or form structurally defined loops responsible for antigen binding. Generally, antibodies contain six hypervariable regions, three in the VH (H1, H2, H3) and three in the VL (L1, L2, L3). Several hypervariable region delineations are in use and are encompassed herein. The hypervariable regions generally consist of amino acid residues from the "complementarity determining regions" or "CDRs" (e.g., around residues 24-34 (L1), 50-56 (L2), and 89-97 (L3) in the VL and around residues 31-35 (H1), 50-65 (H2), and 95-102 (H3) in the VH, as numbered according to the Kabat numbering system; see Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, Md. (1991)), and / or amino acid residues from the "hypervariable loops" (e.g., residues 24-34 (L1), 50-56 (L2), and 89-97 (L3) in VL and 26-32 (H1), 52-56 (H2), and 95-101 (H3) in VH, as numbered according to the Chothia numbering system; Chothia and Lesk, J. Mol. B iol. 196:901-917 (1987)), and / or amino acid residues from the "hypervariable loops" / CDRs (e.g., residues 27-38 (L1), 56-65 (L2), and 105-120 (L3) in VL and 27-38 (H1), 56-65 (H2), and 105-120 (H3) in VH, as numbered according to the IMGT numbering system; Lefranc, MP et al., Nucl. Acids Res. 27:209-212 (1999); Ruiz, M. et al., Nucl. Acids Res. 28:219-221 (2000)).Optionally, the antibody has symmetric insertions at one or more of the following points: 28, 36 (L1), 63, 74-75 (L2), and 123 (L3) in VL, and 28, 36 (H1), 63, 74-75 (H2), and 123 (H3) in VH, when numbered according to Honneger, A. and Plunkthun, AJ (Mol. Biol. 309:657-670 (2001)). The hypervariable regions / CDRs of the antibodies of the invention are preferably defined and numbered according to the IMGT numbering system.

[0048] "Framework" or "FR" residues are those variable domain residues other than the hypervariable region residues as herein defined.

[0049] A "blocking" or "antagonist" antibody is an antibody that inhibits or reduces the biological activity of the antigen to which it binds. Preferred blocking or antagonist antibodies substantially or completely inhibit the biological activity of the antigen.

[0050] An "agonist antibody," as used herein, is an antibody that mimics at least one functional activity of a polypeptide of interest.

[0051] "Binding affinity" generally refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, as used herein, "binding affinity" refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity between a molecule X and a partner Y is generally determined by the dissociation constant (K d) Affinity can be measured by common methods known in the art, including those described herein. Low affinity antibodies generally bind antigens slowly and tend to dissociate easily, whereas high affinity antibodies generally bind antigens faster and tend to remain bound longer. A variety of methods for measuring binding affinity are known in the art, any of which can be used for the purposes of the present invention. Specific illustrative embodiments are described below.

[0052] "K d " or "K d "Values" can be measured by using a surface plasmon resonance assay using a BIAcore™-2000 or Biacore™-3000 (BIAcore, Inc., Piscataway, NJ) at 25°C with an immobilized antigen CM5 chip of approximately 10-50 response units (RU). Briefly, a carboxymethylated dextran biosensor chip (CM5, BIAcore Inc.) is activated with N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions. Antigen is diluted to 5 μg / ml (approximately 0.2 μM) with 10 mM sodium acetate, pH 4.8, and then injected at a flow rate of 5 μl / min to achieve approximately 10 response units (RU) of bound protein. After injection of antigen, 1 M ethanolamine is injected to block unreacted groups. For kinetic measurements, two-fold serial dilutions of antibody or Fab (0.78 nM to 500 nM) are injected at a flow rate of approximately 25 μl / min in PBS containing 0.05% Tween 20 (PBST) at 25 °C. The association rate (k on ) and dissociation rate (k off ) is calculated by simultaneously fitting the association and dissociation sensorgrams using a simple one-to-one Langmuir binding model (BIAcore Evaluation Software, version 3.2). d ) to k off / k onSee, e.g., Chen, Y. et al. (1999) J. Mol Biol 293:865-881. If the on-rate is 10 6 M -1 S -1 If the on-rate exceeds , the on-rate can be determined by using a fluorescence quenching technique that measures the increase or decrease in fluorescence emission intensity (excitation = 295 nm; emission = 340 nm, 16 nm bandpass) at 25 °C of 20 nM anti-antigen antibody (Fab form) in PBS pH 7.2 in the presence of increasing concentrations of antigen as measured in a spectrometer, for example a stop-flow equipped spectrophotometer (Aviv Instruments) or an 8000 series SLM-Aminco spectrophotometer with a stirred cuvette (ThermoSpectronic).

[0053] The "on-rate" or "rate of association" or "association rate" or "k on " can also be determined using the same surface plasmon resonance techniques described above using a Biacore™-2000 or Biacore™-3000 (BIAcore, Inc., Piscataway, NJ) as described above.

[0054] An antibody that "binds" to an antigen of interest, e.g., a polypeptide CCR7 antigen target, is an antibody that binds to the antigen with sufficient affinity to be useful as a therapeutic agent in targeting cells or tissues expressing the antigen and does not significantly cross-react with other proteins. In such embodiments, the extent of binding of the antibody to a "non-target" protein may be less than about 10% of the binding of the antibody to its particular target protein, as determined by fluorescence-activated cell sorting (FACS) analysis or radioimmunoprecipitation (RIA). With respect to the binding of an antibody to a target molecule, the term "specific binding" or "specifically binding to" or "specific for" a particular polypeptide or epitope of a particular polypeptide target refers to binding that is measurably different from non-specific interactions. Specific binding can be measured, for example, by determining the binding of a molecule relative to the binding of a control molecule, which is generally a molecule of similar structure that does not have binding activity. For example, specific binding can be determined by competition with a control molecule similar to the target, e.g., an excess of unlabeled target. In this case, specific binding is indicated when the binding of the labeled target to the probe is competitively inhibited by an excess of unlabeled target. The term "specific binding," or "specifically binding to" or "specific for" a particular polypeptide or epitope of a particular polypeptide target, as used herein, refers to, for example, at least about 10 -4 M, or at least about 10 -5 M, or at least about 10 -6 M, or at least about 10 -7 M, or at least about 10 -8 M, or at least about 10 -9 M, or at least about 10 -10 M, or at least about 10 -11 M, or at least about 10 -12 K against M or more targets d(which can be determined as described above). In one embodiment, the term "specific binding" refers to binding when a molecule binds to a particular polypeptide or epitope of a particular polypeptide without substantially binding to any other polypeptides or polypeptide epitopes.

[0055] Antibody "effector functions" refer to biological activities attributable to the Fc region of an antibody (either a native sequence Fc region or an amino acid sequence variant Fc region) and which vary with antibody isotype. Examples of antibody effector functions include C1q binding and complement dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); antibody-dependent cell-mediated phagocytosis (ADCP); down-regulation of cell surface receptors (e.g., B cell receptor); and B cell activation.

[0056] The term "Fc region" is used herein to define the C-terminal region of an immunoglobulin heavy chain, including native sequence Fc regions and variant Fc regions. Although the boundaries of an immunoglobulin heavy chain Fc region may vary, the human IgG heavy chain Fc region is usually defined to extend from an amino acid residue at position Cys226 or Pro230 to its carboxyl terminus. The C-terminal lysine of the Fc region (residue 447 according to the EU numbering system) may be removed, for example, during antibody production or purification, or by recombinantly engineering the nucleic acid encoding the antibody heavy chain. Thus, a complete antibody composition may include an antibody population with all K447 residues removed, an antibody population with none of the K447 residues removed, and an antibody population with a mixture of antibodies with and without the K447 residue.

[0057] A "functional Fc region" possesses an "effector function" of a native sequence Fc region. Exemplary "effector functions" include C1q binding; CDC; Fc receptor binding; ADCC; phagocytosis; downregulation of cell surface receptors (e.g., B cell receptor, BCR), and the like. Such effector functions generally require that the Fc region be associated with a binding domain (e.g., an antibody variable domain) and can be assessed, for example, using the various assays disclosed in the definitions herein.

[0058] "Antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a form of cytotoxicity in which secreted Ig bound to Fc receptors (FcR) present on certain cytotoxic cells (e.g., natural killer (NK) cells, neutrophils, and macrophages) enables these cytotoxic effector cells to specifically bind to antigen-bearing target cells and subsequently kill the target cells with cytotoxins. Antibodies "arm" the cytotoxic cells and are absolutely required for such killing. NK cells, the primary cells involved in mediating ADCC, express only FcγRIII, whereas monocytes express FcγRI, FcγRII, and FcγRIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-92 (1991). To assess the ADCC activity of a molecule of interest, an in vitro ADCC assay may be performed, such as those described in U.S. Pat. No. 5,500,362 or U.S. Pat. No. 5,821,337. Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively or additionally, the ADCC activity of a molecule of interest may be assessed in vivo, for example in an animal model, such as those disclosed in Clynes et al. (USA) 95:652-656 (1998). WO 2000 / 42072 (Presta) describes antibody variants with improved or diminished binding to FcR. See also, e.g., Shields et al., J. Biol. Chem. 9(2):6591-6604 (2001).

[0059] A "human effector cell" is a leukocyte that expresses one or more FcRs and exerts effector function. Preferably, the cell expresses at least FcγRIII and exerts ADCC effector function. Examples of human leukocytes that mediate ADCC include peripheral blood mononuclear cells (PBMCs), natural killer (NK) cells, monocytes, cytotoxic T cells, and neutrophils, with PBMCs and NK cells being preferred. Effector cells may be isolated from a natural source, such as blood.

[0060] "Complement dependent cytotoxicity" or "CDC" refers to the lysis of target cells in the presence of complement. Activation of the classical complement pathway is initiated by the binding of the first component of the complement system (C1q) to an antibody (of the appropriate subclass) that binds to a cognate antigen. To assess complement activation, a CDC assay may be performed, e.g. as described in Gazzano-Santoro et al. (1996, J. Immunol. Methods 202:163). Antibody variants with altered Fc region amino acid sequences (antibodies with variant Fc regions) and with improved or reduced C1q binding ability are described, e.g., in U.S. Pat. No. 6,194,551 (B1) and WO 1999 / 51642. See also, e.g., Idusogie et al. (2000, J. Immunol. 164:4178-4184). One substitution that improves C1q binding and thereby improves CDC activity is the E333A substitution, which can be advantageously applied to the antibodies of the present invention.

[0061] "Sequence identity" is defined herein as the relationship between sequences determined by comparing two or more amino acid (polypeptide or protein) sequences or two or more nucleic acid (polynucleotide) sequences. In the art, "identity" also means the degree of sequence relatedness between such sequences, as determined by the match between strings of amino acid or nucleic acid sequences, as the case may be. "Similarity" between two amino acid sequences is determined by comparing the amino acid sequence of one polypeptide and its conserved amino acid substitutes with the sequence of a second polypeptide. "Identity" and "similarity" can be readily calculated by known methods. The term "sequence identity" or "sequence similarity" means that two (poly)peptide or two nucleotide sequences, when optimally aligned, preferably over their entire length (at least of the shortest sequence in the comparison), share at least a certain percentage of sequence identity, as defined elsewhere herein, when maximized and minimized by, for example, the ClustalW (1.83), GAP, or BESTFIT programs using default parameters. GAP uses the Needleman and Wunsch global alignment algorithm to align two sequences over their entire length, maximizing the number of matches and minimizing the number of gaps. Generally, the following GAP default parameters are used: gap creation penalty = 50 (nucleotides) / 8 (proteins) and gap extension penalty = 3 (nucleotides) / 2 (proteins). For nucleotides, the default score matrix used is nwsgapdna, and for proteins, the default score matrix is ​​Blosum62 (Henikoff and Henikoff, 1992, PNAS 89, 915-919). A preferred multiple alignment program for aligning the protein sequences of the present invention is ClustalW (1.83), using the blosum matrix and default settings (gap opening penalty: 10, gap extension penalty: 0.05).Sequence alignment and scores for sequence identity percentage can be determined using computer programs such as the GCG Wisconsin Package, version 10.3, available from Accelrys Inc., 9685 Scranton Road, San Diego, CA 92121-3752 USA, or using open source software such as the programs "needle" (using the global Needleman Wunsch algorithm) or "water" (using the local Smith Waterman algorithm) in EmbossWIN, version 2.10.0, using the same parameters as for GAP above or using the default settings (for both "needle" and "water", the initial gap opening penalty is 10.0 and the initial gap extension penalty is 0.5 for both protein and DNA alignments; the initial score matrix is ​​Bloom62 for proteins and DNAFull for DNA). When sequences have substantially different overall lengths, local alignments are preferred, such as local alignments using the Smith Waterman algorithm. Alternatively, the percentage of similarity or identity may be determined by searching public databases using algorithms such as FASTA, BLAST, etc.

[0062] [Detailed Description of the Invention] The present invention is based on the unexpected discovery that, unlike previous reports that BTK inhibitors such as ibrutinib can downregulate or induce complete loss of CCR7 receptor in chronic lymphocytic leukemia (CLL) cells (thus preventing the use of anti-CCR7 antibodies in CLL patients), the inventors studied a large cohort of CLL samples and found no (or only slightly) difference in CCR7 expression levels between CLL patients treated with ibrutinib and those not treated with ibrutinib. Moreover, the CCR7 expression levels in resistant / relapsed patients were comparable or even higher compared to control untreated patients. In addition, the inventors unexpectedly found that anti-CCR7 antibodies completely blocked CCR7 migration in CLL cells, while BTK inhibitors showed only a minor reduction in this process. Finally, the inventors found that the use of anti-CCR7 antibodies effectively killed CLL cells from patients undergoing treatment with BTK inhibitors and from patients with BTK inhibitor relapsed / resistant disease.

[0063] In the same cohort of CLL patients, the inventors observed that CCR7 expression was preserved in CLL patients following treatment with Bcl-2 inhibitors such as venetoclax, and confirmed that in these patients anti-CCR7 in vitro therapy caused inhibition of CCR7 functionality and induced target cell killing.

[0064] This has led to the development of monoclonal antibodies (mAbs) against CCR7, i.e., that recognize an epitope in the CCR7 receptor and are preferably capable of inhibiting CCR7-dependent intracellular signaling and that inhibit CCR7, for treating CLL and other hyperproliferative blood disorders in combination with at least one of a BTK inhibitor and a Bcl-2 inhibitor, as well as for treating such disorders that have relapsed after or become resistant to treatment with at least one of a BTK inhibitor and a Bcl-2 inhibitor. +It opens up the possibility of using antibodies capable of killing tumor cells and / or preventing their migration, activation, proliferation and / or dissemination in vivo.

[0065] Thus, in a first aspect, the present invention relates to an anti-CCR7 antibody for use in the treatment of a hyperproliferative hematological disorder, which is preferably at least one of a) disorders treated with a Bruton's tyrosine kinase (BTK) inhibitor, b) disorders treated with a B-cell lymphoma 2 (Bcl-2) inhibitor, c) disorders treated with a combination of a BTK inhibitor and a Bcl-2 inhibitor, d) disorders that have relapsed after treatment with a BTK inhibitor, e) disorders that have relapsed after treatment with a BTK inhibitor, f) disorders that have relapsed after treatment with a combination of a BTK inhibitor and a Bcl-2 inhibitor, g) disorders that are resistant to treatment with a BTK inhibitor, h) disorders that are resistant to treatment with a Bcl-2 inhibitor, and i) disorders that are resistant to treatment with a combination of a BTK inhibitor and a Bcl-2 inhibitor.

[0066] Thus, in one embodiment, the hyperproliferative blood disorder is treated with a combination of a BTK inhibitor and an anti-CCR7 antibody. In another embodiment, the hyperproliferative blood disorder is treated with a combination of a Bcl-2 inhibitor and an anti-CCR7 antibody. In another embodiment, the hyperproliferative blood disorder is treated with a combination of a BTK inhibitor, a Bcl-2 inhibitor and an anti-CCR7 antibody. In the treatment, the anti-CCR7 antibody can be administered simultaneously, separately or sequentially with the BTK inhibitor and / or the Bcl-2 inhibitor.

[0067] The hyperproliferative hematological disorders treated according to the present invention are disorders in a "subject" or "patient," the term "subject" or "patient" referring to all animals classified as mammals, including but not limited to primates and humans. The subject to be treated is preferably a human, male or female, and may be of any age or race. Treatment of a subject or patient includes first line, second line, or third line treatment.

[0068] The term "combination" as used herein is understood to refer to a combination therapy (as opposed to a monotherapy) in which the treatment includes the use or administration of an anti-CCR7 antibody and the use or administration of at least one of a BTK inhibitor and a Bcl-2 inhibitor. Thus, in the combination therapy of the present invention, the components of the combination can be administered simultaneously, separately, or sequentially. Thus, the components of the combination can be formulated in a single composition, or the components can be formulated in at least two separate formulations. The combination can be a single product that includes a single composition or includes the components formulated in at least two separate formulations. Alternatively, the combination can be at least two different products that can be from one or more suppliers.

[0069] In some embodiments, the hyperproliferative hematological disorder to be treated according to the present invention, preferably with a combination of an anti-CCR7 antibody and at least one of a BTK inhibitor and a Bcl-2 inhibitor, is a disorder in a treatment-naive patient, e.g., a patient who has not received prior treatment or at least prior chemotherapy treatment, chemotherapy treatment having the broad meaning defined below. More preferably, a treatment-naive patient is a patient who has not received prior treatment with a Bcl-2 inhibitor when the anti-CCR7 antibody is administered in combination with a BTK inhibitor, or a patient who has not received prior treatment with a BTK inhibitor when the anti-CCR7 antibody is administered in combination with a Bcl-2 inhibitor. Thus, "pre-treatment naive" should be understood herein as an individual who has not been treated with the respective inhibitor before the start of the respective combination therapy. Combination treatment of a treatment-naive patient with an anti-CCR7 antibody and at least one of a BTK inhibitor and a Bcl-2 inhibitor reduces the risk that the patient will become resistant to one of the components in the treatment and relapse.

[0070] In some embodiments, the hyperproliferative blood disorder treated according to the present invention, preferably with a combination of an anti-CCR7 antibody and at least one of a BTK inhibitor and a Bcl-2 inhibitor, is a disorder in a patient who is at least naive to treatment with at least one of a BTK inhibitor, a Bcl-2 inhibitor, and an anti-CCR7 antibody. However, a patient who is at least naive to treatment with at least one of a BTK inhibitor, a Bcl-2 inhibitor, and an anti-CCR7 antibody may be a patient who is resistant to and / or has relapsed after treatment with a chemotherapeutic agent other than at least one of a BTK inhibitor, a Bcl-2 inhibitor, and an anti-CCR7 antibody.

[0071] Thus, in some embodiments, the patient is resistant to and / or relapses following treatment with a chemotherapeutic agent other than at least one of a BTK inhibitor, a Bcl-2 inhibitor, and an anti-CCR7 antibody. The term "other chemotherapeutic agent" is understood herein to have a broad meaning, including biological therapeutic agents, such as antibodies and other proteins, antisense molecules, gene therapy vectors, and cell therapy.

[0072] In some embodiments, the "other chemotherapeutic agent" is selected from the group consisting of antimitotic agents, alkylating agents, antimetabolites, anthracyclines, vinca alkaloids, plant alkaloids, nitrogen mustards, proteasome inhibitors, intercalating antibiotics, growth factor inhibitors, cell cycle inhibitors, biological response modifiers, antihormones, angiogenesis inhibitors, antiandrogens, DNA interacting agents, purine analogs, topoisomerase I inhibitors, topoisomerase II inhibitors, tubulin interacting agents, hormonal agents, thymidylate synthase inhibitors, non-BTK tyrosine kinase inhibitors, P13K delta tyrosine kinase inhibitors, EGF inhibitors, VEGF inhibitors, CDK inhibitors, SRC inhibitors, c-Kit inhibitors, Her1 / 2 inhibitors, myc inhibitors, anti-tumor antibodies, monoclonal antibodies directed against growth factor receptors, protein kinase modulators, radioisotopes, immunotherapy, glucocorticoids, and combinations thereof.

[0073] In some embodiments, the "other chemotherapeutic agent" is an anti-cancer agent selected from the group consisting of DNA interacting agents such as cisplatin or doxorubicin; topoisomerase II inhibitors such as etoposide; topoisomerase I inhibitors such as CPT-11 or topotecan; naturally occurring or synthetic tubulin interacting agents such as paclitaxel, docetaxel, or epothilones (e.g., ixabepilone); hormonal agents such as tamoxifen; thymidylate synthase inhibitors such as 5-fluorouracil; and antimetabolites such as methotrexate; other tyrosine kinase inhibitors such as Iressa and OSI-774; angiogenesis inhibitors, EGF inhibitors; VEGF inhibitors; CDK inhibitors; SRC inhibitors; c-Kit inhibitors; Her1 / 2 inhibitors, and monoclonal antibodies against growth factor receptors such as Erbitux (EGF) and Herceptin (Her2); other protein kinase modulators, and combinations thereof. Other anti-cancer agents that may be used in the methods of the present invention will be known to those of skill in the art of oncology.

[0074] In some embodiments, the "other chemotherapeutic agents" are selected from the group consisting of proteasome inhibitors, bortezomib (Velcade®), carfilzomib (PR-171), PR-047, disulfiram, lactacystin, PS-519, eponemycin, epoxomicin, aclacinomycin, CEP-1612, MG-132, CVT-63417, (-)-7-methylomuralide, (+ / -)-7-methylomuralide, PS-341, vinylsulfone tripeptide inhibitors, ritonavir, PI-083, lenalidomide, and combinations thereof.

[0075] In some embodiments, the "other chemotherapeutic agent" may be, for example, "CHOP" (a combination including (i) cyclophosphamide, such as Cytoxan, (ii) other topoisomerase II inhibitors, such as doxorubicin or adriamycin, (iii) other vincas, such as vincristine or oncovin, and (iv) a steroid, such as hydrocortisone or prednisolone), "R-CHOP" (a combination including Rituxan, cyclophosphamide, doxorubicin, vincristine, and prednisone), "ICE" (a combination including ifosfamide, carboplatin, and etoposide), "R-ICE" (a combination including Rituxan, ifosfamide, carboplatin, and etoposide), or "R-ICE" (a combination including Rituxan, ifosfamide, carboplatin, and etoposide). and chemotherapy combinations such as "R-ACVBP" (a combination containing rituximab, doxorubicin, cyclophosphamide, vincristine, bleomycin, and prednisone), "DA-EPOCH-R" (a combination of dose-adjusted etoposide, doxorubicin, cyclophosphamide, vincristine, prednisone, and rituximab), "R-bendamustine" (a combination of bendamustine and rituximab), "GemOx or R-GemOx" (a combination of gemcitabine and oxaliplatin with or without rituximab), and "DHAP" (a combination containing dexamethasone, cytarabine, and cisplatin).

[0076] In a preferred embodiment, the "other chemotherapeutic agent" is fludarabine, cyclophosphamide, idelalisib, an anti-CD20 antibody, preferably rituximab, obinituzumab, ocrelizumab, veltuzumab, or ofatumumab, or an anti-CD52 antibody, preferably alemtuzumab, or a combination thereof.

[0077] In another embodiment of the present invention, the hyperproliferative blood disorder is resistant to and / or relapses after treatment with a BTK inhibitor, preferably the BTK inhibitor is as defined herein below. As exemplified herein, the hyperproliferative blood disorder may be resistant to and / or relapses after treatment with a BTK inhibitor, such as ibrutinib, acalabrutinib, and zanbrutinib. The hyperproliferative blood disorder treated according to the present invention may be a disorder that is resistant to and / or relapses after treatment with a BTK inhibitor when the BTK inhibitor is used as the sole agent in the treatment of the disorder (i.e., used as monotherapy). Alternatively, the hyperproliferative blood disorder treated according to the present invention may be a disorder that is resistant to and / or relapses after treatment with a BTK inhibitor when the BTK inhibitor is used in combination with another chemotherapeutic agent (i.e., used as combination therapy). The other chemotherapeutic agent may be "other chemotherapeutic agent" as defined herein above. In a preferred embodiment, the "other chemotherapeutic agent" is one or more of fludarabine, cyclophosphamide, idelalisib, an anti-CD20 antibody, preferably rituximab, obinituzumab, ocrelizumab, veltuzumab, or ofatumumab, or an anti-CD52 antibody, preferably alemtuzumab. In another embodiment, the hyperproliferative hematological disorder treated according to the present invention may be a disorder that is resistant to and / or has relapsed after successive treatments, each treatment being with a different chemotherapeutic agent or combination thereof, one of the treatments including the use of a BTK inhibitor (alone or in combination therapy). In this embodiment, the different chemotherapeutic agent or combination thereof may be "other chemotherapeutic agent" as defined herein above.

[0078] In yet another embodiment of the invention, the hyperproliferative blood disorder is resistant to and / or has relapsed after treatment with a Bcl-2 inhibitor, preferably the Bcl-2 inhibitor being as defined herein below. As exemplified herein, the hyperproliferative blood disorder may be resistant to and / or has relapsed after treatment with a Bcl-2 inhibitor, such as venetoclax. The hyperproliferative blood disorder treated according to the invention may be a disorder that is resistant to and / or has relapsed after treatment with a Bcl-2 inhibitor when the Bcl-2 inhibitor is used as the sole agent in the treatment of the disorder (i.e. used as monotherapy). Alternatively, the hyperproliferative blood disorder treated according to the invention may be a disorder that is resistant to and / or has relapsed after treatment with a Bcl-2 inhibitor when the Bcl-2 inhibitor is used in combination with another chemotherapeutic agent (i.e. used as combination therapy). The other chemotherapeutic agent may be the "other chemotherapeutic agent" as defined herein above. In a preferred embodiment, the "other chemotherapeutic agent" is one or more of fludarabine, cyclophosphamide, ibrutinib, idelalisib, anti-CD20 antibody, preferably rituximab, obinituzumab, ocrelizumab, veltuzumab, or ofatumumab, or anti-CD52 antibody, preferably alemtuzumab. In another embodiment, the hyperproliferative hematological disorder treated according to the present invention may be a disorder that is resistant to and / or has relapsed after successive treatments, each treatment being with a different chemotherapeutic agent or combination thereof, one of the treatments including the use of a Bcl-2 inhibitor (alone or in combination therapy). In this embodiment, the different chemotherapeutic agent or combination thereof may be "other chemotherapeutic agent" as defined herein above.

[0079] In yet another embodiment of the present invention, the hyperproliferative blood disorder is resistant to and / or has relapsed after treatment with a combination of a BTK inhibitor and a Bcl-2 inhibitor, preferably the BTK inhibitor and the Bcl-2 inhibitor are as defined herein below. As exemplified herein, the hyperproliferative blood disorder may be resistant to and / or has relapsed after treatment with a combination of a BTK inhibitor, such as ibrutinib, acalabrutinib, and zanbrutinib, and a Bcl-2 inhibitor, such as venetoclax. The hyperproliferative blood disorder treated according to the present invention may be a disorder that is resistant to and / or has relapsed after treatment with a combination of a BTK inhibitor and a Bcl-2 inhibitor when the combination of a BTK inhibitor and a Bcl-2 inhibitor is used as the sole combination in the treatment of the disorder (i.e., no additional drugs are used in the therapy). Alternatively, the hyperproliferative hematological disorder treated according to the present invention may be a disorder that is resistant to and / or has relapsed after treatment with a combination of a BTK inhibitor and a Bcl-2 inhibitor when the BTK inhibitor and the Bcl-2 inhibitor are used in combination with another chemotherapeutic agent (i.e., used as a combination therapy). The other chemotherapeutic agent may be an "other chemotherapeutic agent" as defined herein above. In a preferred embodiment, the "other chemotherapeutic agent" is one or more of fludarabine, cyclophosphamide, idelalisib, an anti-CD20 antibody, preferably rituximab, obinituzumab, ocrelizumab, veltuzumab, or ofatumumab, or an anti-CD52 antibody, preferably alemtuzumab. In another embodiment, the hyperproliferative hematological disorder treated according to the present invention may be a disorder that is resistant to and / or has relapsed after successive treatments, each treatment being with a different chemotherapeutic agent or combination thereof, one of the treatments including the use of a combination of a BTK inhibitor and a Bcl-2 inhibitor (as such or in combination with another chemotherapeutic agent). In this embodiment, the different chemotherapeutic agent or combination thereof may be an "other chemotherapeutic agent" as defined herein above.

[0080] The hyperproliferative blood disorder treated according to the present invention is one in which the hyperproliferative cells preferably express CCR7 receptor. In particular, when the anti-CCR7 antibody is administered simultaneously, separately, or sequentially with at least a BTK inhibitor, the hyperproliferative cells preferably express Bruton's tyrosine kinase. In particular, when the anti-CCR7 antibody is administered simultaneously, separately, or sequentially with at least a Bcl-2 inhibitor, the hyperproliferative cells preferably express B-cell lymphoma 2 protein. In some embodiments, the hyperproliferative cells express or overexpress at least one of CD20 and CD52. In another embodiment, the hyperproliferative cells lose or reduce the expression of at least CD20, when in some patients the CD20 expression of the hyperproliferative cells is downregulated after BTK treatment but CCR7 is still expressed.

[0081] In one embodiment, the hyperproliferative hematological disorder treated according to the present invention is a disorder in which the hyperproliferative cells are cells of the B cell lineage. In some embodiments, the disorder associated with excessive B cell proliferation is cancer. In some embodiments, the disorder is cancer. In some embodiments, the cancer is a B cell hematological malignancy. In certain embodiments, the B cell hematological malignancy is lymphoma or leukemia.

[0082] In some embodiments, the hematological malignancy is selected from the group consisting of acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), acute monocytic leukemia (AMoL), chronic lymphocytic leukemia (CLL), high-risk CLL, small lymphocytic lymphoma (SLL), high-risk SLL, multiple myeloma (MM), non-Hodgkin's lymphoma (NHL), mantle cell lymphoma (MCL), follicular lymphoma (FL), Waldenstrom's macroglobulinemia (WM), diffuse large B-cell lymphoma (DLBCL), marginal zone lymphoma (MZL), Burkitt's lymphoma (BL), hairy cell leukemia (HCL), Richter's transformation, and T-cell prolymphocytic leukemia (T-PLL).

[0083] In some embodiments, the hematological malignancy is selected from the group consisting of chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), non-Hodgkin's lymphoma (NHL), mantle cell lymphoma (MCL), follicular lymphoma (FL), diffuse large B-cell lymphoma (DLBCL), marginal zone lymphoma (MZL) and T-cell prolymphocytic leukemia (T-PLL).

[0084] In some embodiments, the hematological malignancy is selected from the group consisting of Burkitt's lymphoma, non-Burkitt's high-grade B-cell lymphoma, primary mediastinal B-cell lymphoma (PMBL), immunoblastic large cell lymphoma, precursor B-lymphoblastic lymphoma, B-cell prolymphocytic leukemia, lymphoplasmacytic lymphoma, plasma cell myeloma, plasmacytoma, mediastinal (thymic) large B-cell lymphoma, intravascular large B-cell lymphoma, primary effusion lymphoma, Hodgkin's lymphoma, and lymphomatoid granulomatosis.

[0085] Anti-CCR7 antibody The anti-CCR7 antibody or antigen-binding fragment thereof for use in the present invention may be any antigen-binding protein that specifically binds to CCR7. The antigen-binding protein of the present invention that binds to CCR7 is preferably an anti-CCR7 antibody in the broadest sense defined herein above, including, for example, anti-CCR7 antibodies, antibody fragments, antibody derivatives, antibody muteins, and antibody variants. The anti-CCR7 antibody of the present invention is preferably an isolated antibody. Preferably, the anti-CCR7 antibody of the present invention binds to primate CCR7, more preferably human CCR7. Reference amino acid sequences of human CCR7 are, for example, NP_001288643, NP_001288645, NP_001288646, NP_001288647, NP_001829, NP_001288642, and NP_031745. Amino acids 1-24 of this sequence comprise a membrane translocation signal peptide that is cleaved during expression. Amino acids 25-59 of human CCR7 are Y 32 and Y 41The CCR7 antibody for use in the present invention preferably specifically binds to the N-terminal extracellular domain of CCR7, preferably human CCR7.

[0086] Anti-CCR7 antibodies for use in the present invention are preferably neutralizing antibodies that inhibit CCR7-dependent intracellular signaling, CCR7-dependent function, and / or CCR7 receptor internalization by at least one CCR7 ligand selected from CCL19 and CCL21. The anti-CCR7 antibodies preferably have an IC of 150, 100, 80, 50, 30, 25, 20, 15, 10, 5, or 3 nM or less, as can be determined, for example, in an assay described in the Examples herein, with respect to inhibiting CCR7-dependent intracellular signaling and / or CCR7 receptor internalization by at least one CCR7 ligand selected from CCL19 and CCL21. 50 Alternatively, the antibody has the highest IC 50 is the IC of a reference anti-CCR7 antibody when tested in the same assay 50 Thus, preferably, the anti-CCR7 antibody of the present invention is defined by reference to the IC of the reference anti-CCR7 antibody. 50 IC up to 10, 5, 2, 1.5, 1.2, 1.1, or 1.05 times higher than 50 and the reference anti-CCR7 antibody is a murine anti-CCR7 antibody, the amino acid sequence of the heavy chain variable domain of which is SEQ ID NO: 1 and the amino acid sequence of the light chain variable domain of which is SEQ ID NO: 2.

[0087] The anti-CCR7 antibodies of the present invention preferably inhibit the CCR7-dependent intracellular signaling described above, as can be determined, for example, in the assays described in the Examples herein, without a substantial agonist effect, more preferably without a detectable agonist effect.

[0088] Anti-CCR7 antibodies for use in the present invention preferably have a minimum affinity for the N-terminal extracellular domain of CCR7, preferably human CCR7. The minimum affinity of an antibody is preferably defined herein as the K d Thus, preferably, the anti-CCR7 antibody of the present invention is defined by reference to the K of the reference anti-CCR7 antibody against the N-terminal extracellular domain of human CCR7. d K for the N-terminal extracellular domain of human CCR7 up to 100, 50, 20, 10, 5, 2, 1.5, 1.2, 1.1, or 1.05 times higher than d and the reference anti-CCR7 antibody is a murine anti-CCR7 antibody whose heavy chain variable domain has the amino acid sequence SEQ ID NO: 1 and whose light chain variable domain has the amino acid sequence SEQ ID NO: 2. d Up to 10 times higher than d It is understood herein that an antibody having an affinity at least 1 / 10 that of the reference antibody. Thus, if the reference antibody has an affinity of 1×10 -9 K of M d If the antibody has a -8 K below M d has.

[0089] Examples of anti-CCR7 antibodies having one or more of the above-defined characteristics and suitable for use in the present invention include, for example, the monoclonal antibodies described in U.S. Pat. No. 8,865,170, WO 2009 / 139853, U.S. Pat. No. 20150344580 (WO 2013184200), U.S. Pat. No. 2016031997, U.S. Pat. No. 2017342155 (WO 2014 / 151834), and U.S. Pat. No. 2018237529 (WO 2017 / 025569), all of which are incorporated herein by reference.

[0090] A preferred anti-CCR7 antibody for use in the present invention is an antibody that specifically binds to an epitope comprising or consisting of the amino acid sequence "ZxLFE", where Z is a sulfated tyrosine, x can be any amino acid, and F can be replaced with a hydrophobic amino acid. Thus, the antibody of the present invention preferably specifically binds to an epitope comprising or consisting of the amino acid sequence "ZTLFE" at positions 41-45 of the N-terminal extracellular domain of human CCR7. The antibody is preferably specific for human CCR7. Such a preferred anti-CCR7 antibody preferably has at least a minimum affinity for human CCR7 or a synthetic antigen comprising the "ZTLFE" epitope, preferably the synthetic antigen SYM1899 described in the Examples herein. Thus, preferably, the anti-CCR7 antibody has an affinity of at least 1×10 for the synthetic antigen SYM1899, preferably 1×10 -8 M, 5×10 -9 M, 2×10 -9 M, 1.8×10 -9 M, 1×10 -9 M, 1×10 -10 M, or 1×10 -11 K below M d Alternatively, the minimum affinity of an antibody may be determined by the K of a reference anti-CCR7 antibody when tested in the same assay. d Thus, preferably, the anti-CCR7 antibody of the present invention is defined by reference to the K of the reference anti-CCR7 antibody against human CCR7 or a synthetic antigen comprising the "ZTLFE" epitope (preferably the synthetic antigen SYM1899 described in the Examples herein). d K for that antigen that is up to 10, 5, 2, 1.5, 1.2, 1.1, or 1.05 times higher than d and the reference anti-CCR7 antibody is a murine anti-CCR7 antibody whose heavy chain variable domain has the amino acid sequence SEQ ID NO: 1 and whose light chain variable domain has the amino acid sequence SEQ ID NO: 2. d Up to 10 times higher than d It is understood herein that an antibody having an affinity at least 1 / 10 that of the reference antibody. Thus, if the reference antibody has an affinity of 1×10 -9 K of Md If the antibody has a -8 K below M d has.

[0091] Anti-CCR7 antibodies for use in the present invention preferably have a maximum upper limit of k off The anti-CCR7 antibodies of the present invention bind to human CCR7 or a synthetic antigen comprising the "ZTLFE" epitope (preferably the synthetic antigen SYM1899, SEQ ID NO: 3, described in the Examples herein) with a rate constant of 1×10 -3 , 1×10 -4 , or 1 × 10 -5 s -1 The following k off The antibody has a maximum k off The rate constant is the k of a reference anti-CCR7 antibody when tested in the same assay. off Thus, preferably, the anti-CCR7 antibodies of the present invention are defined by reference to a rate constant k of a reference anti-CCR7 antibody against human CCR7 or a synthetic antigen comprising the "ZTLFE" epitope (preferably the synthetic antigen SYM1899 described in the Examples herein). off The reference anti-CCR7 antibody binds to its antigen at a rate constant that is up to 10, 5, 2, 1.5, 1.2, 1.1, or 1.05 times higher than the rate constant, and the reference anti-CCR7 antibody is a murine anti-CCR7 antibody whose heavy chain variable domain has the amino acid sequence of SEQ ID NO:1 and whose light chain variable domain has the amino acid sequence of SEQ ID NO:2.

[0092] One such preferred antibody for use in the present invention is an antibody having HVRs of a reference mouse anti-human CCR7 antibody, the heavy chain variable domain of which has the amino acid sequence of SEQ ID NO:1 and the light chain variable domain of which has the amino acid sequence of SEQ ID NO:2, as defined in WO2018237529, which is incorporated herein by reference.

[0093] The anti-CCR7 antibody for use in the present invention may be a chimeric antibody, such as a mouse-human antibody, however, preferably the antibody is a humanized or human antibody.

[0094] A humanized antibody for use in the present invention preferably induces little or no immunogenic response against the antibody in a subject to which the antibody is administered. For example, a humanized antibody for use in the present invention induces and / or is expected to induce a substantially reduced level of human anti-mouse antibody response (HAMA) in a host subject compared to an original mouse antibody, e.g., comprising the sequences of SEQ ID NOs: 1 and 2. Preferably, a humanized antibody induces and / or is expected to induce minimal or no human anti-mouse antibody response (HAMA) and / or is expected to induce no human anti-mouse antibody response (HAMA). Most preferably, an antibody of the present invention induces an anti-mouse antibody response at or below a clinically acceptable level.

[0095] Humanization can essentially be performed by substituting hypervariable region sequences for the corresponding sequences of a human antibody according to the method of Winter and coworkers (Jones et al., Nature, 321:522-525 (1986); Reichmann et al., Nature, 332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)). In practice, humanized antibodies are typically human antibodies in which some hypervariable region residues, and possibly some framework region (FR) residues, are substituted by residues from analogous sites in rodent antibodies. The choice of human variable domains, both light and heavy, to be used in making the humanized antibody is crucial to reduce immunogenicity and retain specificity and affinity for the antigen. According to the so-called "best-fit" method, the sequence of the variable domain of a rodent antibody is screened against the entire library of known human variable domain sequences. The human sequence that is closest to the rodent sequence is then accepted as the human framework region (FR) for the humanized antibody (Suns et al., J. Immunol., 151:2296 (1993); Chothia et al., J. Mol. Biol., 196:901 (1987)). Another method uses a particular framework region derived from the consensus sequence of all human antibodies of a particular subgroup of light or heavy chains. The same framework may be used for several different humanized antibodies (Carter et al., Proc. Natl. Acad. Sci. USA, 89:4285 (1992); Presta et al., J. Immunol., 151:2623 (1993)).

[0096] It is further important that the antibody be humanized while retaining high affinity for the antigen and other favorable biological properties. To achieve this goal, according to a preferred method, humanized antibodies are prepared by a method of analysis of the parental sequences and various conceptual humanized products using three-dimensional models of the parental and humanized sequences. The humanized anti-CCR7 antibody of any of the above embodiments of the invention preferably comprises a heavy chain constant region that is an IgG1, IgG2, IgG3, or IgG4 region. The humanized anti-CCR7 antibody of any of the above embodiments of the invention preferably comprises a functional Fc region that possesses at least one effector function selected from the group consisting of C1q binding, complement dependent cytotoxicity; Fc region binding, antibody-dependent cell-mediated cytotoxicity, and phagocytosis.

[0097] A preferred humanized antibody for use in the present invention is an antibody described, for example, in US Patent Publication No. 2018237529, in which the amino acid sequence of the heavy chain variable domain is SEQ ID NO: 4 and the amino acid sequence of the light chain variable domain is SEQ ID NO: 5. A more preferred humanized antibody for use in the present invention is an antibody described, for example, in US Patent Publication No. 2018237529, referred to herein as "CAP-100", in which the amino acid sequence of the heavy chain variable domain is SEQ ID NO: 4, the amino acid sequence of the light chain variable domain is SEQ ID NO: 5, and the amino acid sequence of the heavy chain constant region is SEQ ID NO: 10.

[0098] As an alternative to humanization, human antibodies may be generated. By "human antibody" is meant an antibody containing fully human light and heavy chains and constant regions, produced by any of the known standard methods. For example, transgenic animals (e.g., mice) are available that are capable of producing a full repertoire of human antibodies in the absence of endogenous immunoglobulin production upon immunization. For example, it has been described that homozygous deletion of the antibody heavy chain joining region PH gene in chimeric and germline mutant mice results in complete inhibition of endogenous antibody production. Transfer of the human germline immunoglobulin gene array into such germline mutant mice can result in the production of human antibodies after immunization. See, for example, Jakobovits et al., Proc. Nat. Acad. Sci. USA, 90:255 1 (1993); Jakobovits et al., Nature, 362:255-258 (1993). Alternatively, phage display technology (McCafferty et al., Nature 348:552-553 (1990)) can be used to generate human antibodies and antibody fragments in vitro from donor-derived immunoglobulin variable (V) domain gene repertoires. According to this technique, antibody V domain genes are cloned in frame into either a major or minor coat protein gene of a filamentous bacteriophage, such as M13 or fd, and displayed as functional antibody fragments on the surface of the phage particle. Because the filamentous particle contains a single-stranded DNA copy of the phage genome, selections based on the functional properties of the antibody also result in selection of the gene encoding the antibody exhibiting those functional properties. Thus, the phage mimics some of the properties of the B cell. Phage display can be performed in a variety of formats; for a review of formats, see, e.g., Johnson, Kevin S. and Chiswell, David J., Current Opinion in Structural Biology 3:564-57 1 (1993). Human antibodies can also be generated by in vitro activated B cells or by SCID mice whose immune system has been reconstituted with human cells.Once a human antibody is obtained, its encoding DNA sequence can be isolated, cloned and introduced into an appropriate expression system, i.e., preferably a cell line of mammalian origin, which then expresses and releases the human antibody into the culture medium from which the antibody can be isolated.

[0099] A preferred human antibody for use in the present invention is, for example, an antibody described in US Patent Publication No. 2016031997 (WO 2014 / 151834) in which the amino acid sequence of the heavy chain variable domain is SEQ ID NO: 6 and the amino acid sequence of the light chain variable domain is SEQ ID NO: 7 or 8, or any one of the human anti-CCR7 antibodies described in US Patent Publication No. 20150344580 (WO 2013 / 184200), such as the R707 antibody, in which the amino acid sequence of the heavy chain is SEQ ID NO: 11 and the amino acid sequence of the light chain is SEQ ID NO: 12. Other preferred human antibodies for use in the present invention are human antibodies comprising all of the VH regions of the anti-CCR7 antibodies described in US Patent Publication No. 2013195869 (WO 2012043533) and WO 2018142322.

[0100] Functional fragments of antibodies that bind to the CCR7 receptor included for use within the present invention retain at least one binding and / or regulatory function of the full-length antibody from which the fragment is derived. Preferred functional fragments retain the antigen-binding function of the corresponding full-length antibody (e.g., the ability to bind to a mammalian CCR7 receptor). Particularly preferred functional fragments retain the ability to inhibit one or more functions characteristic of a mammalian CCR7 receptor, such as binding activity and / or blocking signaling activity and / or stimulation of a cellular response. For example, in one embodiment, the functional fragment can inhibit the interaction of CCR7 with one or more of its ligands and / or inhibit one or more receptor-mediated functions.

[0101] In some embodiments, the anti-CCR7 antibody of the present invention comprises a light chain and / or a heavy chain antibody constant region. Any antibody constant region known in the art can be used. The light chain constant region can be, for example, a kappa or lambda type light chain constant region, for example, a human kappa or lambda type light chain constant region. The heavy chain constant region can be, for example, an alpha, delta, epsilon, gamma, or mu type heavy chain constant region, for example, a human alpha, delta, epsilon, gamma, or mu type heavy chain constant region. Thus, the anti-CCR7 antibody of the present invention can have a constant region of any isotype, i.e., a constant region including IgG, IgM, IgA, IgD, and IgE constant regions, as well as IgG1, IgG2, IgG3, or IgG4 constant regions. In one embodiment, the light or heavy chain constant region is a fragment, derivative, variant, or mutein of a naturally occurring constant region. Techniques for deriving antibodies of different subclasses or isotypes from an antibody of interest, i.e., subclass switching, are known. Thus, an IgG antibody may be derived, for example, from an IgM antibody, or vice versa. Such techniques allow the preparation of new antibodies that retain the antigen-binding properties of a given antibody (the parent antibody), but also exhibit biological properties associated with an antibody isotype or subclass different from that of the parent antibody. Recombinant DNA techniques may be used. Cloned DNA encoding a particular antibody polypeptide, for example DNA encoding the constant domain of an antibody of a desired isotype, may be used in such procedures. See also Lantto et al. (2002, Methods Mol. Biol. 178:303-16). Thus, the anti-CCR7 antibodies of the present invention include antibodies comprising one or more variable domain sequences, for example as disclosed herein, and having a desired isotype (e.g., IgA, IgG1, IgG2, IgG3, IgG4, IgM, IgE, and IgD), as well as Fab or F(ab')2 fragments thereof. Furthermore, if IgG4 is desired, it may also be desirable to introduce a point mutation (CPSCP→CPPCP) in the hinge region to mitigate the tendency to form inter-heavy chain disulfide bonds that could introduce heterogeneity into IgG4 antibodies, as described in Bloom et al. (1997, Protein Science 6:407).

[0102] The anti-CCR7 antibodies of the invention preferably comprise a functional Fc region possessing at least one effector function selected from the group consisting of C1q binding, complement dependent cytotoxicity; Fc receptor binding, antibody-dependent cell-mediated cytotoxicity and phagocytosis.

[0103] The anti-CCR7 antibodies of the present invention can be modified to improve effector function, for example to enhance ADCC and / or CDC of the antibody. This can be achieved by introducing one or more amino acid substitutions in the Fc region of the antibody. Preferred substitutions in the Fc region of the antibodies of the present invention are those that improve C1q binding and thereby improve CDC activity, for example as described in Idusogie et al. (2000, J. Immunol. 164:4178-4184). A preferred substitution in the Fc region that improves C1q binding is the E333A substitution.

[0104] Glycosyl groups, which are added to the amino acid backbone of glycoproteins, such as antibodies, are formed by several monosaccharides or monosaccharide derivatives, resulting in a composition that may differ in the same antibody produced in cells from different mammals or tissues. In addition, it has been shown that different compositions of glycosyl groups can affect the efficacy of antibodies in mediating antigen-dependent cell-mediated cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC). These properties can therefore be improved by studying the glycosylation patterns of antibodies from different sources. One example of such an approach is Niwa et al. (2004, Cancer Res, 64(6):2127-33).

[0105] Alternatively or additionally, cysteine ​​residue(s) may be introduced into the Fc region, thereby allowing interchain disulfide bond formation in the Fc region. The homodimeric antibody so generated may have improved internalization capability and / or enhanced complement-mediated cell killing and antibody-dependent cellular cytotoxicity (ADCC). See Caron et al. (1992, J. Exp Med. 176:1191-1195) and Shopes (1992, Immunol. 148:2918-2922). Homodimeric antibodies with enhanced antitumor activity may also be prepared using heterobifunctional cross-linkers as described in Wolff et al. (1993, Cancer Research 53:2560-2565). Alternatively, antibodies with dual Fc regions may be engineered, thereby having enhanced complement lysis and ADCC capabilities. See Stevenson et al. (1989, Anti-Cancer Drug Design 3:219-230). To increase the serum half-life of an antibody, a salvage receptor binding epitope may be incorporated into the antibody (especially an antibody fragment), for example as described in U.S. Pat. No. 5,739,277. As used herein, the term "salvage receptor binding epitope" refers to an epitope in the Fc region of an IgG molecule (e.g., IgG1, IgG2, IgG3, or IgG4) that is responsible for increasing the serum half-life of the IgG molecule in vivo.

[0106] A preferred anti-CCR7 antibody of the invention comprises a heavy chain constant region of human allotype G1m17,1 (see Jefferis and Lefranc (2009) MAbs vol. 1, no. 4, pp. 1-7), which comprises the amino acid sequence of SEQ ID NO: 9. More preferably, the heavy chain constant region of human allotype G1m17,1 in the antibody of the invention comprises an E333A substitution and comprises the amino acid sequence of SEQ ID NO: 10.

[0107] Anti-CCR7 antibodies for use in the present invention can be prepared by any of several conventional techniques. Anti-CCR7 antibodies are typically produced in recombinant expression systems using any technique known in the art. See, for example, Shukla and Thoemmes (2010, "Recent advances in large-scale production of monoclonal antibodies and related proteins", Trends in Biotechnol. 28(5):253-261), Harlow and Lane (1988) "Antibodies: A Laboratory Manual", Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, and Sambrook and Russell (2001) "Molecular Cloning: A Laboratory Manual (3rd ed.)", Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, NY. Any expression system known in the art can be used to produce recombinant polypeptides of the present invention. Generally, a host cell is transformed with a recombinant expression vector containing DNA encoding the desired polypeptide.

[0108] BTK inhibitors The present invention relates to new combinations involving inhibitors of Bruton's tyrosine kinase (BTK), referred to as BTK inhibitors. Such inhibitors are widely known in the art and are commercially available for the treatment of B-cell cancers such as mantle cell lymphoma, chronic lymphocytic leukemia, and Waldenstrom's macroglobulinemia. One example of a commercially available BTK inhibitor is ibrutinib, also known as Imbruvica, which is 1-[(3R)-3-[4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl]piperidin-1-yl]prop-2-en-1-one.

[0109] The activity of BTK inhibitors can be measured using the Immobilized Metal Assay for Phosphorus Chemicals (IMAP). IMAP is a homogeneous fluorescence polarization (FP) assay based on affinity capture of a phosphorylated peptide substrate. IMAP uses a fluorescein-labeled peptide substrate that, when phosphorylated by a protein kinase, binds to so-called IMAP nanoparticles that are derivatized with a trivalent metal complex. Binding causes a change in the rate of molecular motion of the peptide, resulting in an increase in the FP value observed due to the fluorescein label attached to the substrate peptide.

[0110] BTK inhibition can also be determined in B cell lines, such as Ramos cells, or in primary cell assays, such as PBMCs or whole blood from mammals, such as humans, monkeys, rats, or mice, or isolated splenocytes from monkeys, rats, or mice. Inhibition of BTK activity can be measured by measuring anti-IgM-induced MΙΡ1β production (Ramos, PBMCs, splenocytes), H 2 O 2Induced BTK and PLCv2 phosphorylation (Ramos cells), or anti-IgM-induced B cell proliferation or CD86 expression in primary B cells (PBMCs and spleen cells) can be measured. Regulation of BTK activity can also be determined in human, monkey, rat, or mouse mast cells after activation FCER-induced degranulation, cytokine production, and CD63-induced cell surface expression. Furthermore, regulation of BTK activity can be determined in CD14+ monocytes that differentiate into osteoclasts after treatment with M-CSF and are activated by RANKL. The activity of BTK inhibitors can be examined in vivo in mouse spleen cells after administration. In a typical experiment, mice can be sacrificed 3 hours after compound administration. Spleens can be extracted from treated mice for splenocyte isolation. Spleen cells can be plated in 96-well culture plates and stimulated with anti-IgM without further addition of compound. Anti-IgM-induced B cell stimulation and its inhibition by BTK inhibitors can be measured by B cell proliferation, MΙΡ1β production, or CD86 expression in CD19+ splenocyte B cells. Further assays, such as the use of the mouse collagen-induced arthritis model described in WO2013010869, or the rat OVX model described in WO2013010869, are known to those of skill in the art.

[0111] Preferred BTK inhibitors are selective or substantially selective for BTK. More preferably, the BTK inhibitors are selective for Src family kinases or selective for BTK and kinases that have a cysteine ​​residue at the amino acid sequence position of tyrosine kinases that is homologous to the amino acid sequence position of cysteine ​​481 of BTK. The BTK inhibitor is capable of forming a covalent bond with or otherwise interacting with this cysteine.

[0112] BTK inhibitors generally contain a ring system featuring two or more nitrogen atoms in addition to additional affinity elements. In a preferred embodiment, the BTK inhibitor has the general formula (1): [ka] (In the formula, Q 1 and Q 2 are each independently N or CR 4 and preferably, Q 1 and Q 2 One of them is N or CH, and the other is CR 4 and R 1 represents a substituted or unsubstituted aryl group, a substituted or unsubstituted heterocyclic ring, a substituted or unsubstituted fused heterocyclic ring, or a substituted or unsubstituted alkynyl group; R 2 represents a hydrogen atom, a halogen atom, a substituted or unsubstituted lower alkyl group, or a substituted or unsubstituted alkoxy group; R 3 represents a substituted or unsubstituted aryl group, a substituted or unsubstituted heterocyclic ring, or a substituted or unsubstituted fused heterocyclic ring; R 4 represents a hydrogen atom, a substituted or unsubstituted lower alkyl group, a substituted or unsubstituted alkoxy group, a substituted or unsubstituted amino group, or a halogen atom; R 5 represents a hydrogen atom, a substituted or unsubstituted lower alkyl group, or R 1 and R 5 may be bonded to form a saturated or unsaturated 5- or 6-membered ring, thereby forming a polycondensed ring. Is it of

[0113] Alternatively, the BTK inhibitor may be represented by the general formula (2): [ka] (In the formula, Q 3 is an amino group or a lower alkyl group, preferably an amino group or methyl; X is CH, N, O, or S; Y is C(R 11 ), N, O, or S; Z is CH, N, or a bond; A is CH or N; B 1 is N or C(R 12 ) and B 2 is N or C(R 13 ) and B 3 is N or C(R 14 ) and B 4 is N or C(R 15 ) and R 6 is C(=O)R 16 , S(=O)R 17 , S(=O) 2 R 18 , or R 19 is optionally replaced by (C 1~6 ) alkyl, R 7 is H, (C 1~3 ) alkyl, or (C 3~7 ) cycloalkyl; R 8 is H, (C 1~6 ) alkyl, or (C 3~7 ) cycloalkyl) or R 7 and R 8 together with the N and C atoms to which they are attached, one or more fluorine, hydroxyl, (C 1~3 ) alkyl, (C 1~3 ) optionally substituted with alkoxy or oxo (C 3~7 ) heterocycloalkyl, R 9 is H or (C 1~3 ) alkyl, R 10 is H, halogen, cyano, (C 1~4 ) alkyl, (C 1~3 ) alkoxy, any alkyl group optionally substituted with one or more halogens (C 3~6 ) cycloalkyl or R 10 is (C 6~10 ) aryl or (C 2~6) heterocycloalkyl; R 11 is H or (C 1~3 ) alkyl or R 10 and R 11 are taken together, respectively (C 1~3 ) optionally substituted with alkyl or one or more halogens (C 3~7 ) cycloalkenyl or (C 2~6 ) may form a heterocycloalkenyl, R 12 H, halogen, CF 3 , (C 1~3 ) alkyl, or (C 1~3 ) alkoxy, R 13 H, halogen, CF 3 , (C 1~3 ) alkyl, or (C 1~3 ) alkoxy or R 12 and R 13 together with the carbon atoms to which they are attached, (C 6~10 ) aryl or (C 1~9 ) forming a heteroaryl, R 14 is H, halogen, (C 1~3 ) alkyl, or (C 1~3 ) alkoxy, R 15 is H, halogen, (C 1~3 ) alkyl, or (C 1~3 ) alkoxy, R 16 is (C 1~6 ) alkyl, (C 2~6 ) alkenyl, and (C 2~6 )alkynyl, wherein each alkyl, alkenyl, or alkynyl is independently selected from the group consisting of hydroxyl, (C 1~4 ) alkyl, (C 3~7 )cycloalkyl, [(C 1~4 ) alkyl] amino, di[(C 1~4 ) alkyl] amino, (C 1~3 ) alkoxy, (C 3~7) cycloalkoxy, (C 6~10 ) aryl, and (C 3~7 ) heterocycloalkyl, or R 16 (C 1~3 ) alkyl-C(O)-S-(C 1~3 ) alkyl or R 16 is optionally substituted with one or more substituents selected from the group consisting of halogen and cyano (C 1~5 ) heteroaryl, R 17 and R 18 is (C 2~6 ) alkenyl and (C 2~6 ) alkynyl, both of which are hydroxyl, (C 1~4 ) alkyl, (C 3~7 )cycloalkyl, [(C 1~4 ) alkyl] amino, di[(C 1~4 ) alkyl] amino, (C 1~3 ) alkoxy, (C 3~7 ) cycloalkoxy, (C 6~10 ) aryl, and (C 3~7 )heterocycloalkyl, optionally substituted by one or more substituents selected from the group consisting of (C 2~6 ) alkenyl and (C 2~6 ) alkynyl; or (C 1~5 ) heteroaryl; R 19 is halogen, cyano, (C 2~6 ) alkenyl, and (C 2~6 )alkynyl; 2~6 ) alkenyl and (C 2~6 )Alkynyl is either hydroxyl, (C 1~4 ) alkyl, (C 3~7 ) cycloalkyl, (C 1~4 ) alkylamino, di[(C 1~4 ) alkyl] amino, (C1~3 ) alkoxy, (C 3~7 ) cycloalkoxy, (C 6~10 ) aryl, (C 1~5 ) heteroaryl, and (C 3~7 ) heterocycloalkyl, 0 to 2 atoms among X, Y, and Z may be heteroatoms at the same time; when one atom selected from X and Y is O or S, Z is a bond, and the other atom selected from X and Y cannot be O or S; when Z is C or N, Y is C(R 11 ) or N, X is C or N; B 1 , B 2 , B 3 , and B 4 0-2 atoms of Is it of

[0114] Alternatively, the BTK inhibitor may be represented by the general formula (3): [ka] (In the formula, Solid / dashed lines indicate single or double bonds; X 1 is CR 20 or N, X 2 is CR 21 or N, X 3 is CR 22 or N, where X 1 , X 2 , and X 3 0, 1, or 2 of are N; Y 1 and Y 2 is independently selected from CH and N; Y 3 is C or N, Y 4 is CR 25 , N, or NH, where Y 1 , Y 2 , Y 3 , and Y4 one or two of are N; R 20 , R 21 , and R 22 is H, F, Cl, -NH 2 , -NHCH 3 , -N(CH 3 ) 2 , -OH, -OCH 3 , -OCH 2 CH 3 , -OCH 2 CH 2 OH, as well as F, Cl, CN, -NH 2 , -NHCH 3 , -N(CH 3 ) 2 , -OH, -OCH 3 , -OCH 2 CH 3 , and -OCH 2 CH 2 C optionally replaced by OH 1~3 alkyl, R 23 are H, F, Cl, CN, -CH 2 OH, -CH(CH 3 )OH, -C(CH 3 ) 2 OH, -CH(CF 3 )OH, -CH 2 F, -CHF2, -CH 2 CHF 2 , -CF 3 , -C(O)NH 2 , -C(O)NHCH 3 , -C(O)N(CH 3 ) 2 , -NH 2 , -NHCH 3 , -N(CH 3 ) 2 , -NHC(O)CH 3 , -OH, -OCH 3 , -OCH 2 CH 3 , -OCH 2 CH 2OH, cyclopropyl, cyclopropylmethyl, 1-hydroxycyclopropyl, imidazolyl, pyrazolyl, 3-hydroxy-oxetan-3-yl, oxetan-3-yl, and azetidin-l-yl; R 24 is C 1~2 Alkyl, -CH 2 OH, -CH 2 F, -CHF 2 , -CF 3 , -CN, and -CH 2 CH 2 OH or two R 24 the groups form a 3-, 4-, 5-, or 6-membered carbocyclic or heterocyclic ring; or R 24 Groups and R 27 the groups form a 3-, 4-, 5-, or 6-membered carbocyclic or heterocyclic ring; n is 0, 1, 2, 3, or 4; R 25 are H, Cl, and C. 1~2 Alkyl, -CH 2 CH 2 OH, -CH 2 F, -CHF 2 , -CF 3 , -NH 2 , -NHCH 3 , -N(CH 3 ) 2 , -OH, C 1~2 Alkoxy and -OCH 2 CH 2 OH, R 26 is represented by the general formula (3 26 ) [ka] (In the formula, Solid / dashed lines indicate single or double bonds; Q 4 is -CH 2 -, -CH 2 CH 2 -, -CH=CH-, -CH=N-, or -NH-; Q5 , Q 6 , and Q 7 are each independently C or N; Q 8 is C, S, N, or C(halogen), Q 9 is -CH 2 -, -CH 2 CH 2 -, -C(CH 3 ) 2 -, -C(halogen) 2 CH 2 - or a C4-6 cycloalkyl, such as cyclopentyl, attached at two non-adjacent carbon atoms, preferably at the 1st and 3rd carbon atoms. This is the part R 27 -H, -CH 3 , -S(O) 2 CH 3 , cyclopropyl, azetidin-3-yl, oxetan-3-yl, and morpholin-4-yl; Z is CR 28 or N, R 28 are H, F, Cl, and C. 1~2 Alkyl, -CH 2 CH 2 OH, -NH 2 , -NHCH 3 , -N(CH 3 ) 2 , -OH, C 1~2 Alkoxy and -OCH 2 CH 2 OH) Is it of

[0115] Alternatively, the BTK inhibitor may be represented by the general formula (4): [ka] (In the formula, Solid / dashed lines indicate single or double bonds; Q 10 , Q 11 , Q 12 , and Q 13are each independently N or NH depending on the valence, or C or CH depending on the valence, where Q 10 , Q 11 , Q 12 , and Q 13 at least one of is N or NH and at least one of is C or CH; Q 14 NH 2 or CH 3 and L a CH 2 , O, NH, or S; A r is a substituted or unsubstituted aryl or substituted or unsubstituted heteroaryl; Y is an optionally substituted group selected from the group consisting of alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl; Z is C(=O), OC(=O), NR 29 C(=O), C(=S), S(=O) x , OS(=O) x , or NR 29 S(=O) x where x is 1 or 2; R 29 is H or (C1-6) alkyl; R 30 and R 31 are each independently H or R 30 and R 31 come together to form a bond) Is it of

[0116] Alternatively, the BTK inhibitor may be represented by the general formula (5): [ka] (In the formula, Q 15 NH 2 or CH 3 and L b O, S, SO, SO 2, NH, C(O), CH 2 O, OCH 2 , C.H. 2 or CH(OH), R 32 is a halogen, C 1~4 Alkyl, C 1~4 Alkoxy, C 1~4 Haloalkyl or C 1~4 represents haloalkoxy; Ring 1 is halogen, C 1~4 Alkyl, C 1~4 Alkoxy, Nitrile, C 1~4 Haloalkyl and C 1~4 haloalkoxy, wherein when two or more substituents are present on ring 1, these substituents may form a 4-7 membered ring group together with the atoms of ring 1 to which these substituents are bonded; Ring 2 is 1 to 3 KR 33 represents a 4- to 7-membered saturated heterocycle optionally substituted by K is a bond, C 1~4 Alkylene, C(O), C(O)CH 2 , C.H. 2 C(O), C(O)O, or SO 2 represents R 33 is C 1~4 Alkyl, C 2~4 Alkenyl, or C 2~4 Each of these represents an alkynyl group, NR 34 R 35 , halogen, CONR 36 R 37 , CO 2 R 38 , and OR 39 and optionally substituted with 1 to 5 substituents each independently selected from the group consisting of R 34 and R 35 are each independently H or OR 40 or CONR 41 R 42 C may be substituted 1~4R represents an alkyl group; 34 and 35 may form, together with the nitrogen atom to which they are attached, a 4- to 7-membered nitrogen-saturated heterocycle which may be substituted by an oxo group or OH, R 36 and R 37 are independently H, C 1~4 alkyl or phenyl; R 38 , R 40 , R 41 , and R 42 are each independently H or C 1~4 represents an alkyl group, R 39 , H, C 1~4 represents an alkyl, phenyl, or benzotriazolyl group; nn represents an integer from 0 to 4. m represents an integer of 0 to 2; If n is 2 or more, any R 32 may be the same or different from each other) Is it of

[0117] Alternatively, the BTK inhibitor may be represented by the general formula (6): [ka] (In the formula, Solid / dashed lines are either single or double bonds, Ring A is a 5-membered heteroaryl or a 5,6-membered bicyclic heteroaryl, where CONH 2 is attached to a 5-membered heteroaryl, each of which is optionally substituted by one or more A'; A' is -NHR 40 or R 44 and R 40 ,H,-R 41 , -R 41 -R 42 -R 43 , -R 41 -R 43 , or -R 42 -R43 and R 41 is an aryl, heteroaryl, cycloalkyl, heterocycloalkyl, or heteroaryl fused to a heterocycloalkyl, each of which may be selected from one or more R 1’ or R 1” is optionally replaced by Each R 1’ are independently halo, nitro, cyano, lower alkylsulfonamido, S(O) 2 or oxo, Each R 1” are independently lower alkyl, cycloalkyl, heterocycloalkyl, lower alkoxy, amino, or amido, each of which is represented by one or more R 1’’’ is optionally replaced by Each R 1’’’ is independently hydroxy, halo, amino, alkylamino, dialkylamino, or heterocycloalkyl; R 42 are -C(=O), -C(=O)O, -C(=O)NR 2’ , -NHC(=O)O, -C(R 2’ ) 2 , O, -C(=NH)NR 2 > or -S(=O) 2 and Each R 2’ is independently H or lower alkyl; R 43 is H or R 44 and R 44is lower alkyl, lower haloalkyl, lower alkoxy, amino, lower alkylamino, lower dialkylamino, aryl, arylalkyl, alkylaryl, heteroaryl, alkylheteroaryl, heteroarylalkyl, cycloalkyl, alkylcycloalkyl, cycloalkylalkyl, heterocycloalkyl, alkylheterocycloalkyl, heterocycloalkylalkyl, bicyclic cycloalkyl, bicyclic heterocycloalkyl, spirocycloalkyl, or spiroheterocycloalkyl, each of which is optionally substituted by one or more lower alkyl, halo, lower alkylamino, lower dialkylamino, hydroxy, hydroxy lower alkyl, lower alkoxy, halo, nitro, amino, amido, acyl, cyano, oxo, guanidino, hydroxylamino, carboxy, carbamoyl, carbamate, halo lower alkoxy, or halo lower alkyl, where two lower alkyl groups may be taken together to form a ring; Q' is CH or N; X 2 is CH, N, or N(X 3 ) and X 3 is lower alkyl, Y 5 is H, halogen, or lower alkyl; Y 6 is Y 6a , Y 6b , Y 6c , or Y 6d and Y 6a is H or a halogen, Y 6b is lower alkyl optionally substituted with one or more substituents selected from the group consisting of lower haloalkyl, halogen, hydroxy, amino, cyano, and lower alkoxy; Y 6c is lower cycloalkyl optionally substituted with one or more substituents selected from the group consisting of lower alkyl, lower haloalkyl, halogen, hydroxy, amino, cyano, and lower alkoxy; Y 6dis amino optionally substituted by one or more lower alkyl, alkoxy lower alkyl, or hydroxy lower alkyl; Y 7 is H, halogen, or lower alkyl; Y 8 is H, halogen, lower alkyl, lower haloalkyl, lower alkoxy, or lower hydroxyalkyl; Y 9 is H, lower alkyl, or lower hydroxyalkyl. Is it of

[0118] Alternatively, the BTK inhibitor may be represented by the general formula (7): [ka] (In the formula, A 2 is N or CR 49 are independently selected from R 45 , H, L 2a -(substituted or unsubstituted alkyl), L 2a -(substituted or unsubstituted cycloalkyl), L 2a -(substituted or unsubstituted alkenyl), L 2a -(substituted or unsubstituted cycloalkenyl), L 2a -(substituted or unsubstituted heterocycle), L 2a -(substituted or unsubstituted heteroaryl), or L 2a -(substituted or unsubstituted aryl), where L 2a is a bond, O, S, S(=O), S(=O) 2 , C(=O), substituted or unsubstituted C 1~6 Alkylene, or substituted or unsubstituted C 2~6 alkenylene), R 46 and R 47 is independently selected from H, lower alkyl, and substituted lower alkyl; R 48 L 3a -X a -L 4a-G, where L 3a is optional and, when present, is a bond or an optionally substituted group selected from alkylene, heteroalkylene, arylene, heteroarylene, alkylarylene, alkylheteroarylene, or alkylheterocycloalkylene; X a is optional and, if present, is a bond, O, C(=O), S, S(=O), S(=O) 2 , N.H., N.R. 53 , NHC(O), C(O)NH, NR 53 C(O), C(O)NR 53 , S(=O) 2 NH, NHS(=O) 2 , S(=O) 2 NR 53 , N.R. 53 S(=O) 2 , OC(O)NH, NHC(O)O, OC(O)NR 53 , N.R. 53 C(O)O, C(H)=NO, ON=CH, NR 54 C(O)NR 54 , heteroarylene, arylene, NR 54 C(=NR 55 )NR 54 , N.R. 54 C(=NR 55 ), C(=NR 55 )NR 54 ,OC(=NR 55 ), or C(=NR 55 )O, L 4a is optional and, if present, is a bond, substituted or unsubstituted alkylene, substituted or unsubstituted cycloalkylene, substituted or unsubstituted alkenylene, substituted or unsubstituted alkynylene, substituted or unsubstituted arylene, substituted or unsubstituted heteroarylene, substituted or unsubstituted heterocyclene; Or L 3a , X a , and L 4aare taken together to form a nitrogen-containing heterocyclic ring or an optionally substituted group selected from alkyl, heteroalkyl, aryl, heteroaryl, alkylaryl, alkylheteroaryl, or alkylheterocycloalkyl; G is [ka] (In the formula, R b is H, substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, and R 51 and R 52 is H, R 50 is H, substituted or unsubstituted C 1~4 Alkyl, substituted or unsubstituted C 1~4 Heteroalkyl, C 1~8 Alkylaminoalkyl, C 1~8 Hydroxyalkylaminoalkyl, C 1~8 Alkoxyalkylaminoalkyl, substituted or unsubstituted C 3~6 Cycloalkyl, substituted or unsubstituted C 1~8 Alkyl C 3~6 Cycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted C 2~8 Heterocycloalkyl, substituted or unsubstituted heteroaryl, C 1~4 Alkyl (aryl), C 1~4 Alkyl (heteroaryl), C 1~8 Alkyl ether, C 1~8 Alkylamide, or C 1~4 Alkyl(C 2~8 heterocycloalkyl) or Or R 50 and R 52 is H, R 51 is H, substituted or unsubstituted C 1~4 Alkyl, substituted or unsubstituted C 1~4 Heteroalkyl, C 1~8 Alkylaminoalkyl, C 1~8 Hydroxyalkylaminoalkyl, C 1~8Alkoxyalkylaminoalkyl, substituted or unsubstituted C 3~6 Cycloalkyl, substituted or unsubstituted C 1~8 Alkyl C 3~6 Cycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted C 2~8 Heterocycloalkyl, substituted or unsubstituted heteroaryl, C 1~4 Alkyl (aryl), C 1~4 Alkyl (heteroaryl), C 1~8 Alkyl ether, C 1~8 Alkylamide, or C 1~4 Alkyl(C 2~8 heterocycloalkyl) or Or R 51 and R 52 come together to form a bond) is selected from the group consisting of R 49 H, halogen, -L 6a -(substituted or unsubstituted C 1~3 alkyl), -L 6a -(substituted or unsubstituted C 2~4 alkenyl), -L 6a -(substituted or unsubstituted heteroaryl), or -L 6a -(substituted or unsubstituted aryl), where L 6a is a bond, O, S, S(=O), S(=O) 2 , NH, C(O), NHC(O)O, OC(O)NH, NHC(O), or C(O)NH; R 53 is selected from among H, substituted or unsubstituted lower alkyl, and substituted or unsubstituted lower cycloalkyl; Each R 54 are independently H, substituted or unsubstituted lower alkyl, or substituted or unsubstituted lower cycloalkyl, or two R 54 the groups together can form a 5-, 6-, 7-, or 8-membered heterocyclic ring; or R 54 and R 55 can be taken together to form a 5-, 6-, 7-, or 8-membered heterocyclic ring; or R55 is H, S(=O) 2 R 56 , S(=O) 2 NH 2 , C(O)R 56 , CN, NO 2 , heteroaryl, or heteroalkyl). Is it of

[0119] Alternatively, the BTK inhibitor may be represented by the general formula (8): [ka] (In the formula, X 4 represents CH or N; R 56 NH 2 ,CONH 2 , or H, R 57 Hal, Ar 1b , or Het 1b represents R 58 is NR 60 [C(R 60 ) 2 ] n Het 2b , N.R. 60 [C(R 60 ) 2 ] nnn Cyc, Het 2b , O[C(R 60 ) 2 ] nnn Ar 2b , N.R. 60 [C(R 60 ) 2 ] nnn Ar 2b , O[C(R 60 ) 2 ] nnn Het 2b , N.R. 60 (CH 2 ) p NR 60 R 61 , O(CH 2 ) p NR 60 R61 , or NR 60 (CH 2 ) p CR 62 R 63 NR 60 R 61 represents R 59 H, CH 3 , or NH 2 represents R 60 represents H or alkyl having 1, 2, 3 or 4 C atoms, R 61 is N(R 60 ) 2 CH 2 CH=CHCONH, Het 3b CH 2 CH=CHCONH, CH 2 =CHCONH(CH 2 ) nnn , Het 4b (CH 2 ) nnn COHet 3b -Diyl-CH 2 CH=CHCONH, HC≡CCO, CH 3 C≡CCO, CH 2 =CH-CO, CH 2 =C(CH 3 )CONH,CH 3 CH=CHCONH(CH 2 ) nnn , N≡CCR 52 R 53 CONH(CH 2 ) nnn , Het 4b NH(CH 2 ) p COHet 3b -Diyl-CH 2 CH=CHCONH, Het 4b (CH 2 )pCONH(CH 2 CH 2 O) p (CH 2 ) p COHet 3b -Diyl-CH 2 CH = CHCONH, CH2 = CHSO2 , A b CH=CHCO, CH 3 CH=CHCO, Het 4b (CH 2 ) p CONH(CH 2 ) p Het 3b -Diyl-CH 2 CH=CHCONH, Ar 3b CH=CHSO 2 , C.H. 2 =CHSO 2 NH or N(R 60 )CH 2 CH=CHCO, R 62 , R 63 together represent an alkylene having 2, 3, 4 or 5 C atoms, Ar 1b represents phenyl or naphthyl, each of which is unsubstituted or 61 ,Hal,(C.H. 2 ) nnn NH 2 ,CONHAr 3b , (CH 2 ) nnn NHCOA b , O(CH 2 ) nnn Ar 3b , O.Cyc b , A b , COHet 3b , O.A. b , and / or OHet 3b (CH2) mono-, di-, or tri-substituted; Ar 2b represents phenyl, naphthyl, or pyridyl, each of which is unsubstituted or 61 , Hal, OAr 3b , (CH 2 ) nnn NH 2 , (CH 2 ) nnn NHCOA b and / or Het 3b is mono-, di- or trisubstituted by Ar3b is unsubstituted or is OH, OA b , Hal, CN, and / or A b represents phenyl mono-, di- or trisubstituted by Het 1b Even if unsubstituted, R 61 , O(CH 2 ) nnn Ar 3b , and / or (CH 2 ) nnn Ar 3b represents a mono- or bicyclic saturated, unsaturated or aromatic heterocycle having 1 to 4 N, O and / or S atoms, optionally mono-, di- or trisubstituted by Het 2b Even if unsubstituted, R 61 , Het 3b , Cyc b SO 2 , OH, Hal, COOH, OA b , C.O.A. b , COHet 3b , Cyc b CO, SO 2 represents a mono- or bicyclic saturated heterocycle having 1-4 N, O and / or S atoms, optionally mono-, di- or tri-substituted by ═O, Het 3b represents a monocyclic unsaturated, saturated or aromatic heterocycle having 1 to 4 N, O and / or S atoms, which may be unsubstituted or mono-, di- or tri-substituted by Hal, A and / or ═O, Het 4b Even if it is non-substitutional, A b , NO 2 represents a bi- or tricyclic unsaturated, saturated or aromatic heterocycle having 1 to 4 N, O and / or S atoms, which may be mono-, di-, tri- or tetra-substituted by , Hal and / or ═O, Cyc b is unsubstituted or R 61 and / or OH mono- or disubstituted cyclic alkyl having 3, 4, 5 or 6 C atoms, which may contain a double bond, Ab may have 1 to 7 H atoms replaced with F and / or Cl, and / or may have 1 or 2 non-adjacent CH 2 and / or CH groups may be replaced by O, NH, and / or N 1~10 represents an alkyl group, Hal represents F, Cl, Br or I; nnn represents 0, 1, 2, 3, or 4, p represents 1, 2, 3, 4, 5, or 6. Is it of

[0120] Alternatively, the BTK inhibitor may be represented by the general formula (9): [ka] (In the formula, Solid / dashed lines are either single or double bonds, X c -Y c -Z c is NCC and R 65 exists or is a CNN and R 65 does not exist, R 64 is a 3-8 membered N-containing ring, where N is unsubstituted or substituted by R67; R 65 is H or lower alkyl, in particular methyl, ethyl, propyl, or butyl; or R 64 and R 65 together with the atom to which they are attached, represent cycloalkyl, saturated or unsaturated heterocycle, aryl, and unsubstituted or at least one substituent L c -R 67 A 4- to 8-membered ring, preferably a 5- or 6-membered ring, is formed from a heteroaryl ring substituted by R 66 is, at each occurrence, independently halogen, alkyl, S-alkyl, CN, or OR 68 and v is 1, 2, 3, or 4, preferably 1 or 2; L c is a bond, NH, heteroalkyl, or heterocyclyl; R 67 COR 69 , CO 2 R 69 , or SO 2 R 69 where R 69 is substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, R 68 is H, or unsubstituted or substituted heteroalkyl, alkyl, cycloalkyl, saturated or unsaturated heterocyclyl, aryl, or heteroaryl. Is it of

[0121] Alternatively, the BTK inhibitor may be represented by the general formula (10): [ka] (In the formula, Ring G is a 5- or 6-membered aromatic ring containing 0-3 N, S, or O heteroatoms; Each W is independently -(CH 2 )- or -C(O)-, L d is a bond, CH 2 , N.R. 81 , O, or S; The solid / dashed lines are either single or double bonds; if they are double bonds, R 74 and R 76 does not exist, md is 0 or an integer from 1 to 4; mm is 0 or an integer from 1 to 4, where, when mm is 2 or more, each R 71 may be different, pd is 0 or an integer from 1 to 2, where, when pd is 0, md is not 0, and when pd is 2 or more, each R 75 and each R 76 may be different, R70 , R 73 , R 74 , R 75 , and R 76 are each independently H, halogen, heteroalkyl, alkyl, alkenyl, cycloalkyl, aryl, saturated or unsaturated heterocyclyl, heteroaryl, alkynyl, -CN, -NR 82 R 83 , OR 82 , -COR 82 , -CO 2 R 82 , -CONR 82 R 83 , -C(=NR 82 )NR 83 R 84 , -NR 82 COR 83 , -NR 82 CONR 83 R 84 , -NR 82 CO 2 R 83 , -SO 2 R 82 , -NR 82 SO 2 NR 83 R 84 , or -NR 82 SO2R 83 where alkyl, alkenyl, alkynyl, cycloalkyl, heteroaryl, aryl, and saturated or unsaturated heterocyclyl are each independently selected from the group consisting of at least one substituent R 85 and optionally replaced by (R 83 and R 84 ), or (R 83 and R 85 ), or (R 85 and R 86 ), or (R when pd is 2 85 and R 85 ) together with the atoms to which they are attached form at least one substituent R 85 may form a ring selected from cycloalkyl, saturated or unsaturated heterocycle, aryl, and heteroaryl rings, optionally substituted by R 71is halogen, alkyl, -S-alkyl, -CN, -NR 82 R 83 , -OR 82 , -COR 82 , -CO 2 R 82 , -CONR 82 R 83 , -C(=NR 82 )NR 83 R 84 , -NR 82 COR 83 , -NR 82 CONR 83 R 84 , -NR 82 CO2R 83 , -SO 2 R 82 , -NR 82 SO 2 NR 83 R 84 , or -NR 82 SO 2 R 83 and R 81 is H or lower alkyl; R 82 , R 83 , and R 84 are each independently H, heteroalkyl, alkyl, alkenyl, alkynyl, cycloalkyl, saturated or unsaturated heterocyclyl, aryl, or heteroaryl, where (R 82 and R 83 ) and / or (R 83 and R 84 ), together with the atom(s) to which they are attached, each represent at least one substituent R 85 may form a ring selected from cycloalkyl, saturated or unsaturated heterocycle, aryl, and heteroaryl rings, optionally substituted by R 85 is halogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocyclyl, oxo, -CN, -OR86 , -NR 86 R 87 , -COR 86 , -CO 2 R 86 , -CONR 86 R 87 , -C(=NR 86 )NR 87 R 88 , -NR 86 COR 87 , -NR 86 CONR 86 R 87 , -NR 86 CO 2 R 87 , -SO 2 R 86 , -SO 2 Aryl, -NR 86 SO 2 NR 87 R 88 , or -NR 86 SO 2 R 87 where R 86 , R 87 , and R 88 are independently hydrogen, halogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocyclyl; (R 86 and R 87 ) and / or (R 87 and R 88 ), together with the atom to which they are attached, can form a ring selected from cycloalkyl, saturated or unsaturated heterocycle, aryl, and heteroaryl rings. Is it of or a stereoisomer, tautomer, or pharma- ceutically acceptable salt thereof.

[0122] In a preferred embodiment, the BTK inhibitor is of the general formula (1) having the variables defined above, or a stereoisomer, tautomer, or pharma- ceutically acceptable salt thereof.

[0123] In a preferred embodiment, the BTK inhibitor is of the general formula (2) having the variables defined above, or a stereoisomer, tautomer, or pharma- ceutically acceptable salt thereof.

[0124] In a preferred embodiment, the BTK inhibitor is of the general formula (3) having the variables defined above, or a stereoisomer, tautomer, or pharma- ceutically acceptable salt thereof.

[0125] In a preferred embodiment, the BTK inhibitor is of the general formula (4) having the variables defined above, or a stereoisomer, tautomer, or pharma- ceutically acceptable salt thereof, more preferably ibrutinib.

[0126] In a preferred embodiment, the BTK inhibitor is of the general formula (5) having the variables defined above, or a stereoisomer, tautomer, or pharma- ceutically acceptable salt thereof.

[0127] In a preferred embodiment, the BTK inhibitor is of the general formula (6) having the variables defined above, or a stereoisomer, tautomer, or pharma- ceutically acceptable salt thereof.

[0128] In a preferred embodiment, the BTK inhibitor is of the general formula (7) having the variables defined above, or a stereoisomer, tautomer, or pharma- ceutically acceptable salt thereof.

[0129] In a preferred embodiment, the BTK inhibitor is of the general formula (8) having the variables defined above, or a stereoisomer, tautomer, or pharma- ceutically acceptable salt thereof.

[0130] In a preferred embodiment, the BTK inhibitor is of the general formula (9) having the variables defined above, or a stereoisomer, tautomer, or pharma- ceutically acceptable salt thereof.

[0131] In a preferred embodiment, the BTK inhibitor is of the general formula (10) having the variables defined above, or a stereoisomer, tautomer, or pharma- ceutically acceptable salt thereof.

[0132] A wide variety of pharma- ceutically acceptable salts can be formed from the BTK inhibitors, including, for example, acid addition salts formed by reacting ibrutinib with organic acids, including aliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxylalkanoic acids, alkanedioic acids, aromatic acids, aliphatic and aromatic sulfonic acids, amino acids, and the like, including, for example, acetic acid, trifluoroacetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, and the like; and acid addition salts formed, for example, by reacting ibrutinib with inorganic acids, including, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, hydroiodic acid, hydrofluoric acid, phosphorous acid, and the like. HCl addition salts of the BTK inhibitors are preferred.

[0133] Preferred halogens are fluorine, chlorine, and bromine, more preferably fluorine and bromine, and most preferably fluorine. The aryl group portion of the substituted or unsubstituted aryl group is preferably an aryl group having 6 to 14 carbon atoms, specific examples of which include phenyl, naphthyl, and indenyl. The heterocyclic ring portion of the substituted or unsubstituted heterocyclic ring is preferably an alicyclic heterocyclic group or an aromatic heterocyclic group. Examples of the alicyclic heterocyclic group include a 3- to 8-membered heterocyclic group having at least one heteroatom selected from a nitrogen atom, a sulfur atom, and an oxygen atom. Specific examples of the alicyclic heterocyclic group include pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, and thiomorpholinyl. Examples of the aromatic heterocyclic group include a 5- or 6-membered monocyclic aromatic heterocyclic group having at least one heteroatom selected from a nitrogen atom, a sulfur atom, and an oxygen atom. Specific examples of the alicyclic heterocyclic group include imidazolyl, pyrazolyl, thienyl, thiazolyl, and pyridyl. The heterocyclic fused ring portion of the substituted or unsubstituted heterocyclic fused ring may be, for example, a fused heterocyclic ring group having at least one heteroatom selected from a nitrogen atom, a sulfur atom, and an oxygen atom, which is a fused bicyclic group having a 3-8 member ring. Specific examples thereof include tetrahydroisoquinolyl, benzothiophenyl, benzimidazolyl, benzoxazolyl, benzothiazolyl, indolyl, isoquinolyl, and phthalimide. The lower alkyl group portion of the substituted or unsubstituted lower alkyl group may be any of a linear, branched, and cyclic alkyl group having 1 to 3 carbon atoms, specific examples of which include a methyl group, an isopropyl group, and a cyclopropyl group. The alkoxy group portion of the substituted or unsubstituted alkoxy group may be any of a linear, branched, and cyclic alkyl group having 1 to 3 carbon atoms, specific examples of which include a methoxy group, an ethoxy group, an isopropyloxy group, and a cyclopropyloxy group. More specifically, the substituted or unsubstituted amino group may be any of amino groups having a linear, branched, or cyclic alkyl group having 1 to 3 carbon atoms, and specific examples thereof include an amino group, a methylamino group, and a dimethylamino group. The substituted or unsubstituted amino group is preferably an amino group.The alkynyl group portion of the substituted or unsubstituted alkynyl group may be any of linear or branched groups having 2 to 6 carbon atoms, specific examples of which include an ethynyl group, a propargyl group, and a 2-butynyl group. The substituted portion of the substituted or unsubstituted alkynyl group may be any of a substituted or unsubstituted aryl ring, a substituted or unsubstituted heterocyclic ring, or a substituted or unsubstituted fused heterocyclic ring, specific examples of which include an aryl group.

[0134] In a preferred embodiment, the BTK inhibitor has one or more of any kind of substituents at any chemically possible position as a substituent of a substituted or unsubstituted aryl group, a substituted or unsubstituted heterocyclic ring, a substituted or unsubstituted heterocyclic fused ring, a substituted or unsubstituted lower alkyl group, a substituted or unsubstituted alkoxy group, or a substituted or unsubstituted amino group. When the above groups have two or more substituents, each substituent may be the same or different, and examples of the substituents include a halogen atom, a substituted or unsubstituted alkyl group, a substituted or unsubstituted alkoxy group, a substituted or unsubstituted amino group, a nitro group, a cyano group, a hydroxy group, a substituted or unsubstituted alkylamino group, a substituted or unsubstituted carbamoyl group, a carboxyl group, a formyl group, an acetyl group, a benzoyl group, and a substituted or unsubstituted acylamino group. 5 and R 1 Examples of polycondensed rings that can be formed by combining with the above to form a saturated or unsaturated 5- or 6-membered ring include 3- to 8-membered condensed heterocyclic groups having heteroatoms such as nitrogen, sulfur, and oxygen atoms. Specific examples thereof include oxoisoquinolyl, oxodihydroisoquinolyl, oxophthalazinyl, and oxothienopyrrolyl.

[0135] (C 1~2 ) Alkyl means an alkyl group having 1 to 2 carbon atoms, and is methyl or ethyl; (C 1~3 ) Alkyl means a branched or unbranched alkyl group having 1 to 3 carbon atoms, and is methyl, ethyl, propyl, or isopropyl; 1~4) Alkyl means a branched or unbranched alkyl group having 1 to 4 carbon atoms, including methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, and tert-butyl; (C 1~3 ) Alkyl groups are preferred; 1~5 ) Alkyl means a branched or unbranched alkyl group having 1 to 5 carbon atoms, e.g., methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, and isopentyl; (C 1~4 )Alkyl groups are preferred. 1~6 ) Alkyl means a branched or unbranched alkyl group having 1 to 6 carbon atoms, such as methyl, ethyl, propyl, isopropyl, butyl, tert-butyl, n-pentyl, and n-hexyl. 1~5 ) alkyl groups are preferred, (C 1~4 ) alkyl is most preferred.

[0136] (C 1~2 ) Alkoxy means an alkoxy group having 1 to 2 carbon atoms, the alkyl portion of which has the same meaning as defined above; 1~3 ) Alkoxy means an alkoxy group having 1 to 3 carbon atoms, the alkyl portion of which has the same meaning as defined above; 1~2 )Alkoxy groups are preferred. 1~4 Alkoxy means an alkoxy group having 1 to 4 carbon atoms, the alkyl portion of which has the same meaning as defined above. 1~3 ) alkoxy groups are preferred, (C 1~2 ) Alkoxy groups are most preferred.

[0137] (C 2~4 ) Alkenyl means a branched or unbranched alkenyl group having 2 to 4 carbon atoms, for example, ethenyl, 2-propenyl, isobutenyl, or 2-butenyl. 2~6) Alkenyl means a branched or unbranched alkenyl group having 2 to 6 carbon atoms, such as ethenyl, 2-butenyl, and n-pentenyl; (C 2~4 ) alkenyl groups are most preferred.

[0138] (C 2~4 ) Alkynyl means a branched or unbranched alkynyl group having 2 to 4 carbon atoms, for example, ethynyl, 2-propynyl, or 2-butynyl; (C 2~6 ) Alkynyl means a branched or unbranched alkynyl group having 2 to 6 carbon atoms, for example, ethynyl, propynyl, n-butynyl, n-pentynyl, isopentynyl, isohexynyl, or n-hexynyl. 2~4 ) Alkynyl groups are preferred.

[0139] (C 3~6 ) Cycloalkyl means a cycloalkyl group having 3 to 6 carbon atoms, such as cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl; 3~7 ) Cycloalkyl means a cycloalkyl group having 3 to 7 carbon atoms, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, or cycloheptyl; 2~6 Heterocycloalkyl means a heterocycloalkyl group having 2 to 6 carbon atoms, preferably 3 to 5 carbon atoms, and 1 or 2 heteroatoms selected from N, O, and / or S, which may be bonded via a heteroatom or a carbon atom if possible; preferred heteroatoms are N or O; also preferred are piperidine, morpholine, pyrrolidine, and piperazine; most preferred are (C 2~6 ) heterocycloalkyl is pyrrolidine; heterocycloalkyl groups may be attached via a heteroatom, if possible; (C 3~7 )Heterocycloalkyl means a heterocycloalkyl group having 3 to 7 carbon atoms, preferably 3 to 5 carbon atoms, and 1 or 2 heteroatoms selected from N, O, and / or S. Preferred heteroatoms are N or O; preferred (C 3~7) Heterocycloalkyl groups are azetidinyl, pyrrolidinyl, piperidinyl, homopiperidinyl, or morpholinyl; more preferred (C 3~7 ) heterocycloalkyl groups are piperidine, morpholine, and pyrrolidine; heterocycloalkyl groups may be attached via a heteroatom where possible; (C 3~7 ) cycloalkoxy means a cycloalkyl group having 3 to 7 carbon atoms and the same meaning as defined above, attached via a ring carbon atom to an exocyclic oxygen atom; (C 6~10 ) Aryl means an aromatic hydrocarbon group having 6 to 10 carbon atoms, for example, phenyl, naphthyl, tetrahydronaphthyl, or indenyl; 6~10 ) the aryl group is phenyl; (C 1~5 ) Heteroaryl means a substituted or unsubstituted aromatic group having 1 to 5 carbon atoms and 1 to 4 heteroatoms selected from N, O, and / or S; 1~5 )Heteroaryl may be optionally substituted; preferred (C1-5)heteroaryl groups are tetrazolyl, imidazolyl, thiadiazolyl, pyridyl, pyrimidyl, triazinyl, thienyl, or furyl, more preferred (C 1~5 ) heteroaryl is pyrimidyl; 1~9 ) Heteroaryl means a substituted or unsubstituted aromatic group having 1 to 9 carbon atoms and 1 to 4 heteroatoms selected from N, O, and / or S; 1~9 ) Heteroaryl may be optionally substituted; preferred (C 1~9 ) heteroaryl groups are quinoline, isoquinoline, and indole; [(C 1~4 [(C alkyl)]amino means an amino group monosubstituted with an alkyl group having 1 to 4 carbon atoms as defined above; 1~4 ) alkyl] amino group is methylamino; 1~4[(C alkyl)]amino means an amino group disubstituted by alkyl group(s) each containing 1 to 4 carbon atoms and having the same meaning as defined above; 1~4 ) alkyl] amino group is dimethylamino; 1~3 ) alkyl-C(O)-S-(C 1~3 ) Alkyl means alkyl-carbonyl-thio-alkyl groups, each alkyl group having 1 to 3 carbon atoms and as previously defined; 3~7 )Cycloalkenyl means a cycloalkenyl group having 3 to 7 carbon atoms, preferably 5 to 7 carbon atoms; 3~7 ) The cycloalkenyl group is cyclopentenyl or cyclohexenyl; the cyclohexenyl group is most preferred; (C 2~6 ) Heterocycloalkenyl means a heterocycloalkenyl group having 2 to 6 carbon atoms, preferably 3 to 5 carbon atoms and one heteroatom selected from N, O and / or S; 2~6 ) Heterocycloalkenyl groups are oxycyclohexenyl and azacyclohexenyl groups. When in a definition of a substituent it is indicated that "all alkyl groups" of said substituent are optionally substituted, all alkyl groups also include the alkyl portion of alkoxy groups.

[0140] Depending on the ring or moiety formed, valence is conserved for the indicated atom, e.g., nitrogen may carry a hydrogen when present in X or Y. The term "substituted" means that one or more hydrogens bonded to the indicated atom or atoms are replaced with one selected from the indicated group, provided that the replacement does not exceed the normal valence of the indicated atom under the existing circumstances, and that the replacement results in a stable compound. Combinations of substituents and / or variables are permissible only if such combinations result in stable compounds. A "stable compound" or "stable structure" is defined as a compound or structure that is sufficiently robust to survive isolation to a useful degree of purity from a reaction mixture, and formulation into a drug product containing an active pharmaceutical ingredient with efficacy. The term "optionally substituted" means optional substitution with a specified group, radical, or moiety.

[0141] BTK inhibitors of general formula (1) and their synthesis are described in EP 2824099, WO 2013161848, U.S. Pat. No. 8,450,335, U.S. Pat. No. 8,609,679, and WO 2013063401. Preferred BTK inhibitors of general formula (1) are selected from the group consisting of compound 1 in Table 1 shown below, compound 2 in Table 1 shown below, and the compounds disclosed in Tables 1-1 to 1-27 of EP 2824099, more preferably compound 1 or 2 in Table 1 shown below.

[0142] BTK inhibitors of general formula (2) and their synthesis are described in WO 2013010869, U.S. Patent Application Publication No. 2017136014, and U.S. Patent Application Publication No. 2017095471. Preferred BTK inhibitors of general formula (2) are compounds 3, 4, 5, or 6 in Table 1 shown below, and the compounds disclosed in WO 2013010869, page 9, line 9 to page 11, line 10, preferably compounds 3, 4 (acalabrutinib), 5, or 6 in Table 1.

[0143] BTK inhibitors of general formula (3) and their synthesis are described in WO2013067260. A preferred BTK inhibitor of general formula (3) is described in R 7 R, shown as 26 Preferred BTK inhibitors of general formula (3) are compound 7 in Table 1 shown below, as well as the compounds disclosed in Tables 1 and 2 of WO2013067260, preferably compound 4 in Table 1 shown below.

[0144] BTK inhibitors of general formula (4) and their synthesis are described in U.S. Patent Application Publication No. 2015352116. A preferred BTK inhibitor of general formula (4) is compound 8 in Table 1 shown below, (R)-1-(3-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)prop-2-en-1-one, 1-[(3R)-3-[4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl]piperidin-1-yl]prop-2-en-1-one, (S)-1-(3-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)prop-2-en-1-one, preferably compound 8 in Table 1 shown below. Compound 8 is ibrutinib.

[0145] BTK inhibitors of general formula (5) and their synthesis are described in U.S. Patent Application Publication No. 2015125446, WO 2013 / 081016, WO 2011 / 152351, and U.S. Patent Application Publication No. 2017136014. Preferred BTK inhibitors of general formula (5) are compound 9 in Table 1 shown below, and compounds disclosed in WO 2013 / 081016 and WO 2011 / 152351, preferably compound 9 in Table 1.

[0146] BTK inhibitors of general formula (6) and their synthesis are described in WO2012156334. A preferred BTK inhibitor of general formula (6) is compound 10 in Table 1 shown below, and the compounds disclosed in claim 8 of WO2012156334, preferably compound 10 in Table 1.

[0147] BTK inhibitors of general formula (7) and their synthesis are described in U.S. Patent Application Publication No. 2016303130, U.S. Patent Application Publication No. 2017209462, U.S. Patent Application Publication No. 2016022684, WO 2013191965, and WO 2017023815. Preferred BTK inhibitors of general formula (7) are compounds 8 or 11 in Table 1 shown below, (R)-1-(3-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)prop-2-en-1-one, 1-[(3R)-3-[4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl]piperidin-1-yl]prop-2-en-1-one, (S)-1-(3-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)prop-2-en-1-one, (S)-1-(3-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)prop-2-en-1-one, and the compounds disclosed in embodiment L of WO2013191965 starting on page 43, preferably compound 8 or 11 of Table 1.

[0148] BTK inhibitors of general formula (8) and their synthesis are described in WO2012170976. Preferred BTK inhibitors of general formula (8) are compound 12 in Table 1 shown below and the compounds disclosed in claim 9 of WO2012170976, preferably compound 12 in Table 1 shown below.

[0149] BTK inhibitors of general formula (9) and their synthesis are described in U.S. Patent Application Publication No. 2017 / 0136014. A preferred BTK inhibitor of general formula (9) is compound 13 in Table 1, shown below.

[0150] BTK inhibitors of general formula (10) and their synthesis are described in WO 2014173289. Preferred BTK inhibitors of general formula (10) are compounds 14 and 15 in Table 1 shown below, and the compounds disclosed in Table 3 of paragraph

[0643] of WO 2014173289, preferably compounds 14 and 15 in Table 1 shown below.

[0151] In a preferred embodiment, the BTK inhibitor is selected from Table 1.

[0152] [Table 1] JPEG0007676372000014.jpg216149

[0153] In some embodiments, the BTK inhibitor is selected from the group consisting of ibrutinib, zanbrutinib (BGB-3111), PCI-45292, PCI-45466, AVL-101 / CC-101 (Avila Therapeutics / Celgene Corporation), AVL-263 / CC-263 (Avila Therapeutics / Celgene Corporation), AVL-292 / CC-292 (Avila Therapeutics / Celgene Corporation), AVL-291 / CC-291 (Avila Therapeutics / Celgene Corporation), CNX774 (Avila Therapeutics), BMS-488516 (Bristol-Myers Squibb), BMS-509744 (Bristol-Myers Squibb), CGI-1746 (CGI Pharma / Gilead Sciences), CGI-560 (CGI Pharma / Gilead Sciences), CTA-056, GDC-0834 (Genentech), HY-11066 (further, CTK4I7891, HMS3265G21, HMS3265G22, HMS3265H21, HMS3265H22, 439574-61-5, AG-F-54930), ONO-4059 (Ono Pharmaceutical Co., Ltd.), ONO-WG37 (Ono Pharmaceutical Co., Ltd.), PLS-123 (Peking University), RN486 (Hoffmann-La Roche), HM71224 (Hanmi Pharmaceutical Company Limited), or LFM-A13. In preferred embodiments, the BTK inhibitor is ibrutinib, zanbrutinib (BGB-3111), or acalabrutinib (compound 4 in Table 1), of which ibrutinib is most preferred.

[0154] Bcl-2 inhibitors The term "Bcl-2 inhibitors" as used herein refers to compounds that selectively target, decrease or inhibit at least one activity of Bcl-2. The B-cell lymphoma 2 (Bcl-2) family of proteins are key regulators of the mitochondrial (also called "intrinsic") pathway of apoptosis. See Denial, NN and Korsmeyer, SJ Cell (2004) 116, 205-219. Misregulation of Bcl-2 is implicated in a wide variety of cancers, particularly hematological cancers such as follicular lymphoma, diffuse large cell lymphoma, and chronic lymphocytic leukemia. Adams, JM and Cory, S. Science (1998) 281, 1322-1326. As used herein, "Bcl-2" refers to B-cell lymphoma 2, an anti-apoptotic protein that has been implicated in many types of cancer, including chronic lymphocytic leukemia, melanoma, breast carcinoma, prostate carcinoma, and lung carcinoma. There are several targeted and selective Bcl-2 inhibitors, including oblimersen, navitoclax, and venetoclax. In a preferred embodiment, the Bcl-2 inhibitor is an antisense oligonucleotide or a small molecule that targets Bcl-2. In a more preferred embodiment, the Bcl-2 inhibitor is a small molecule. In another more preferred embodiment, the Bcl-2 inhibitor is an antisense oligonucleotide that targets Bcl-2. In a preferred embodiment, the Bcl-2 inhibitor is not an antibody.

[0155] Antisense oligonucleotide Bcl-2 inhibitors Antisense oligonucleotides targeting Bcl-2 can reduce Bcl-2 activity and thus act as Bcl-2 inhibitors. Preferred examples are oblimersen and PNT2258. In a more preferred embodiment, the antisense oligonucleotide Bcl-2 inhibitor is oblimersen. In another more preferred embodiment, the antisense oligonucleotide Bcl-2 inhibitor is PNT2258. In a preferred embodiment, the antisense oligonucleotide Bcl-2 inhibitor is encapsulated in a liposomal formulation, more preferably in a smarticle as described in Tolcher et al., 2013, DOI:10.1007 / s00280-013-2361-0.

[0156] Antisense DNA or RNA strands are non-coding and complementary to the coding strand (which is the template for producing RNA or protein, respectively). Antisense drugs are short sequences of RNA that hybridize with and inactivate mRNA, preventing it from being made into protein. Human lymphoma cell growth (with t(14;18) translocation) is inhibited by antisense RNA such as oblimersen, which targets the start codon region of Bcl-2 mRNA. Multiple in vitro studies led to the identification of oblimersen (also known as genasense), which is complementary to the first six codons of Bcl-2 mRNA. Antisense oligonucleotides have shown successful results in phase I / II trials for lymphoma. Preferred antisense oligonucleotide inhibitors are discussed in Dias and Stein (2002, Eur. J. Pharm. Biopharm.) DOI: 10.1016 / S0939-6411(02)00060-7. PNT2258 is a liposomal formulation of an antisense oligonucleotide Bcl-2 inhibitor developed by ProNAi Therapeutics, Inc. (see, e.g., Ebrahim et al., 2016, DOI:10.18632 / oncotarget.9872, or Tolcher et al., cited above).

[0157] Small molecule Bcl-2 inhibitors Examples of small molecules that inhibit Bcl-2 are ABT-737, and navitoclax (ABT-263), and venetoclax (ABT-199). Abbott Laboratories developed ABT-737 and navitoclax, part of a group of BH3 mimetic small molecule inhibitors (SMIs) that target Bcl-2 family proteins but not A1 or Mcl-1. ABT-737 and navitoclax are superior to previous Bcl-2 inhibitors, endowed with higher affinity for Bcl-2, Bcl-xL, and Bcl-w. Multiple in vitro studies have shown that primary cells from patients with B-cell malignancies are sensitive to ABT-737, making it the preferred Bcl-2 inhibitor. ABT-737 does not directly induce apoptosis, but rather enhances the effect of apoptotic signals, resulting in single-agent mechanism-based killing of cells in small cell lung carcinoma and lymphoma lines. In animal models, ABT-737 improves survival, induces tumor shrinkage, and cures a high percentage of mice. In preclinical studies utilizing patient xenografts, ABT-737 has shown efficacy for treating lymphoma and other blood cancers.

[0158] Abbvie has developed venetoclax (ABT-199), a highly selective inhibitor that inhibits Bcl-2 but not Bcl-xL or Bcl-w. A clinical trial studied the effect of venetoclax, a BH3 mimetic designed to block the function of the Bcl-2 protein, on patients with chronic lymphocytic leukemia (CLL) (Roberts et al., 2016, doi:10.1056 / NEJMoa1513257). Good responses were reported and no thrombocytopenia was observed, making venetoclax a highly preferred Bcl-2 inhibitor. Venetoclax was approved by the US FDA in April 2016 as a second-line treatment for CLL associated with 17-p deletion. In June 2018, the FDA extended the approval to all patients with CLL or small lymphocytic lymphoma, regardless of the presence or absence of 17p deletion, although still as a second-line treatment.

[0159] In a preferred embodiment, the Bcl-2 inhibitor has the general formula B1 [ka] (wherein the definitions of the variables (represented by letters with numerical and / or alphabetical superscripts) are as provided in claim 1 of WO2011149492). These compounds and their synthesis are known from WO2011149492. Preferred compounds of general formula B1 have the variables defined on page 76, line 19 to page 85, line 26, more preferably on page 84, line 28 to page 93, line 2 of WO2011149492.

[0160] In a preferred embodiment, the Bcl-2 inhibitor of general formula B1 is of general formula B2 [ka] (wherein the definitions of the variables (represented by letters with numerical and / or alphabetical superscripts) are as provided in EP 2 435 432 (B1)

[0007] , more preferably as provided in claim 2 of EP 2 435 432 (B1). Such Bcl-2 inhibitors and the preparation thereof are known from EP 2 435 432 (B1). A preferred compound of the general formula B2 is the compound named in EP 2435432(B1) in

[0008] or a pharma- ceutically acceptable salt thereof, a more preferred compound of the general formula B2 is the compound named in EP 2435432(B1) in

[0009] to

[0028] or a pharma- ceutically acceptable salt thereof, and a most preferred compound of the general formula B2 is 4-(4-{[2-(4-chlorophenyl)-4,4-dimethylcyclohex-1-en-1-yl]methyl}piperazin-1-yl)-N-({3-nitro-4-[(tetrahydro-2H-pyrazin-1-yl)methyl}piperazin-1-yl)-N-( and 4-(4-{[2-(4-chlorophenyl)-4,4-dimethylcyclohex-1-en-1-yl]methyl}piperazin-1-yl)-N-[(4-{[(trans-4-hydroxy-4-methylcyclohexyl)methyl]amino}-3-nitrophenyl)sulfonyl]-2-(1H-pyrrolo[2,3-b]pyridin-5-yloxy)benzamide or a therapeutically acceptable salt thereof. In a preferred embodiment, the compound of general formula B2 is 4-(4-{[2-(4-chlorophenyl)-4,4-dimethylcyclohex-1-en-1-yl]methyl}piperazin-1-yl)-N-({3-nitro-4-[(tetrahydro-2H-pyran-4-ylmethyl)amino]phenyl}sulfonyl)-2-(1H-pyrrolo[2,3-b]pyridin-5-yloxy)benzamide or a therapeutically acceptable salt thereof.In a preferred embodiment, the compound of general formula B2 is 4-(4-{[2-(4-chlorophenyl)-4,4-dimethylcyclohex-1-en-1-yl]methyl}piperazin-1-yl)-N-[(4-{[(trans-4-hydroxy-4-methylcyclohexyl)methyl]amino}-3-nitrophenyl)sulfonyl]-2-(1H-pyrrolo[2,3-b]pyridin-5-yloxy)benzamide or a therapeutically acceptable salt thereof.

[0161] In preferred embodiments, the Bcl-2 inhibitor is ABT-199 (venetoclax), ABT-737, ABT-263 (navitoclax), or PNT 2258. In more preferred embodiments, the Bcl-2 inhibitor is venetoclax or PNT 2258. In other preferred embodiments, the Bcl-2 inhibitor is 4-(4-{[2-(4-chlorophenyl)-5,5-dimethyl-1-cyclohexen-1-yl]methyl}-1-piperazinyl)-N-[(4-{[(2R)-4-(4-morpholinyl)-1-(phenylsulfanyl)-2-butanyl]amino}-3-[(trifluoromethyl)sulfonyl]phenyl)sulfonyl]-benzamide (navitoclax or ABT-263); tetrocarcin A; antimycin; gossypol (preferably (-)-gossypol acetic acid known as AT101); obatoclax (preferably obatoclax is its mesylate salt); ethyl 2-amino-6-bromo-4-(1-cyano-2-ethoxy-2-oxoethyl)-4H-chromene-3-carboxylate (HA14-1); oblimersen; Bak BH3 peptides; 4-[4-[(4'-chloro[1,T-biphenyl]-2-yl)methyl]-1-piperazinyl]-A / -[[4-[[(1R)-3-(dimethylamino)-1-[(phenylthio)methyl]propyl]amino]-3-nitrophenyl]sulfonyl]-benzamide (ABT-737); 2-((1H-pyrrolo[2,3-b]pyridin-5-yl)oxy)-4-(4-((4' -chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,T-biphenyl]-2-yl)methyl)piperazin-1-yl)-N-((3-nitro-4-(((tetrahydro-2H-pyran-4-yl)methyl)amino)phenyl)sulfonyl)benzamide (venetoclax); and S55746 (BCL201), or a pharma- ceutical acceptable salt thereof. S55746 is known from clinical trials for patients with chronic lymphocytic leukemia (CLL), B-cell non-Hodgkin's lymphoma, or multiple myeloma (see, for example, clinicaltrials.gov / ct2 / show / NCT02920697).

[0162] The structural formula of venetoclax is C 45 H 50 ClN 7 O 7 Venetoclax binds to two hydrophobic pockets of Bcl-2, generating hydrogen bonds between the azaindole nitrogen of venetoclax and Asp103 and Arg107 of BLC-2. This electrostatic interaction prevents the inhibitory effect of Bcl-2 on BAK and BAX, allowing the BAK and BAX proteins to initiate the intrinsic pathway of apoptosis (Scheffold et al., 2018, supra; Moia et al., 2018, supra). Despite selectively binding to Bcl-2 with high affinity, the drug also binds with significantly lower affinity to the pro-apoptotic BCL-XL and BCL-W, but has no reported activity against the pro-apoptotic protein MCL-1 (Scheffold et al., 2018, supra; Moia et al., 2018, supra).

[0163] A preferred Bcl-2 structure is shown below: These compounds are known in the art.

[0164] [Table 2] JPEG0007676372000018.jpg202149 JPEG0007676372000019.jpg79149

[0165] In a preferred embodiment, the Bcl-2 inhibitor is selected from compounds 1-11 in the table above, more preferably from compounds 1, 2, 3, 4, 5, 6, 7, 9, and 10, even more preferably from compounds 1, 2, 3, 4, 5, 6, 7, and 10, even more preferably from compounds 1, 2, 3, 4, and 5, and most preferably from compounds 1, 2, and 5.

[0166] In a preferred embodiment, a Bcl-2 inhibitor or a composition comprising such a Bcl-2 inhibitor is administered for at least one cycle of 5 days, and even more preferably, such Bcl-2 inhibitor is a small molecule, most preferably venetoclax.

[0167] Pharmaceutical Compositions In another aspect, the present invention relates to a pharmaceutical composition comprising at least one of an anti-CCR7 antibody (or antigen-binding fragment thereof) as defined herein, a BTK inhibitor as defined herein, and a Bcl-2 inhibitor as defined herein, for use in the present invention. The pharmaceutical composition preferably comprises at least one of a BTK inhibitor, a Bcl-2 inhibitor, and an anti-CCR7 antibody, or a pharmaceutical derivative or prodrug of these active ingredients, together with a pharma- ceutically acceptable carrier, adjuvant, or vehicle for administration to a subject. Said pharmaceutical composition can be used in a method of treatment by administration of an effective amount of the composition to a subject in need thereof, as described herein below. The term "subject" is used interchangeably with the term "recipient" herein and refers to all animals classified as mammals, as used herein, including, but not limited to, primates and humans. The subject is preferably a human male or female of any age or race. Treatment of a patient includes first-line, second-line, or third-line treatment.

[0168] The term "pharmaceutically acceptable carrier" as used herein is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, that are compatible with pharmaceutical administration (see, for example, "Handbook of Pharmaceutical Excipients", Rowe et al., 7th ed., 2012, www.pharmpress.com). The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, its use in the composition is contemplated. Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed and include, for example, buffers, such as phosphate, citric acid, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl, or benzyl alcohol; alkyl parabens, such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, zeolite, or the like. 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 (e.g., Zn-protein complexes); and / or non-ionic surfactants, such as TWEEN™, PLURONICS™, or polyethylene glycol (PEG).

[0169] The antibody of the present invention may be formulated in the same pharmaceutical composition as the BTK inhibitor and the Bcl-2 inhibitor, or each active agent may be formulated in its own pharmaceutical composition. The preferred route of administration will determine the preferred formulation for each active agent. If parenteral is the preferred route of administration for the antibody, while oral is the preferred route of administration for at least some of the BTK inhibitors and Bcl-2 inhibitors, the antibody is preferably formulated in a different pharmaceutical composition from the inhibitor. Administration of the antibody and at least one of the BTK inhibitor and the Bcl-2 inhibitor may be simultaneous or sequential, and may be effective in either order.

[0170] Additional active compounds can also be incorporated into the pharmaceutical composition of the present invention. Thus, in certain embodiments, the pharmaceutical composition of the present invention may also contain two or more active compounds necessary for the particular indication being treated, preferably two or more active compounds with complementary activities that do not adversely affect each other. For example, it may be desirable to provide additional chemotherapeutic agents, cytokines, analgesics, or immunomodulatory agents, such as immunosuppressants or immunostimulants. The effective amount of such other active agents depends, among other things, on the amount of the antibody of the present invention present in the pharmaceutical composition, the type of disease or disorder or treatment, etc.

[0171] In some embodiments, the antibody of the present invention is prepared with a carrier that can protect the compound from rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems, such as liposomes. Biodegradable, biocompatible polymers, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid, can be used. Methods for the preparation of such formulations will be clear to those skilled in the art. Liposomal suspensions, including targeted liposomes, can also be used as pharma- ceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,811 or U.S. Patent Publication No. 2011305751, which are incorporated herein by reference.

[0172] The route of administration of the antibody (or fragment thereof) of the present invention may be oral, parenteral, inhalation, or topical. The term "parenteral" as used herein includes intravenous, intraarterial, intralymphatic, intraperitoneal, intramuscular, subcutaneous, rectal, or vaginal administration. The intravenous form of parenteral administration is preferred. By "systemic administration" is meant oral, intravenous, intraperitoneal, and intramuscular administration. Of course, the amount of antibody required for therapeutic or prophylactic effect may vary with the antibody selected, the nature and severity of the condition being treated, and the patient. In addition, the antibody may be suitably administered by pulse infusion, for example with declining doses of the antibody. Preferably, the dosage is given by injections, most preferably intravenous or subcutaneous injections, depending in part on whether the administration is brief or chronic.

[0173] Thus, in certain embodiments, the pharmaceutical composition of the present invention may be in a form suitable for parenteral administration, for example, a sterile solution, suspension, or lyophilized formulation in an appropriate unit dosage form. Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions, and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EM (BASF, Parsippany, NJ), or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy needle passage exists. The composition must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier may be, for example, a solvent or dispersion medium containing water, ethanol, a pharma- ceutically acceptable polyol, for example, glycerol, propylene glycol, liquid polyethylene glycol, and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of a coating agent such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition.

[0174] Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

[0175] Sterile injection solutions can be prepared by incorporating the required amount of active compound (e.g., BTK inhibitor and / or Bcl-2 inhibitor or anti-CCR7 antibody) into a suitable solvent with one or a combination of the above-listed ingredients as required, followed by filtration sterilization. In general, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and other required ingredients from the above-listed ingredients. In the case of sterile powders for the preparation of sterile injection solutions, the preferred method of preparation is vacuum drying and freeze-drying, which obtains a powder of the active ingredient and any additional desired ingredients from its solution that has previously been sterile-filtered.

[0176] In certain embodiments, the pharmaceutical composition is administered intravenously (IV) or subcutaneously (SC). Suitable excipients, such as bulking agents, buffers, or surfactants, may be used. The mentioned formulations can be prepared using standard methods for preparing parenterally administered compositions, which are well known in the art and described in detail in various sources, including, for example, "Remington: The Science and Practice of Pharmacy" (ed. Allen, LV, 22nd edition, 2012, www.pharmpress.com).

[0177] It is particularly advantageous to formulate pharmaceutical compositions, i.e. parenteral compositions, into dosage unit forms for easy administration and uniformity of dosage.Dosage unit forms, as used herein, refer to physically discrete units suitable as unitary dosages for subjects to be treated, each unit containing a predetermined amount of active compound (the antibody of the present invention) calculated to produce a desired therapeutic effect in association with the required pharmaceutical carrier.The specifications for the dosage unit forms of the present invention are dependent on and directly depend on the unique characteristics of the active compound and the specific therapeutic effect to be achieved, as well as the limitations inherent in the technical field of compounding such active compounds for the treatment of individuals.

[0178] In general, the effective dosage of the antibody of the invention may depend on the relative efficacy of the compound selected, the severity of the disorder being treated, and the body weight of the affected individual. However, the active compound may be administered, typically once or more than once per day, for example 1, 2, 3, or 4 times per day, at a typical total daily dosage ranging from 0.001 to 1,000 mg / kg body weight / day, preferably from about 0.01 to about 100 mg / kg body weight / day, and most preferably from about 0.05 to 10 mg / kg body weight / day. More specifically, for the use of the invention, the anti-CCR7 antibody is preferably administered at a dosage of 1 to 1000, 2 to 500, 5 to 200, 10 to 100, 20 to 50, or 25 to 35 mg / kg body weight / day, preferably administered in multiple doses every 1, 2, 4, 7, 14, or 28 days.

[0179] In addition to administering the antibody to a patient, the present application contemplates administering the antibody by gene therapy. WO 96 / 07321 relates to the use of gene therapy to generate intracellular antibodies.

[0180] The pharmaceutical compositions can be included in a container, package, or dispenser device together with instructions for administration.

[0181] The antibodies and pharmaceutical compositions of the invention may be used together with other drugs to provide combination therapy, which may form part of the same composition or may be provided as separate compositions for administration at the same or different times.

[0182] In this document and the claims, the verb "comprise" and its conjugations are used in an open-ended sense to mean that the items following the word are included but not to exclude items not specifically mentioned. In addition, reference to an element by the indefinite article "a" or "an" does not exclude the possibility that more than one element is present, unless the context clearly requires that only one element is present. Thus, the indefinite article "a" or "an" typically means "at least one."

[0183] The terms "about" or "approximately," when used in connection with a numerical value (e.g., about 10), preferably mean that the value may be 5 or 10% more or less than the stated value (10).

[0184] All patent and literature references cited herein are incorporated by reference in their entirety.

[0185] The present invention is further described by the following examples, which should not be construed as limiting the scope of the invention. [Brief description of the drawings]

[0186] [Figure 1A] Graph showing that CCR7 expression is maintained in patients treated with BTK inhibitors. CCR7 expression is slightly decreased (ibrutinib) or unchanged (acalbrutinib or zanbrutinib) in CLL patients treated with BTK inhibitors. CCR7 and CD20 expression was determined in primary CLL cells from peripheral blood samples obtained from naïve patients (n=125), ibrutinib-treated patients (OT, n=44), patients with ibrutinib relapsed / resistant disease (RR, n=16), acalabrutinib-treated patients (ACALA, n=5), or zanbrutinib-treated patients (ZANA, n=4). A) Surface CCR7 (or CD20) expression was analyzed in terms of relative median intensity of fluorescence (RMIF, relative to unrelated isotype control). [Figure 1B] B) Surface CCR7 (or CD20) expression was analyzed in terms of the percentage of malignant cells expressing the CCR7 (or CD20) receptor. [Figure 2A]Graphs showing that CCR7 expression in malignant cells is high regardless of current or previous treatment. A) Graphs show CCR7 expression from one representative naïve (treatment-naïve) CLL patient, one representative CLL patient receiving ibrutinib (OT), one representative CLL patient who failed ibrutinib (RR), and one representative CLL patient receiving venetoclax. In each patient, CCR7 expression is displayed as a frequency histogram compared to matched unrelated controls. Marker regions were located at the base to these unrelated controls. Each histogram shows the intensity of fluorescence of cells labeled with anti-CCR7-PE. The percentage of CCR7 positive cells (within the marker) is also shown. [Figure 2B] B) Changes in CCR7 and CD20 expression in four CLL patients who initiated treatment with the BTK inhibitor ibrutinib. For each patient, CCR7 expression (determined as RMIF) is shown before (N) and after (Y) ibrutinib treatment. [Figure 3A] Graph showing that CCR7 expression is maintained in patients treated with Bcl-2 inhibitors. CCR7 expression is maintained in essentially all CLL cells from patients treated with venetoclax. CCR7 and CD20 expression was determined in CLL cells from peripheral blood samples obtained from naïve patients (n=125) or patients undergoing venetoclax treatment (OT, n=11). A) Surface CCR7 (or CD20) expression was analyzed in terms of relative median intensity of fluorescence (RMIF, relative to unrelated isotype control). [Figure 3B] B) Surface CCR7 and CD20 expression was analyzed in terms of the percentage of malignant cells expressing the CCR7 and CD20 receptors. [Figure 4]Graph showing that treatment with ibrutinib (in vivo) does not neutralize CCR7-mediated migration in CLL cells. A) Comparative analysis of migration index (% of input) between CLL cells obtained from naïve treatment-naïve patients (N, n=7, black bars) and CLL patients receiving ibrutinib (OT, n=10, grey bars). Migration was achieved by exposure of CLL cells to the CCR7 ligands CCL19 (1 μg / ml) or CCL21 (1 μg / ml). Migration was tested in a chemotaxis assay in which cells were placed in nude transwell chambers. The assay was performed for 4 hours at 37° C. Basal migration was defined as spontaneous migration not mediated by a chemotactic stimulus (no chemokines were added at this point). B) Comparative analysis of migration index (% of input) in CLL cells from treatment-naïve patients (n=7) exposed to different final concentrations of ibrutinib (0, i.e. vehicle / DMSO; 0.01, 0.1, 1, 10 μM) for 1 h, followed by exposure to the CCR7 ligands CCL19 (1 μg / ml) or CCL21 (1 μg / ml). Migration was tested in a chemotaxis assay in which cells were placed in nude transwell chambers. The assay was performed for 4 h at 37° C. (ibrutinib was present for the entire migration time). Basal migration was defined as spontaneous migration not mediated by a chemotactic stimulus (no chemokines were added at this point). As a positive control, cells without ibrutinib exposure were used (black bars). In A and B, bars represent the mean ± standard error of the mean (SEM). ns, not significant; *, p<0.05, **, p<0.01. [Diagram 5]Graph showing that treatment with 0.1 μM ibrutinib for 24 hours (in vitro) does not neutralize CCR7-mediated migration in CLL cells. Comparative analysis of migration index (% of input) in CLL cells obtained from naïve, treatment-naïve patients (n=6) exposed to CCR7 ligands CCL19 (1 μg / ml) or CCL21 (1 μg / ml) after 24 hours of incubation with ibrutinib at a final concentration of 0 (vehicle / DMSO) or 0.1 μM. Migration was tested in a chemotaxis assay in which cells were placed in nude transwell chambers. Migration assays were performed for 4 hours at 37° C. Basal migration was defined as spontaneous migration not mediated by a chemotactic stimulus (no chemokines were added at this point). As a positive control, cells without ibrutinib exposure were used (black bars). Bars represent the mean ± standard error of the mean (SEM). ns, not significant; *, p<0.05. [Figure 6] Graph showing that treatment with anti-CCR7 antibody CAP-100 neutralizes CCR7-mediated migration of CLL cells from patients treated with BTK inhibitors or Bcl-2 inhibitors. Specific blockade of CCR7-ligand interaction, expressed as the reduction in % of input cells migrating, is shown for CLL cells obtained from patients receiving venetoclax (A) or ibrutinib (B). CLL cells were incubated with different final concentrations (100, 10, 1, 0.1, 0.01, 0 μg / ml) of anti-CCR7 antibody (CAP-100). Cells were then placed in transwell chambers and exposed to chemokines CCL19 (white bars) and CCL21 (gray bars) at 1 μg / ml. Migration assays in naked transwell chambers were performed for 4 hours at 37° C. Basal migration is spontaneous migration not mediated by a chemotactic stimulus, in this respect no chemokine or mAb is added. Maximal migration is observed with the addition of chemokine but in the absence of CAP-100 (point 0). [Figure 7]Graph showing the effect of the combination of ibrutinib and anti-CCR7 antibody CAP-100 on migration induced by CCR7 ligand. Comparative analysis of migration index (% of input) in CLL cells obtained from treatment-naïve patients (n=5) incubated with anti-CCR7 antibody (CAP-100, 10 μg / ml), ibrutinib (0.1 μM), or the combination of both compounds (CAP-100 10 μg / ml, ibrutinib 0.1 μM) and then subjected to chemotaxis assay towards CCR7 ligand [CCL19 (1 μg / ml) or CCL21 (1 μg / ml), in nude transwell chambers]. Basal migration was defined as spontaneous migration not mediated by chemotactic stimuli (no chemokine was added in this respect; open bars). As a positive control, cells without preincubation with anti-CCR7 antibody or ibrutinib were used (closed bars). Bars represent the mean ± standard error of the mean (SEM). ns, not significant; *, p<0.05. [Figure 8-1] Graph showing that CCR7 expression in CLL cells from patients under treatment with BTK or Bcl-2 inhibitors can effectively induce cell death upon binding of anti-CCR7 antibodies. ADCC activity was assayed in one patient undergoing treatment with ibrutinib (A), one patient undergoing treatment with zanbrutinib (B), one patient undergoing venetoclax (C), and one patient with ibrutinib relapsed / resistant disease (D). In all cases, CLL cells were incubated in the presence of an unrelated matched isotype control or incubated with anti-CD20 antibody (rituximab, RTX) or anti-CCR7 antibody (CAP-100). Antibodies were tested at different final concentrations (0, 0.01, 0.1, 1, 10, 100 μg / ml). To perform ADCC, isolated PBMCs (peripheral blood mononuclear cells) were used as effector cells at a fixed E:T ratio of 10:1. The percentage of CLL cells killed by ADCC was determined by flow cytometry based on uptake of 7-aminoactinomycin D (7-AAD). The percentage of antibody-induced specific lysis is shown. [Figure 8-2]Graph showing that CCR7 expression in CLL cells from patients under treatment with BTK or Bcl-2 inhibitors can effectively induce cell death upon binding of anti-CCR7 antibodies. ADCC activity was assayed in one patient undergoing treatment with ibrutinib (A), one patient undergoing treatment with zanbrutinib (B), one patient undergoing venetoclax (C), and one patient with ibrutinib relapsed / resistant disease (D). In all cases, CLL cells were incubated in the presence of an unrelated matched isotype control or incubated with anti-CD20 antibody (rituximab, RTX) or anti-CCR7 antibody (CAP-100). Antibodies were tested at different final concentrations (0, 0.01, 0.1, 1, 10, 100 μg / ml). To perform ADCC, isolated PBMCs (peripheral blood mononuclear cells) were used as effector cells at a fixed E:T ratio of 10:1. The percentage of CLL cells killed by ADCC was determined by flow cytometry based on uptake of 7-aminoactinomycin D (7-AAD). The percentage of antibody-induced specific lysis is shown. EXAMPLES

[0187] introduction The chemokine receptor CCR7 controls the migration of certain immune cell subsets to lymph nodes, where CCR7 further contributes to the organization and activation of immune cells (Legler et al., Int J Biochem Cell Biol. 2014;54:78-82). As well as being of lymphoid origin, several hematological cancers with lymph node metastasis express CCR7 (Lopez-Giral et al., J Leukoc Biol. 2004;76(2):462-71). In this disease, CCR7 expression correlates with bulky lymphadenopathy, aggressive disease, and short survival (Legler et al., 2014; supra). Specifically, in B cell malignancies, CCR7 expression contributes to prolonging tumor cell residence in LNs and directing tumor cells to niches where they can provide tumor-promoting stimuli (Rehm et al., Blood, 2011;118(4):1020-33).

[0188] Recently, some reports disclosed that ibrutinib treatment of CLL cells resulted in downregulation of surface CCR7, which in turn caused subsequent impairment in CCR7-mediated migration and adhesion (Patrussi et al., 2015; supra, 75(19):4153-63; de Rooij et al., 2012; supra). As a result of these papers, it was proposed that one of the off-target effects mediated by ibrutinib was to reduce lymph node migration, caused in part by the restoration of increased surface CCR7 in CLL cells to levels normally expressed in B cells. Similarly, a positive feedback of expression profile has been reported for CCR7 and Bcl-2 (Kim et al., 2005; supra).

[0189] Based on the findings described, the combination of a BTK inhibitor and / or a Bcl-2 inhibitor with an anti-CCR7 antibody would not be expected to improve the therapeutic inhibition of malignant cell migration to lymph nodes (or other SLOs). Moreover, based on the previously reported loss of CCR7 expression, therapeutic approaches aimed at targeting CCR7 to kill tumor cells would be hindered and would not be expected to improve the individual therapeutic efficacy of BTK or Bcl-2 inhibitors.

[0190] Herein, we demonstrate the effect of BTK or Bcl-2 inhibitors on CCR7 expression in CLL patients and perform several approaches to examine whether these compounds may adversely affect CCR7-driven functions (e.g., migration) and whether these compounds may prevent effective target cell killing mediated by anti-CCR7 antibodies as a result of the lack of CCR7 expression on the target cell surface.

[0191] Materials and Methods sample Leukemia cells from patients were isolated from freshly drawn peripheral blood using Ficoll-Paque Plus density gradient centrifugation (Amersham Biosciences). Isolated cells were maintained for short-term culture in RPMI1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS), 2 mM L-glutamine, and 100 U / mL penicillin / 100 μg / mL streptomycin at 37°C and 5% CO2. Normal peripheral blood mononuclear cells (PBMCs) were obtained from adult blood buffy coats after Ficoll gradient and maintained in complete medium as described above. In all cases, patients and healthy donors signed informed consent in accordance with the Declaration of Helsinki. Experimental procedures were approved by the Institutional Review Board of the Hospital de la Princesa.

[0192] Three types of patient samples (n=196) were included in this study: 1) Naïve patients (i.e., patients not previously treated with either a BTK inhibitor or a Bcl-2 inhibitor). 2) Patients undergoing treatment with ibrutinib (or other BTK inhibitors). 3) Ibrutinib R / R patients.

[0193] CCR7 expression Flow cytometric analysis of CCR7 and CD20 expression on CLL cells was performed using a four-color monoclonal antibody panel: CD19-APC-H7 (BD Biosciences), CD3-FITC (BD Biosciences), CD5-APC (BD Biosciences), and either CCR7-PE (R&D Systems) or CD20-PE (R&D Systems). + CD3 - CD5 + CLL population. Appropriate matched isotype controls (IC) conjugated with PE were included (R&D Systems). 6Cells (in approximately 50 μl PB) were incubated with antibodies for 15-20 min at room temperature (RT). Cells were lysed with BD FACS™ lysis solution (BD Biosciences) for 10 min at RT and centrifuged at 1,800 rpm for 2 min. Cells were then washed with 2 ml of Dulbecco's phosphate-buffered saline (PBS) (Lonza) and finally centrifuged at 1,800 rpm for 2 min. Data acquisition was performed on a BD FACSCanto™ II flow cytometer (BD Biosciences). A minimum of 10,000 CLL cells were analyzed using BD FACSDIVA™ software. Results are expressed as both the percentage of CCR7 and CD20 positive cells [receptor %-control %] and the median relative fluorescence intensity (RMFI) of CCR7 and CD20 expression compared to IC [MIF(receptor) / MIF(control)].

[0194] Chemotaxis assay (migration) PBMCs were isolated by centrifugation in a Biocoll separation solution density gradient centrifugation (Merck Millipore). PBMCs were washed twice with saline solution (Fresenius) and centrifuged at 1200 for 10 min. Cells were cultured at 5 × 10 6 Cells were suspended at a concentration of 10 ... 5Cells were suspended in RPMI-1640, 0.1% BSA was added to the upper chamber, and chemokines CCL19 or CCL21 (1 μg / ml, Peprotech) were added to the lower well. After 4 hours at 37° C. and 5% CO, cells in the lower chamber were harvested and stained with anti-CD3-PE (BD Biosciences) and anti-CD5-APC (BD Biosciencies). Migrated cells were counted for 60 seconds in a BD FACSCanto™ II flow cytometer. The percentage of migrated cells (% of input) was calculated according to the following formula: 100×(number of cells in the lower chamber / number of cells added to the upper chamber). After calculating the % of input, the % of inhibition was estimated by the following formula: % of inhibition=[(% of input without mAb-% of input with mAb×100] / [% of input without mAb]. In addition, results are presented as % input relative to the maximum effect mediated by each chemokine (CK), calculated according to % input relative to CK=100×(% input / % input with CK).

[0195] Antibody-dependent cell-mediated injury (ADCC) ADCC assays were performed as described in SR-HPM-1026. Briefly, target tumor cells were incubated with medium alone (RPMI + 10% FBS) or in the presence of different final concentrations of IC, rituximab, alemtuzumab, or CAP-100 antibodies for 30 min at 37°C. Unbound antibodies were washed away (1800 rpm, 2 min / 2 times) and cells were incubated for 10 min at 37°C. 5Cells were plated at 100 / well in p96 U-bottom plates. Human PBMCs from healthy donors were obtained by Ficoll density gradient centrifugation. Effector cells were labeled with Calcein-UV cell tracker (Invitrogen) according to the manufacturer's protocol and stimulated with recombinant human IL-2 (500UI / ml, StemCell Technologies). Different effector:target (E:T) ratios were used [10:1 for dose response assay, (5:1, 25:1, 50:1 for E:T assay)]. After 6 hours, cells were stained with 7AAD, CD3-PE, and CD5-APC and analyzed by flow cytometry. Cell tracker - CD5 + CD3 - The percentage of specific lysis in CLL cells was determined by uptake of 7-AAD (BD Pharmingen) and calculated according to the following formula: % specific lysis = 100 x (ER-SR) / (MR-SR), where ER, SR, and MR represent experimental cell death, spontaneous cell death, and maximum cell death.

[0196] statistical analysis Statistics followed the following steps: 1) Homogeneity was tested using KS and / or Shapiro-Wilk and / or D'Agostino-Pearson normality tests. 2) Equality of variance was verified using Bartlett's test. 3) For parametric variables, group means were compared using ANOVA (one-way analysis of variance followed by Dunnett's multiple comparison test) or two-sample t-test. 4) For nonparametric variables, group medians were compared using the Kruskal-Wallis (followed by Dunn's multiple comparison test) and two-sample Mann-Whitney U tests. When paired samples were analyzed, Wilcoxon or Friedman tests were used. 5) All data were analyzed using Graph-Pad Prism5 (GraphPad Software, San Diego, CA). All tests were two-sided. P<0.05 was considered statistically significant. Means and standard errors of the mean (SEM) are shown for each group unless otherwise stated.

[0197] For the calculation of EC50 values, GraphPad Prism 5.0 (GraphPad Software, Inc.) was used. For this purpose, a global nonlinear regression, i.e., dose-response stimulation equation in robust fit mode (standard slope) was selected.

[0198] result Surface expression of CCR7 is maintained in CLL patients treated with BTK or Bcl-2 inhibitors.

[0199] Since previous papers reported substantial loss of surface CCR7 in CLL cells treated with ibrutinib, the first objective of this study was to validate these results in a larger cohort of CLL patients. Therefore, surface CCR7 was measured in 125 samples obtained from naive patients (patients who had not been previously treated with either a BTK inhibitor or a Bcl-2 inhibitor), 44 samples obtained from patients who were on ibrutinib treatment at the time of decision, and 16 samples obtained from patients who had discontinued ibrutinib treatment due to the development of relapsed / resistant disease. Remarkably, we observed that CCR7 expression was maintained during treatment with BTK inhibitors. As can be seen in Figures 1A-B and 2A, we observed that patients receiving ibrutinib as their current treatment continued to show significant surface CCR7 expression. In general, a slight but significant downregulation was observed in patients treated with ibrutinib, but nevertheless, approximately 100% of CLL cells maintained surface expression (determined as the percentage of CCR7-expressing cells) and also high surface levels (determined as RMIF). In fact, surface levels in treated patients were still above 200 arbitrary units. In other words, expression was still 200-fold higher than negative unrelated isotype controls. No changes in CCR7 cell surface expression were observed in patients who were being treated with other BTK inhibitors, such as acalabrutinib or zanbrutinib. Finally, in the case of ibrutinib R / R CLL cells, CCR7 surface levels were comparable (or even higher) than those of naive patients (Figures 1A-B and 2A).

[0200] Interestingly, in the case of CD20, a receptor known to be downregulated or lost by ibrutinib treatment, we confirmed that BTK inhibitors caused substantial downregulation of CD20 surface levels. For example, treatment caused a complete loss of CD20 in some patients as determined by a decrease in the percentage of CD20 positive cells compared to naive patients (Figures 1A-B). Thus, in Figure 2B, we show how ibrutinib treatment caused a consistent downregulation of CD20 surface levels in four (4 / 4) patients compared to the time when ibrutinib treatment was started (Y), while in the case of CCR7, the overall picture remained essentially constant, showing a large decrease in one patient (1 / 4), a mild decrease in another (1 / 4), but an increase in the other two patients (2 / 4). Finally, in striking contrast to CCR7, CD20 expression in ibrutinib R / R CLL cells was even lower than in patients receiving ibrutinib (Figures 1A and 2B).

[0201] Surface expression of CCR7 is maintained in CLL patients treated with Bcl-2 inhibitors.

[0202] In CLL cells obtained from patients under treatment with venetoclax, we observed that approximately 100% of CLL cells maintained surface expression (determined as the percentage of CCR7 expressing cells) and also maintained high surface levels (determined as RMIF). As with ibrutinib, surface levels in venetoclax treated patients were still approximately 150-250 fold higher than negative unrelated isotype controls (Figures 2A and 3A-B).

[0203] Treatment with BTK inhibitors does not neutralize CCR7-mediated migration After it was determined that BTK inhibitors did not induce significant loss of CCR7 on the CLL cell surface, we aimed to confirm that these compounds could adversely affect CCR7-mediated functions due to additional mechanisms that could impair migration induced by CCR7 ligands. To this end, a comparative analysis of the in vitro chemotactic response of CLL cells obtained from naive patients (N) and patients treated with ibrutinib (OT) was performed (Figure 4A). In both groups of patients, the basal migration index was comparable, and in both groups, CLL cells migrated significantly toward CCR7 ligands. Although a slight decrease was observed in the migration index of the OT group, these values ​​were not significantly different from the N group, indicating that BTK inhibitors had only a marginal effect on CCR7-induced migration. Furthermore, the migration index in the OT group did not reach basal levels, indicating that BTK inhibitor treatment does not completely neutralize migration induced by CCR7.

[0204] To further confirm this, in vitro chemotaxis assays were performed in transwell chambers. In these assay settings, CLL cells from naïve patients were incubated with increasing concentrations of ibrutinib (0, 0.01, 0.1, 1, 10 μM) for 1 h before exposure to CCR7 ligand (black bars). As can be seen in Figure 4B, the migration index with ibrutinib pretreatment was comparable to the control migration induced by CCR7 ligand. Only when administered at 0.1 μM, ibrutinib showed a moderate (but not significant) effect on migration towards CCL21.

[0205] As the lack of ibrutinib inhibition could be related to the short incubation time used (1 h before chemotaxis + additional 4 h during the chemotaxis assay), it was decided to perform new experiments in which naïve CLL cells were incubated with 0.1 μM ibrutinib for 24 h before testing chemotaxis. Again, as shown in Figure 5, no effect of ibrutinib was seen on the CCR7-mediated migration of CLL cells towards CCL19, and a small, non-significant effect was observed on the migration index towards CCL21.

[0206] Taken together, these results showed that CCR7-induced CLL cell migration was not significantly affected by prior in vivo or in vitro treatment with ibrutinib. In other words, the effects mediated by BTK inhibitors proved insufficient to suppress CCR7-mediated migration, and agents blocking CCR7 are required to achieve complete neutralization of CCR7-mediated migration to LNs. Nevertheless, the utility of anti-CCR7 as a blocking agent in patients receiving ibrutinib was not addressed. Moreover, as seen in Figure 1A, the slight downregulation of CCR7 surface levels was likely to negatively affect the activity of CAP-100 in this group of ibrutinib-treated patients. For these reasons, we tested the neutralizing activity of CAP-100 in CLL cells obtained from 10 CLL patients treated with ibrutinib. As can be seen in Figure 6, CAP-100 showed a clear dose-response inhibitory activity against CLL cells obtained from one patient undergoing venetoclax treatment (Figure 6A) or one patient who had failed ibrutinib treatment (Figure 6B). In both cases, the migration index to CCL19 or CCL21 reached basal levels after treatment with 10 or 100 μg / ml of antibody. These results confirmed that CLL cells from these groups of patients still responded to CCR7 ligands and that anti-CCR7 therapy is probably the best means of impairing CCR7-induced migration.

[0207] We further tested whether the combination of ibrutinib with an anti-CCR7 antibody such as CAP-100 could have an additive or synergistic neutralizing effect on CCR7 ligand-induced migration of CLL cells. For this purpose, CLL cells from naive patients were incubated with ibrutinib (0.1 μM) as a single agent, CAP-100 (10 μg / ml) as a single agent, and a combination of both compounds, followed by exposure to CCL19 or CCL21. The choice of compound concentrations was based on previous findings of ibrutinib (Figure 4B) and CAP-100 (Figure 6) as inhibitors of CCR7-mediated migration. As can be seen in Figure 7, ibrutinib as a monotherapy had no effect on CCR7-induced migration, thus confirming the results shown in Figure 4B and Figure 5. As expected, a reduction in migration to basal levels was achieved upon treatment with CAP-100, thus confirming that CAP-100 is a potent agent for the inhibition of migration elicited by CCR7 ligands. When CCL21 was used as the ligand, the combination of ibrutinib with CAP-100 showed a moderate, but not significant, increase in the neutralizing activity obtained by the anti-CCR7 antibody as a single agent, thus suggesting that a potential synergistic effect could be achieved by combining both compounds (Figure 7). Although further validation is required, this effect appeared to be specific for CCL21, since a similar outcome was not observed when CLL cells treated with such a combination were exposed to CCL19.

[0208] CCR7 expression in CLL cells from patients under treatment with BTK or Bcl-2 inhibitors can effectively induce cell death upon binding of anti-CCR7 antibodies.

[0209] In antibody-based therapy, high target surface levels are essential to achieve effective killing activity mediated by effector immune mechanisms such as ADCC, ADCP, or CDC. In fact, the proximity of two antibodies that bind to the target is necessary to successfully complete any of the listed mechanisms

[12] . Our results on expression demonstrated that surface CCR7 levels were slightly reduced after treatment with BTK inhibitors, although no reduction was observed in the percentage of tumor cells expressing the receptor. Therefore, we determined whether the reduction in RMIF observed in patients treated with BTK inhibitors could adversely affect the killing activity mediated by anti-CCR7 antibodies. To this end, a series of ADCC assays were performed in which target CLL cells from patients on ibrutinib treatment (Figure 8A), zanbrutinib treatment (Figure 8B), venetoclax treatment (Figure 8C), or ibrutinib R / R patients (Figure 8D) were incubated with increasing concentrations of CAP-100, rituximab (used as a reference therapeutic antibody), or IC, and then the cells were incubated with effector immune cells obtained from healthy donors at a fixed effector:target ratio of 10:1.

[0210] As seen in FIG. 8, CAP-100 demonstrated strong ADCC activity in all four groups of patients. Notably, CAP-100 was superior to rituximab. These results confirm that in the current setting, the slight reduction in surface CCR7 levels observed in patients treated with BTK or Bcl-2 inhibitors did not adversely affect antibody-mediated immune effector mechanisms such as ADCC. These results further confirm the utility of BTK inhibitor or Bcl-2 inhibitor-based combinations with anti-CCR7 antibodies such as CAP-100. Further embodiments are as follows. [Embodiment 1] 1. An anti-CCR7 antibody for use in the treatment of a hyperproliferative hematological disorder, the disorder comprising: a) disorders treated with at least one of a Bruton's tyrosine kinase (BTK) inhibitor and a B-cell lymphoma 2 (Bcl-2) inhibitor; b) the disorder has recurred following treatment with at least one of a BTK inhibitor and a Bcl-2 inhibitor; and c) A disorder that is resistant to treatment with at least one of a BTK inhibitor and a Bcl-2 inhibitor. an anti-CCR7 antibody, [Embodiment 2] The anti-CCR7 antibody of embodiment 1, administered simultaneously, separately or sequentially with at least one of a BTK inhibitor and a Bcl-2 inhibitor. [Embodiment 3] The anti-CCR7 antibody of embodiment 1 or 2, wherein the hyperproliferative hematological disorder is a disorder in which the hyperproliferative cells are cells of the B-cell lineage, preferably, the hyperproliferative hematological disorder is a B-cell hematological malignancy, more preferably lymphoma or leukemia. [Embodiment 4] The anti-CCR7 antibody of embodiment 4, wherein the hematological malignancy is selected from the group consisting of acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), acute monocytic leukemia (AMoL), chronic lymphocytic leukemia (CLL), high-risk CLL, small lymphocytic lymphoma (SLL), high-risk SLL, multiple myeloma (MM), non-Hodgkin's lymphoma (NHL), mantle cell lymphoma (MCL), follicular lymphoma (FL), Waldenstrom's macroglobulinemia (WM), diffuse large B-cell lymphoma (DLBCL), marginal zone lymphoma (MZL), Burkitt's lymphoma (BL), hairy cell leukemia (HCL), Richter's transformation, and T-cell prolymphocytic leukemia (T-PLL). [Embodiment 5] IC of 100 nM or less for inhibiting at least one of CCR7-dependent intracellular signaling and CCR7 receptor internalization by at least one CCR7 ligand selected from CCL19 and CCL21 50 The anti-CCR7 antibody according to any one of embodiments 1 to 4, comprising: [Embodiment 6] The anti-CCR7 antibody of embodiment 5, which inhibits CCR7-dependent intracellular signaling without substantial agonistic effects. [Embodiment 7] The anti-CCR7 antibody is a 20-fold increase over the K d K for the N-terminal extracellular domain of human CCR7 is up to 20-fold higher than d and the reference anti-CCR7 antibody is a murine anti-CCR7 antibody, the heavy chain variable domain of which has an amino acid sequence of SEQ ID NO: 1 and the light chain variable domain of which has an amino acid sequence of SEQ ID NO: 2. [Embodiment 8] The anti-CCR7 antibody of any one of embodiments 1 to 7, which is a chimeric, humanized, or human antibody. [Embodiment 9] The anti-CCR7 antibody of embodiment 8, which is an antibody having the HVR of an anti-human CCR7 antibody in which the amino acid sequence of the heavy chain variable domain is SEQ ID NO: 1 and the amino acid sequence of the light chain variable domain is SEQ ID NO: 2. [Embodiment 10] The anti-CCR7 antibody of any one of embodiments 1 to 9, wherein the BTK inhibitor is ibrutinib, zanbrutinib, or acalabrutinib, and the Bcl-2 inhibitor is venetoclax or navitoclax. [Embodiment 11] The anti-CCR7 antibody of any one of embodiments 1 to 10, wherein the hyperproliferative hematological disorder is a disorder in a treatment-naive patient. [Embodiment 12] The anti-CCR7 antibody of any one of embodiments 1 to 10, wherein the hyperproliferative blood disorder is a disorder in a patient who is treatment-naive with at least one of a BTK inhibitor, a Bcl-2 inhibitor, and an anti-CCR7 antibody. [Embodiment 13] The anti-CCR7 antibody of embodiment 12, wherein the hyperproliferative hematological disorder is resistant to and / or relapses following treatment with a chemotherapeutic agent other than a BTK inhibitor, a Bcl-2 inhibitor, and an anti-CCR7 antibody. [Embodiment 14] The anti-CCR7 antibody of any one of embodiments 1 to 10, wherein the hyperproliferative hematological disorder is resistant to and / or relapses following treatment with at least one of a BTK inhibitor and a Bcl-2 inhibitor, and chemotherapeutic agents other than a BTK inhibitor, a Bcl-2 inhibitor, and an anti-CCR7 antibody. [Embodiment 15] The anti-CCR7 antibody of embodiment 13 or 14, wherein the chemotherapeutic agent is one or more of fludarabine, cyclophosphamide, idelalisib, an anti-CD20 antibody, preferably rituximab, obinituzumab, ocrelizumab, veltuzumab, or ofatumumab, or an anti-CD52 antibody, preferably alemtuzumab.

Claims

1. A pharmaceutical composition comprising an anti-CCR7 antibody for use in treating a hyperproliferative blood disorder in a subject, comprising: the subject being treated with at least one of a Bruton's tyrosine kinase (BTK) inhibitor and a B-cell lymphoma 2 (Bcl-2) inhibitor; the anti-CCR7 antibody is administered simultaneously, separately or sequentially with at least one of a BTK inhibitor and a Bcl-2 inhibitor; The pharmaceutical composition, wherein the BTK inhibitor is selective for BTK and the Bcl-2 inhibitor is selective for Bcl-2.

2. The pharmaceutical composition of claim 1, wherein the hyperproliferative blood disorder is a disorder in which the hyperproliferative cells are cells of the B cell lineage.

3. The pharmaceutical composition of claim 1 or 2, wherein the hyperproliferative blood disorder is a B-cell hematological malignancy.

4. 4. The pharmaceutical composition of claim 3, wherein the B-cell hematological malignancy is selected from the group consisting of acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), acute monocytic leukemia (AMoL), chronic lymphocytic leukemia (CLL), high-risk CLL, small lymphocytic lymphoma (SLL), high-risk SLL, multiple myeloma (MM), non-Hodgkin's lymphoma (NHL), mantle cell lymphoma (MCL), follicular lymphoma (FL), Waldenstrom's macroglobulinemia (WM), diffuse large B-cell lymphoma (DLBCL), marginal zone lymphoma (MZL), Burkitt's lymphoma (BL), hairy cell leukemia (HCL), Richter's transformation, and T-cell prolymphocytic leukemia (T-PLL).

5. The anti-CCR7 antibody has an IC of 100 nM or less for inhibiting at least one of CCR7-dependent intracellular signaling and CCR7 receptor internalization induced by at least one CCR7 ligand selected from CCL19 and CCL21. ５０ The pharmaceutical composition according to any one of claims 1 to 4, comprising:

6. The pharmaceutical composition described in claim 5, wherein the anti-CCR7 antibody inhibits CCR7-dependent intracellular signaling without a substantial agonist effect.

7. The anti-CCR7 antibody has a K ｄ K for the N-terminal extracellular domain of human CCR7 is up to 20-fold higher than ｄ and the reference anti-CCR7 antibody is a murine anti-CCR7 antibody, the amino acid sequence of the heavy chain variable domain of which is SEQ ID NO: 1 and the amino acid sequence of the light chain variable domain of which is SEQ ID NO:

2.

8. The pharmaceutical composition according to any one of claims 1 to 7, which is a chimeric, humanized or human antibody.

9. The pharmaceutical composition described in claim 8, wherein the anti-CCR7 antibody is an antibody having the HVR of an anti-human CCR7 antibody in which the amino acid sequence of the heavy chain variable domain is SEQ ID NO: 1 and the amino acid sequence of the light chain variable domain is SEQ ID NO:

2.

10. A pharmaceutical composition described in any one of claims 1 to 9, wherein the BTK inhibitor is ibrutinib, zanbrutinib or acalabrutinib, and the Bcl-2 inhibitor is venetoclax or navitoclax.

11. The BTK inhibitor is a compound 1 to 15 in Table 1. 【Table 1】 is selected from The Bcl-2 inhibitor is selected from the group consisting of compounds 1 to 11 in Table 2. 【Table 2】 The pharmaceutical composition according to any one of claims 1 to 9, wherein the pharmaceutical composition is selected from the group consisting of 12. The pharmaceutical composition of any one of claims 1 to 11, wherein the subject is resistant to treatment with a chemotherapeutic agent other than a BTK inhibitor, a Bcl-2 inhibitor, and an anti-CCR7 antibody and / or has relapsed after said treatment.

13. The pharmaceutical composition of any one of claims 1 to 11, wherein the hyperproliferative blood disorder is resistant to and / or recurs after treatment with at least one of a BTK inhibitor and a Bcl-2 inhibitor, and a chemotherapeutic agent other than a BTK inhibitor, a Bcl-2 inhibitor, and an anti-CCR7 antibody.

14. The pharmaceutical composition of claim 12 or 13, wherein the chemotherapeutic agent is one or more of fludarabine, cyclophosphamide, idelalisib, an anti-CD20 antibody and an anti-CD52 antibody.

15. The pharmaceutical composition of claim 14, wherein the anti-CD20 antibody is rituximab, obinituzumab, ocrelizumab, veltuzumab or ofatumumab, and the anti-CD52 antibody is alemtuzumab.

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