Combination cancer therapy including anti-CCR9 antibody and vincristine

Abstract

The present invention provides an anti-CCR9 antibody molecule for use in a method for treating cancer in a mammalian subject, wherein the anti-CCR9 antibody molecule is administered simultaneously, sequentially or separately with a chemotherapeutic agent selected from the group consisting of vincristine, docetaxel, paclitaxel, nanoparticle albumin-bound paclitaxel, and vinblastine, and the anti-CCR9 antibody molecule and the chemotherapeutic agent are not conjugated together. Related methods of treatment are also provided.

Description

[Technical field]

[0001] This application claims priority to European Patent Application Publication No. 22382093.7, filed February 3, 2022, the contents and elements of which are incorporated herein by reference for all purposes.

[0002] [1] FIELD OF THEINVENTION [2] The present invention relates to therapeutic molecules and combinations thereof for the treatment of cancer. [Background technology]

[0003] [3] Background of the invention [4] Chemokines are a family of small structurally related proteins that bind to seven-transmembrane G protein-coupled receptors, mostly with chemotactic functions, and currently consist of 44 members in humans. Chemokines and their receptors play essential roles in organogenesis and lymphocyte trafficking, both in homeostatic and inflammatory conditions. Chemokines generate soluble or immobilized gradients and play a wide variety of roles, including inducing cell motility, stimulating cell growth, activation, or differentiation, or regulating mammalian organogenesis (Zlotnik and Yoshie 2000; Raman, Sobolik-Delmaire et al. 2011). With regard to cells of the immune system, chemokines play important roles in homeostasis and in both innate and adaptive immunity by controlling leukocyte trafficking and recruitment.

[0004] [5] Chemokines exert their biological effects by interacting with specific receptors present on the cell surface. Structurally, these receptors belong to the G protein-coupled seven-transmembrane domain receptor (GPCR) superfamily (Bachelerie, Ben-Baruch et al., 2014). This system is characterized by a certain redundancy, in that the same chemokine can bind to multiple receptors and vice versa, which are important molecules in the activation of various genes and trigger various cellular responses, including chemotaxis, cell survival and proliferation (DeVries, Kelvin et al., 2006). There is a strong association between aberrant tumor cell expression of chemokine receptors, such as CXCR4 or CCR7, and cancer progression, organ-selective metastasis, and poor prognosis. At the same time, they have great potential for use in tumor-targeted therapy (Weitzenfeld and Ben-Baruch 2014), and cancer-associated chemokines could promote tumor growth, angiogenesis, and chemoresistance (Lazennec and Richmond 2010; Mukaida and Baba 2012; Sarvaiya, Guo et al. 2013).

[0005] [6] The human chemokine receptor CCR9 (GenBank accession number U45982) is a member of this superfamily of receptors identified by Zaballos et al. (Zaballos, Gutierrez et al. 1999) (EMBL database accession number AJ132337) and Youn et al. (Youn, Kim et al. 1999). Under physiological conditions, expression of CCR9 has been described to be highly restricted in thymocytes (Zaballos, Gutierrez et al. 1999; Carramolino, Zaballos et al. 2001), infiltrating immune cells of the small intestine (Kunkel, Campbell et al. 2000), and in a small population of circulating memory T cells (Zabel, Agace et al. 1999), and plasmacytoid dendritic cells (Wendland, Czeloth et al. 2007).

[0006] [7] Unlike other chemokine receptors, CCR9 binds to a single ligand called CCL25 (chemokine ligand 25, or TECK), which is secreted by epithelial and dendritic cells from the thymus and small intestinal crypt epithelium and activates intracellular signaling pathways related to cell survival and migration upon binding to its receptor (Wurbel, Malissen et al., 2006). CCR9-CCL25 interaction is a key regulator of thymocyte migration in the thymus and cell homing to the intestinal tract. Recent insights into the mechanisms of CCL25 / CCR9 implicate them in tumor chemoresistance and metastasis (Tu, Xiao et al., 2016), indicating the potential application of CCR9 in targeted therapy.

[0007] [8] In cancer, CCR9 expression is increased on CD4+ helper T cells in acute and chronic lymphocytic leukemia (Qiuping, Qun et al., 2003) and other types of hematological malignancies such as follicular lymphoma and diffuse B-cell lymphoma (Wu, Doan et al., 2014). At the same time, aberrant expression of CCR9 has been described in some solid tumors such as prostate, breast, pancreatic, and melanoma cancers (Letsch, Keilholz et al., 2004; Amersi, Terando et al., 2008; Johnson, Singh et al., 2010; Singh, Stockard et al., 2011; Heinrich, Arrington et al., 2013; Gupta, Sharma et al., 2014). This expression of CCR9 is beneficial for cells because binding to its ligand CCL25 activates various signaling pathways, such as PI3K / Akt, increases cell survival in transformed T lymphocytes and resistance to apoptosis mediated by this signaling pathway in various types of cancer (Sharma, Singh et al. 2010; Johnson-Holiday, Singh et al. 2011), activates the JNK1 anti-apoptotic pathway, and enhances proliferation in leukemic cells, especially T-lineage acute lymphoblastic leukemia (T-ALL), by activating Notch1 (Mirandola, Chiriva-Internati et al. 2012). Furthermore, moderate levels of CCR9 expression have been observed in T-cell chronic lymphocytic leukemia (T-CLL) CD4+ cells. Notably, when CCR9 was internalized by T-ALL CD4+ T cells, it abolished the chemotactic and adhesive abilities of leukemic cells, indicating that CCR9 is closely related to leukemic cell invasion and metastasis.

[0008] [9] The PI3K / AKT pathway is one of the most important signaling pathways related to numerous physiological functions of cells and the development of certain diseases (Thorpe, Yuzugullu et al., 2015). Sharma et al. found that the interaction of CCR9 with its natural ligand CCL25 exerts an anti-apoptotic effect by upregulating the levels of PI3K, AKT, ERK1 / 2, and GSK-3β, while downregulating pro-apoptotic proteins such as caspase-3 levels in pancreatic cancer cells, thereby inhibiting cell apoptosis. However, a PI3K inhibitor (wortmannin) can significantly downregulate these CCR9-mediated anti-apoptotic proteins in pancreatic cancer cells, suggesting that the anti-apoptotic effect of CCR9 is mainly regulated by PI3K. In addition, CCL25 can inhibit the cytotoxic effect of etoposide (an antitumor drug) in tumor-bearing mice. However, this inhibition of cytotoxicity is abolished when CCR9 / CCL25 interaction is blocked using CCR9 monoclonal antibody (Sharma, Singh et al., 2010). Moreover, the combination of CCL25 neutralizing antibody and etoposide in the treatment of tumor burden causes significant tumor regression compared to monotherapy. These results indicate that the mechanism by which the CCR9 / CCL25 axis regulates PI3K / AKT-dependent anti-apoptotic signaling in pancreatic cancer cells may be related to low numbers of apoptotic cells and poor chemotherapy response. Matrix metalloproteinases (MMPs) constitute a class of zinc-dependent endogenous proteases that play a key role in tissue remodeling and degradation of various proteins in the extracellular matrix (Mittal, Patel et al., 2016), promote cell proliferation, migration, and differentiation, and play a role in apoptosis, angiogenesis, tissue repair, and immune response (Raffetto and Khalil, 2008). Furthermore, MMPs are also involved in key steps in the processes of cancer cell invasion and metastasis (Yoon, Park et al. 2003).

[0009]

[10] These results suggest that CCR9 / CCL25 expression and activation promotes cancer cell migration, invasion, and MMP expression, which together affect pancreatic cancer metastasis (Singh, Singh et al., 2004). Taken together, these findings suggest that knocking out or blocking CCR9 / CCL25 may have beneficial effects on the clinical treatment of pancreatic cancer.

[0010]

[11] CCR9 is highly expressed in breast cancer MDA-MB-231 cell line. Activation of CCR9 / CCL25 signaling did not significantly increase the migration of MDA-MB-231 cells, but it could significantly promote the invasion of MDA-MB-231 cells (Zhang, Sun et al. 2016). Activation of CCR9 / CCL25 signaling increased the invasion of MDA-MB-231 cells by significantly upregulating MMP-1 expression and moderately upregulating MMP-2 and MMP-11 expression. Of note, downregulation of E-cadherin and upregulation of N-cadherin and vimentin are involved in increased motility and migration of cancer cells (Cavallaro and Christofori 2004; Berx and van Roy 2009). Nevertheless, CCR9 / CCL25 signaling not only reduced the expression of E-cadherin, but also slightly reduced the expression of N-cadherin and vimentin, which may partially explain why CCR9 / CCL25 signaling had little effect on the migration of MDA-MB-231 cells. These results indicate that CCR9 / CCL25 signaling promotes breast cancer cell invasion by regulating multiple epithelial-mesenchymal transition (EMT) markers and that CCR9 / CCL25 signaling may be a potential target for blocking breast cancer cell invasion.

[0011]

[12] CCR9 / CCL25 interaction has been shown to promote breast cancer cell (MDA-MB-231) proliferation and upregulate anti-apoptotic signaling mediated by the PI3K / AKT survival pathway and independent of FAK (Johnson-Holiday, Singh et al., 2011). In addition, another study also found that CCR9 expression was significantly increased in moderately and poorly differentiated breast cancer tissues compared to expression levels in non-neoplastic breast tissues. Interestingly, CCR9 expression was significantly higher in poorly differentiated breast cancer tissues than in moderately differentiated breast cancer tissues. Similarly, CCR9 was highly expressed in the high-grade breast cancer MDA-MD-231 cell line compared to expression levels in the less aggressive breast cancer MCF-7 cell line. In summary, CCR9 is functionally significantly expressed in breast cancer tissues and cells, and CCL25 activation promotes breast cancer cell migration and invasion as well as MMP expression, which are key components of breast cancer metastasis.

[0012]

[13] Specific therapeutic approaches to treat human CCR9+ tumors growing in xenogeneic models are limited to the use of toxin-conjugated ligands (CCL25-PE38 fusion proteins) (Hu, Zhang et al. 2011) or ligand-specific antibodies, alone or in combination with the cytotoxic agent etoposide (Sharma, Singh et al. 2010). In these strategies, the CCL25-CCR9 interaction is aimed at eliminating tumor cells, and although results are limited, they provide evidence that CCR9 is a potential target for cancer immunotherapy.

[0013]

[14] Given the paucity of CCR9-targeting therapies, the industry remains in need of agents that specifically recognize CCR9 for the diagnosis, prognosis, and / or treatment of diseases or conditions associated with cells expressing CCR9. Recently, it was found that triple-negative breast cancer (TNBC) in both humans and mice does not express CCL25 (Chen, Cong, et al., 2020). Furthermore, another study found that the success of T cell-based cancer immunotherapy is limited by tumor resistance to killing by cytotoxic T lymphocytes (Brightman, Naradikian, et al., 2020). Tumor-immune resistance is mediated by cell surface ligands that engage immune inhibitory receptors on T cells (Borst, Ahrends, et al., 2018). These ligands are potent targets for therapeutic inhibition. CCR9 is expressed in many cancers and exerts a strong immunomodulatory effect on T cell responses in numerous tumors (Khandelwal, Breinig, et al., 2015). Unlike PD-L1, which inhibits T cell receptor signaling, CCR9 regulates STAT signaling in T cells, leading to decreased T helper 1 cytokine secretion and reduced cytotoxicity. Furthermore, inhibition of CCR9 expression on tumor cells promoted immunotherapy of human tumors by tumor-specific T cells in vivo. In summary, these studies elucidate the immunobiological role of the CCR9 / CCL25 axis in cancer immunity and immunotherapy, providing a solid experimental basis for further research on cancer immunotherapy.

[0014]

[15] WO 2015 / 075269 discloses anti-CCR9 antibodies and their use in the treatment of cancers such as T-cell acute lymphoblastic leukemia (T-ALL), prostate cancer, breast cancer, melanoma, ovarian cancer, colorectal cancer, and lung cancer. WO 2015 / 075269 also discloses combination therapy of anti-CCR9 antibodies with CCR9 antagonists such as those described in US Patent Application Publication No. 2005 / 0049286 and CCL25-PE38 fusion proteins. The anti-CCR9 antibodies and other agents may form part of the same composition or be provided as separate compositions for simultaneous or separate administration. WO 2015 / 075269 also discloses immunoconjugates in which the anti-CCR9 antibodies are conjugated to or in the form of fusion proteins with another therapeutic agent, such as a chemotherapy agent including vincristine.

[0015]

[16] There is an urgent and unmet medical need for effective therapies to treat cancer. Despite the advances described above, there is still a need for treatment regimens that further extend survival for the treatment of cancer, including T-cell acute lymphoblastic leukemia (T-ALL) and pancreatic cancer. The present invention seeks to address this need and further provides related advantages, as described in detail herein. Summary of the Invention [Means for solving the problem]

[0016]

[17] BRIEF DESCRIPTION OF THE PREFERRED EMBODIMENTS

[18] The present invention relates generally to therapeutic agents and their uses for the treatment of cancer, particularly cancers such as T-ALL, in mammalian subjects.

[0017]

[19] As described herein, the inventors surprisingly found that combination treatment of tumor-bearing mice with a CCR9-targeting antibody (SRB1) in combination with the chemotherapeutic agent vincristine caused a significant increase in median survival, not only compared to controls, but also compared to treatment groups receiving only SRB1 antibody or only vincristine. The results described herein indicate a synergistic relationship between SRB1 and vincristine in terms of antitumor efficacy. Furthermore, a significant increase in median survival was also achieved with a triple combination treatment using SRB1 antibody, vincristine, and the corticosteroid dexamethasone. Without wishing to be bound by a particular theory, the inventors believe that the synergistic relationship for antitumor efficacy observed with SRB1 antibody and vincristine is also exhibited by other anti-CCR9 antibodies in combination with other vinca alkaloids described herein.

[0018]

[20] As defined herein, the proposed combination dosing schedule and optimal administration form are determined by the different chemical compositions and inherent different half-lives of the compounds: anti-CCR9 antibody molecules and chemotherapeutic agents. Therefore, separate and / or sequential administration is recommended (as appropriate) according to the following clinical guidelines. The formation of antibody-drug conjugates is considered less preferred because the optimal frequency of administration of both agents may differ, since antibodies have a long half-life and can be administered no more frequently than once a week, whereas chemotherapeutic agents may require more frequent administration to maintain high concentrations.

[0019]

[21] Thus, in a first aspect, the present invention provides an anti-CCR9 antibody molecule for use in a method of treating cancer in a mammalian subject, wherein the anti-CCR9 antibody molecule is administered simultaneously, sequentially or separately with a chemotherapeutic agent, such as a chemotherapeutic agent selected from a vinca alkaloid (e.g., vincristine), docetaxel, paclitaxel, nanoparticle albumin-bound paclitaxel, and vinblastine, and the anti-CCR9 antibody and the chemotherapeutic agent are not conjugated together. In certain embodiments, the anti-CCR9 antibody molecule may be for use in a method further comprising the simultaneous, sequential or separate administration of a corticosteroid, such as dexamethasone.

[0020]

[22] In some embodiments, the anti-CCR9 antibody molecule comprises an anti-CCR9 monoclonal antibody or antigen-binding fragment thereof that specifically binds to CCR9. In particular, the anti-CCR9 monoclonal antibody or antigen-binding fragment thereof may be selected from the group consisting of Fv, Fab, F(ab')2, Fab', scFv, scFv-Fc, minibody, nanobody, and diabody.

[0021]

[23] In some embodiments, the antibody or antigen-binding fragment thereof can include:

[24] Heavy chain complementarity determining region 1 (CDR-H1) containing the amino acid sequence: NFWMN (SEQ ID NO: 1) or KFWMN (SEQ ID NO: 2);

[25] Heavy chain complementarity determining region 2 (CDR-H2) containing the amino acid sequence: EIRLKSNNYATHYAESVKG (SEQ ID NO:3);

[26] heavy chain complementarity determining region 3 (CDR-H3) containing the amino acid sequence DGWFAY (SEQ ID NO:4);

[27] Light chain complementarity determining region 1 (CDR-L1) comprising the amino acid sequence: RSSQSLLHSNGNTYVQ (SEQ ID NO:5) or RSSQSLVHSNGNTYLN (SEQ ID NO:6);

[28] Light chain complementarity determining region 2 (CDR-L2) comprising the amino acid sequence: KVSNRFP (SEQ ID NO: 7) or KVSNRFS (SEQ ID NO: 8); and

[29] Light chain complementarity determining region 3 (CDR-L3) comprising the amino acid sequence: AQSTHVPRT (SEQ ID NO:9) or SQSTHFPRT (SEQ ID NO:10).

[0022]

[30] In certain embodiments, the six CDR sequences may be selected according to the "91R" antibody disclosed in Figure 9 of WO 2015 / 075269, which is incorporated herein by reference. In this case, the CDR sequences are as follows: CDR-H1 is SEQ ID NO: 1, CDR-H2 is SEQ ID NO: 3, CDR-H3 is SEQ ID NO: 4, CDR-L1 is SEQ ID NO: 5, CDR-L2 is SEQ ID NO: 7, and CDR-L3 is SEQ ID NO: 9.

[0023]

[31] In certain embodiments, the six CDR sequences may be selected according to the "92R" antibody disclosed in Figure 9 of WO 2015 / 075269, which is incorporated herein by reference. In this case, the CDR sequences are as follows: CDR-H1 is SEQ ID NO:2, CDR-H2 is SEQ ID NO:3, CDR-H3 is SEQ ID NO:4, CDR-L1 is SEQ ID NO:6, CDR-L2 is SEQ ID NO:8, and CDR-L3 is SEQ ID NO:10.

[0024]

[32] In some embodiments, the antibody or antigen-binding fragment thereof comprises:

[33] Amino acid sequence:

[0025] [ka]

[0026] or

[0027] [ka]

[0028] or

[0029] [ka]

[0030] a heavy chain variable region comprising:

[34] Amino acid sequence:

[0031] [ka]

[0032] or

[0033] [ka]

[0034] or

[0035] [ka]

[0036] a light chain variable region comprising Includes.

[0037]

[35] In particular, the antibody or antigen-binding fragment thereof may comprise the VH and VL of SEQ ID NOs: 11 and 14, or SEQ ID NOs: 12 and 15, or SEQ ID NOs: 13 and 16.

[0038]

[36] In some embodiments, the antibody or antigen-binding fragment thereof has a binding affinity dissociation constant K D It indicates 500nM, 250nM, 100nM, 10nM or less. D The value may be determined by any suitable method. D The value may be as determined by surface plasmon resonance (SPR) (e.g., BIACORE). In certain cases, the binding affinity constant K D may be as determined or estimated by whole cell-based antibody binding determinations.

[0039]

[37] In some embodiments, the antibody or antigen-binding fragment thereof exhibits CCR9-specific binding in the presence of human CCL25 at a concentration of 10 μg / mL, as measured by flow cytometry. The inventors have found that certain anti-CCR9 antibodies, such as "91R" and "92R" as disclosed in WO 2015 / 075269, exhibit CCR9 binding that is essentially unaffected by the presence of the ligand CCL25. This is in contrast to anti-CCR9 antibodies, such as "3C3" as disclosed in WO 00 / 53635, which exhibit significant sensitivity to the presence of CCL25. Indeed, at physiologically relevant concentrations of CCL25, the binding of antibody "3C3" to CCR9 is effectively abolished. The "SRB1" antibody, having VH and VL of SEQ ID NOs: 13 and 16, respectively, has also been found to exhibit CCR9 binding in the presence of CCL25. Thus, like "91R" and "92R," "SRB1" is preferred for use in the treatment of conditions in which CCL25 levels may be elevated, such as the treatment of certain cancers, as described in detail herein.

[0040]

[38] In some embodiments, the antibody or antigen-binding fragment thereof is + It shows lymphocyte depletion. The inventors have observed antibody-mediated complement-dependent cytotoxicity (CDC) using anti-CCR9 antibodies such as "91R", "92R" and "SRB1" as described above. In certain embodiments, the anti-CCR9 antibody or antigen-binding fragment thereof may be an antibody isotype or subisotype that shows or is engineered to show CDC effector function (e.g., for human IgG, hIgG3>hIgG1>hIgG2>hIgG4; for mouse, mIgG2a>mIgG1, IgM also shows good CDC).

[0041]

[39] In some embodiments, the chemotherapeutic agents include docetaxel, paclitaxel, nanoparticle albumin-bound paclitaxel, vinblastine, and / or vincristine.

[0042]

[40] In some embodiments, the chemotherapeutic agent comprises a vinca alkaloid, such as vincristine, vinblastine, vindesine, vinorelbine, vincaminol, vineridin, and / or vinburnine.

[0043]

[41] In some embodiments, the simultaneous, sequential, or separate administration further comprises administration of a corticosteroid, such as dexamethasone.

[0044]

[42] In some embodiments, a vinca alkaloid (e.g., vincristine) and a corticosteroid (e.g., dexamethasone) are both administered along with the anti-CCR9 antibody molecule.

[0045]

[43] In some embodiments, the subject is a mammal (preferably a human) diagnosed with cancer or at risk for developing cancer. In some embodiments, the subject has been or is being treated with an anti-cancer therapy (e.g., anti-cancer drug therapy, surgical therapy, and / or radiation therapy). In certain cases, the subject's cancer may have recurred, metastasized, and / or become resistant to first-line therapy.

[0046]

[44] In some embodiments, the subject cancer is a hematological tumor.

[0047]

[45] In some embodiments, the subject's cancer is selected from the group consisting of T-cell acute lymphoblastic leukemia (T-ALL), T-cell lineage lymphoma, and acute myeloid leukemia (AML).

[0048]

[46] In a second aspect, the present invention provides a chemotherapeutic agent for use in a method of treating cancer in a mammalian subject, the chemotherapeutic agent being administered simultaneously, sequentially or separately with an anti-CCR9 antibody molecule, the chemotherapeutic agent being selected from the group consisting of vinca alkaloids (e.g., vincristine), docetaxel, paclitaxel, nanoparticle albumin-bound paclitaxel, and vinblastine, and the chemotherapeutic agent and the anti-CCR9 antibody molecule are not conjugated together. In some embodiments, the method may further comprise administering simultaneously, sequentially or separately a corticosteroid such as dexamethasone.

[0049]

[47] An anti-CCR9 antibody molecule may be defined according to the first aspect of the invention.

[0050]

[48] ​​The chemotherapeutic agent may be defined according to the first aspect of the invention.

[0051]

[49] The subject and / or the cancer may be defined according to the first aspect of the invention.

[0052]

[50] In a third aspect, the present invention provides a method of treating cancer in a mammalian subject, comprising administering to a subject in need thereof, simultaneously, sequentially, or separately, a therapeutically effective amount of an anti-CCR9 antibody molecule and a chemotherapeutic agent selected from the group consisting of a vinca alkaloid (e.g., vincristine), docetaxel, paclitaxel, nanoparticle albumin-bound paclitaxel, and vinblastine, wherein the anti-CCR9 antibody molecule and the chemotherapeutic agent are not conjugated together. In some embodiments, the method may further comprise administering simultaneously, sequentially, or separately a corticosteroid, such as dexamethasone.

[0053]

[51] An anti-CCR9 antibody molecule may be defined according to the first aspect of the invention.

[0054]

[52] The chemotherapeutic agent may be defined according to the first aspect of the invention.

[0055]

[53] The subject and / or the cancer may be defined according to the first aspect of the invention.

[0056]

[54] In a fourth aspect, the present invention provides the use of an anti-CCR9 antibody molecule in the preparation of a medicament for use in a method of treating cancer in a mammalian subject, said method being defined according to the third aspect of the invention.

[0057]

[55] In a fifth aspect, the present invention provides the use of a chemotherapeutic agent in the preparation of a medicament for use in a method of treating cancer in a mammalian subject, the method being defined according to the third aspect of the invention and the chemotherapeutic agent being defined according to the first aspect of the invention.

[0058]

[56] The present invention includes combinations of aspects and preferred features described except where such combinations are expressly impermissible or expressly avoided.

[0059]

[57] These and further aspects and embodiments of the present invention are described in further detail below with reference to the accompanying examples and figures. [Brief description of the drawings]

[0060]

[58] BRIEF DESCRIPTION OF THE DRAWINGS [Figure 1]

[59] Figure 1 shows the optimization of SRB1 antibody dose (16 mg / kg) and its effect on survival of NSG mice xenografted with MOLT4-GFP. (A) Experimental design. Briefly, NSG mice were implanted with MOLT-4 cells (1x106 cells / mouse; IV) on day 0, and from day 4, 16 mg / kg SRB1 or isotype was administered IP once a week for 4 weeks, and the mice were monitored daily. (B) Cumulative survival of mice. [Diagram 2]

[60] Figure 2 shows the in vivo efficacy of the anti-CCR9 antibody SRB1 (4 mg / kg) in combination with a suboptimal dose of SRB1 and chemotherapy in NSG mice bearing MOLT4-GFP xenografts. (A) NSG mice were implanted with MOLT-4 cells (1 × 106 cells / mouse; IV) on day 0 and received 4 mg / kg SRB1, PBS, or isotype IP on days 4, 10, 17, and 24. (B) In addition to the groups described in (A), mice received vincristine 0.6 mg / kg on day 5 or dexamethasone 50 μg / mouse / dose on days 5–9 (C), or both (D). [Diagram 3]

[61] Figure 3 shows the effect of the anti-CCR9 antibody SRB1 (4 mg / kg) in combination with chemotherapy on the survival of NSG mice bearing MOLT4-GFP xenografts. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0061]

[62] Detailed Description of the Invention

[63] Aspects and embodiments of the present invention are described herein. Further aspects and embodiments will be apparent to those skilled in the art. All documents mentioned in this text are incorporated herein by reference.

[0062]

[64] For the avoidance of doubt, the theoretical explanations provided herein are provided for the purpose of enhancing the understanding of the reader, and the inventors do not wish to be bound by any of these theoretical explanations.

[0063]

[65] Any section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0064]

[66] Throughout this specification, including the claims which follow, unless the context indicates otherwise, the words "comprise" and "include", and variations such as "comprises", "comprising", and "including", should be understood to imply the inclusion of a stated integer or step or group of integers or steps, but not the exclusion of any other integer or step or group of integers or steps.

[0065]

[67] It should be noted that, as used in the specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context dictates otherwise. Ranges may be expressed herein as from "about" one particular value and / or to "about" another particular value. When such a range is expressed, another aspect includes from the one particular value and / or to the other particular value. Similarly, when values ​​are expressed as approximations, by use of the antecedent "about," it is to be understood that the particular value forms another aspect. The term "about" with respect to numerical values ​​is optional and may mean, for example, + / - 10%.

[0066]

[68] CCR9

[69] Chemokines are a family of small, structurally related proteins that bind to seven-transmembrane G protein-coupled receptors. Chemokines and their receptors play essential roles in organogenesis and lymphocyte trafficking, both under homeostatic and inflammatory conditions.

[0067]

[70] The amino acid sequence of CCR9 (CC chemokine receptor type 9) has been published under UniProt accession number P51686 (see second edition dated September 5, 2006), the entire contents of which are incorporated herein by reference.

[0068]

[71] Anti-CCR9 antibody molecule

[72] The anti-CCR9 antibody molecule binds, preferably specifically, to CCR9 (e.g., human CCR9). In certain embodiments, the anti-CCR9 antibody molecule comprises a monoclonal antibody or antigen-binding fragment thereof that specifically binds to CCR9. In this context, "specifically binds" can be distinguished from non-specific binding by the degree of binding affinity and / or binding selectivity, where the binding affinity for CCR9 is greater than for other antigens. The anti-CCR9 antibody or antigen-binding fragment thereof can be selected from the group consisting of Fv, Fab, F(ab')2, Fab', scFv, scFv-Fc, minibody, nanobody, and diabody.

[0069]

[73] In some embodiments, the antibody or antigen-binding fragment thereof comprises:

[74] Heavy chain complementarity determining region 1 (CDR-H1) containing the amino acid sequence: NFWMN (SEQ ID NO:1) or KFWMN (SEQ ID NO:2);

[75] Heavy chain complementarity determining region 2 (CDR-H2) containing the amino acid sequence: EIRLKSNNYATHYAESVKG (SEQ ID NO:3);

[76] heavy chain complementarity determining region 3 (CDR-H3) containing the amino acid sequence DGWFAY (SEQ ID NO:4);

[77] Light chain complementarity determining region 1 (CDR-L1) containing the amino acid sequence: RSSQSLLHSNGNTYVQ (SEQ ID NO:5) or RSSQSLVHSNGNTYLN (SEQ ID NO:6);

[78] Light chain complementarity determining region 2 (CDR-L2) comprising the amino acid sequence: KVSNRFP (SEQ ID NO:7) or KVSNRFS (SEQ ID NO:8); and

[79] Light chain complementarity determining region 3 (CDR-L3) comprising the amino acid sequence: AQSTHVPRT (SEQ ID NO:9) or SQSTHFPRT (SEQ ID NO:10).

[0070]

[80] In certain embodiments, the six CDR sequences may be selected according to the "91R" antibody disclosed in Figure 9 of WO2015 / 075269, which is incorporated herein by reference. In this case, the CDR sequences are as follows: CDR-H1 is SEQ ID NO: 1, CDR-H2 is SEQ ID NO: 3, CDR-H3 is SEQ ID NO: 4, CDR-L1 is SEQ ID NO: 5, CDR-L2 is SEQ ID NO: 7, and CDR-L3 is SEQ ID NO: 9.

[0071]

[81] In certain embodiments, the six CDR sequences may be selected according to the "92R" antibody disclosed in Figure 9 of WO2015 / 075269, which is incorporated herein by reference. In this case, the CDR sequences are as follows: CDR-H1 is SEQ ID NO:2, CDR-H2 is SEQ ID NO:3, CDR-H3 is SEQ ID NO:4, CDR-L1 is SEQ ID NO:6, CDR-L2 is SEQ ID NO:8, and CDR-L3 is SEQ ID NO:10.

[0072]

[82] In some embodiments, the antibody or antigen-binding fragment thereof comprises:

[83] Amino acid sequence:

[0073] [ka]

[0074] or

[0075] [ka]

[0076] or

[0077] [ka]

[0078] a heavy chain variable region comprising:

[84] Amino acid sequence:

[0079] [ka]

[0080] or

[0081] [ka]

[0082] or

[0083] [ka]

[0084] A light chain variable region comprising:

[0085]

[85] In particular, the antibody or antigen-binding fragment thereof may comprise the VH and VL of SEQ ID NOs:11 and 14, or SEQ ID NOs:12 and 15, or SEQ ID NOs:13 and 16.

[0086]

[86] In some embodiments, the antibody or antigen-binding fragment thereof has a binding affinity dissociation constant K D It indicates 500nM, 250nM, 100nM, 10nM, or less. D The value may be determined by any suitable method. D The value may be as determined by surface plasmon resonance (SPR) (e.g., BIACORE). In certain cases, the binding affinity constant K D may be as determined or estimated by whole cell-based antibody binding determinations.

[0087]

[87] As used herein, the term "antibody molecule" of the present invention includes antibodies, or antigen-binding fragments thereof, including full-length antibodies (e.g., IgG), as well as antigen-binding fragments thereof, such as Fab, Fab', F(ab')2, Fv fragments, human antibodies, humanized antibodies, chimeric antibodies, antibodies of non-human origin, recombinant antibodies, and immunoglobulin-derived polypeptides produced by genetic engineering techniques, such as single-chain Fv (scFv), diabodies, heavy chains or fragments thereof, light chains or fragments thereof, VH or dimers thereof, VL or dimers thereof, disulfide-stabilized Fv fragments (dsFv), molecules with single-chain variable region domains (Abs), minibodies, scFv-Fc, and fusion proteins comprising antibodies, or any other modified structure of an immunoglobulin molecule that contains an antigen recognition site of a desired specificity. The antibody of the present invention may also be a bispecific antibody. An antibody fragment may refer to an antigen-binding fragment. The antibody includes any class of antibody, i.e., IgA, IgD, IgE, IgG (or a subclass thereof), and IgM, and the antibody need not be of any particular class. Additionally, the antibody of the present invention may be conjugated to an additional compound, such as a therapeutic agent, a toxin, etc.

[0088]

[88] In certain embodiments, the anti-CCR9 antibody or antigen-binding fragment thereof may be any anti-CCR9 antibody disclosed in WO 2015 / 075269, the contents of which are expressly incorporated by reference herein.

[0089]

[89] Target

[90] As used herein, a subject is a mammal, preferably a human. The subject may be a non-human mammal, such as a farm animal or livestock or a research animal. The subject may be male or female. The subject may be a patient. The subject may have been diagnosed with a disease or condition requiring treatment, may be suspected of having such a disease or condition, or may be at risk of developing such a disease or condition. In particular, the subject may have cancer, or may be suspected of having cancer, or may be at risk of developing cancer. The subject may have been or may be undergoing treatment with an anti-cancer therapy (e.g., anti-cancer drug therapy, surgical therapy, and / or radiation therapy). In certain cases, the subject's cancer may have recurred, metastasized, and / or acquired resistance to first-line therapy. The subject may have, in particular, a hematological tumor. The subject may have, in particular, T-cell acute lymphoblastic leukemia (T-ALL), T-cell lineage lymphoma, or acute myeloid leukemia (AML).

[0090]

[91] Combination therapy

[92] As described in detail herein, the inventors found that combination treatment of tumor-bearing mice with a CCR9-targeting antibody (SRB1) and the chemotherapeutic agent vincristine caused a significant increase in median survival, not only compared to controls, but also compared to treatment groups receiving only SRB1 antibody or only vincristine. The results described herein indicate a synergistic relationship between SRB1 and vincristine in terms of antitumor efficacy. Furthermore, a significant increase in median survival was also achieved with a triple combination treatment using SRB1 antibody, vincristine, and the corticosteroid dexamethasone. Without wishing to be bound by a particular theory, the inventors believe that the synergistic relationship for antitumor efficacy observed with SRB1 antibody and vincristine is also exhibited by anti-CCR9 antibodies in combination with other vinca alkaloids or in combination with other chemotherapeutic agents identified herein.

[0091]

[93] The combination therapy as contemplated herein differs from the antibody-drug conjugate approach disclosed in WO2015 / 075269, since the combination therapy of the present invention includes an anti-CCR9 antibody molecule administered simultaneously, sequentially, or separately with the chemotherapeutic agent identified herein. That is, the combination therapy includes the administration of at least two separate agents that are not conjugated or covalently linked to each other. These agents may be administered together as a mixture in a single pharmaceutical composition, or may be administered separately as two different pharmaceutical compositions. Advantageously, the anti-CCR9 antibody molecule (e.g., naked anti-CCR9 antibody or anti-CCR9 antibody-drug conjugate) may be administered at different times, in different amounts or ratios, or at different frequencies, from the chemotherapeutic agent. In certain embodiments contemplated herein, the frequency of administration of the chemotherapeutic agent may be greater than that of the anti-CCR9 antibody molecule.

[0092]

[94] In some embodiments, the dosing regimen in humans for the anti-CCR9 antibody molecule and the chemotherapeutic agent, e.g., vincristine, may be as follows:

[95] According to clinical guidelines, anti-CCR9 antibody immunotherapy is usually administered intravenously, by mouth, or by injection at a fixed dose of between 200 and 400 mg / week for four doses. For vincristine, the recommended dosing schedule is up to 2 mg / m2 per week. 2 The patient was given four intravenous infusions (Gilbar, Chambers et al. 2015; Hoelzer, Bassan et al. 2016; Majem, Juan et al. 2019; Mikhael, Ismaila et al. 2019; (CADTH) 2019; Compendium-EMC 2020; England-NHS 2020; (ASHP) updated 2020).

[0093]

[96] The chemotherapeutic agent or agents for combination therapy with the anti-CCR9 antibody molecule may be selected from the group consisting of docetaxel, paclitaxel, nanoparticle albumin-bound paclitaxel, vinblastine, or vincristine. In some embodiments, the chemotherapeutic agent comprises a vinca alkaloid, such as vincristine, vinblastine, vindesine, vinorelbine, vincaminol, vineridin, and vinburnine.

[0094]

[97] In some embodiments, the compositions for the uses and methods of the invention further comprise a corticosteroid, such as dexamethasone. In some embodiments, a vinca alkaloid (e.g., vincristine) and a corticosteroid (e.g., dexamethasone) are both administered with the anti-CCR9 antibody molecule.

[0095]

[98] Pharmaceutical Compositions and Treatments

[99] The anti-CCR9 antibody molecules of the present invention and pharma- ceutical acceptable compositions thereof may be administered to a patient by any number of different routes, including intravenous, cutaneous or subcutaneous, nasal, intramuscular, transepithelial, intraperitoneal, and oral administration.

[0096]

[0100] The composition of the present invention may be formulated as a pharmaceutical composition, which may be in the form of a solid or liquid composition. Such compositions generally contain some kind of carrier, for example, a solid carrier or a liquid carrier such as water, petroleum, animal or vegetable oil, mineral oil or synthetic oil. Physiological saline or glycols such as ethylene glycol, propylene glycol or polyethylene glycol may be included. Such compositions and preparations generally contain at least 0.1 wt% of the anti-CCR9 antibody molecule of the present invention.

[0097]

[0101] In addition to one or more anti-CCR9 antibody molecules of the present invention, optionally in combination with another active ingredient, the composition can contain one or more pharma- ceutically acceptable excipients, carriers, buffers, stabilizers, isotonicity agents, preservatives or antioxidants, or other substances known to those skilled in the art. Such substances must be non-toxic and must not interfere with the effectiveness of the active ingredient. The exact nature of the carrier or other substances may depend on the route of administration.

[0098]

[0102] Preferably, the pharmaceutical composition is administered to an individual in a prophylactically effective amount or a therapeutically effective amount (although in some cases prophylaxis is considered to be treatment), which is sufficient to show benefit to the individual. Typically, this causes a therapeutically useful activity that benefits the individual. The actual amount of the compound administered and the rate and time course of administration depend on the nature and severity of the condition being treated. Treatment indication, such as determining dosage, is within the responsibility of general practitioners and other physicians, and typically takes into account the disorder being treated, the condition of the individual patient, the site of delivery, the method of administration, and other factors known to physicians. Examples of the techniques and procedures described above can be found in Handbook of Pharmaceutical Additives, 2nd Edition (edited by M. Ash and I. Ash), 2001 (Synapse Information Resources, Inc., Endicott, New York, USA); Remington's Pharmaceutical Sciences, 20th Edition, 2000, published by Lippincott, Williams & Wilkins; and Handbook of Pharmaceutical Excipients, 2nd Edition, 1994. By way of example, the compositions are preferably administered to a patient at a dosage of between about 0.01 and 100 mg of active compound per kg of body weight, more preferably about 0.5 to 10 mg / kg of body weight.

[0099]

[0103] The following are offered by way of example and not by way of limitation to the scope of the claims. EXAMPLES

[0100]

[0104] Working Example

[0105] method

[0106] In vivo efficacy of anti-CCR9 antibody SRB1 in NSG mice bearing MOLT4-GFP xenografts - Combination of SRB1 with chemotherapeutic agents

[0107] 1x10 in 100 μl of PBS into the tail vein of an NSG mouse 6 MOLT4-GFP cells were injected intravenously (day 0) and 400 μg (16 mg / kg) of each monoclonal antibody was administered four times (SRB1 or isotype control, at weekly intervals, on days 4, 10, 17, and 24). Animals were sacrificed on day 28. The total number of tumor cells in the spleen and bone marrow was determined by fluorescence microscopy after analysis by flow cytometry, which detects tumor cells by GFP expression. The presence of tumor cell accumulation in the spleens of these mice was also analyzed.

[0101]

[0108] In addition, the effect of SRB1 treatment on the survival of animals (10 animals / group) bearing MOLT4-GFP xenografts was determined by using SRB1 (4 mg / kg) administered four times (once a week) or an isotype control. In addition, other chemotherapeutic agents were used: Vincristine: 0.6 mg / kg / mouse and Dexamethasone: 50 μg / administration / mouse, according to this procedure: MOLT4-GFP cells were injected intravenously on day 0 (1 × 10 6 cells / mouse).

[0102] · Treat with intravenous vincristine (0.6mg / kg) on ​​day 5.

[0103] Treatment with dexamethasone (50 μg / mouse / dose) on days 5–9 (including both). This treatment was given alone or in combination with vincristine.

[0104] · Intraperitoneal treatment with SRB1 (100 μg / mouse / dose) on days 11, 18, 25, and 32.

[0105] · Intraperitoneal treatment with isotype control (100 μg / mouse / dose) on days 11, 18, 25, and 32.

[0106]

[0109] The results were analyzed by the Kaplan-Meier test (further details provided herein).

[0107]

[0110] Example 1 - In vivo survival of an orthotopic mouse model following treatment with specific antibody-drug combinations

[0111] As shown in Figures 1-3 and in the table below, increased animal survival was observed when using 4 mg / kg SRB1 versus control (IC50 70 days versus 48 days). The combination of SRB1 with the vinca alkaloid vincristine extends mouse survival to a median survival of 306.2 days ("vincristine+SRB1"). Additionally, the triple combination of SRB1+vincristine+dexamethasone also showed increased survival, with a median survival of 232.1 days.

[0112]

[0108] [Table 1]

[0109]

[0113] It is clear that the combination of SRB1 and vincristine significantly extends median survival (66.2 and 67.9 days compared to 306.2 days, respectively) above the merely additive effect on survival observed with SRB1 or vincristine administered alone. Thus, the results demonstrate the synergistic combination of an anti-CCR9 antibody (SRB1) and a vinca alkaloid therapeutic (vincristine) with or without additional corticosteroid therapy (dexamethasone) in the treatment of cancer, specifically acute lymphoblastic leukemia (ALL).

[0110]

[0114] All references cited in this specification are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication or patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety.

[0111]

[0115] The features disclosed in the foregoing description, or in the following claims, or the accompanying drawings, expressed in a particular form or in terms of means for performing a disclosed function or a method or process for obtaining a disclosed result, can, where appropriate, be utilized separately or in any combination of such features to realize the invention in various of its forms.

[0112]

[0116] While the present invention has been described in conjunction with the exemplary embodiments set forth above, many equivalent modifications and variations will be apparent to those skilled in the art upon reading this disclosure. Accordingly, the exemplary embodiments of the invention set forth above are considered to be illustrative and not limiting. Various changes can be made to the described embodiments without departing from the spirit and scope of the invention.

[0113] References

[0114] [Table 2-1]

[0115] [Table 2-2]

[0116] [Table 2-3]

[0117] [Table 2-4]

Claims

1. 1. A pharmaceutical composition for use in a method for treating cancer in a mammalian subject, said pharmaceutical composition comprising an anti-CCR9 antibody molecule, said pharmaceutical composition being administered simultaneously, sequentially or separately with a chemotherapeutic agent selected from vincristine, docetaxel, paclitaxel, nanoparticle albumin-bound paclitaxel, and vinblastine; the anti-CCR9 antibody and the chemotherapeutic agent are not conjugated together; The pharmaceutical composition.

2. A pharmaceutical composition for use as described in claim 1, wherein the method further comprises administering dexamethasone simultaneously, sequentially, or separately.

3. The pharmaceutical composition for use according to claim 1 , wherein the anti-CCR9 antibody molecule comprises a monoclonal antibody or an antigen-binding fragment thereof that specifically binds to CCR9.

4. The anti-CCR9 monoclonal antibody or antigen-binding fragment thereof is an Fv, Fab, F(ab') ２ 4. The pharmaceutical composition for use according to claim 3, wherein the antibody is selected from the group consisting of: Fab', scFv, scFv-Fc, minibody, nanobody, and diabody.

5. the monoclonal antibody or antigen-binding fragment thereof Heavy chain complementarity determining region 1 (CDR-H1) comprising the amino acid sequence: NFWMN (SEQ ID NO: 1) or KFWMN (SEQ ID NO: 2); Heavy chain complementarity determining region 2 (CDR-H2) comprising the amino acid sequence: EIRLKSNNYATHYAESVKG (SEQ ID NO: 3); Heavy chain complementarity determining region 3 (CDR-H3) containing the amino acid sequence: DGWFAY (SEQ ID NO: 4); a light chain complementarity-determining region 1 (CDR-L1) comprising the amino acid sequence: RSSQSLLHSNGNTYVQ (SEQ ID NO: 5) or RSSQSLVHSNGNTYLN (SEQ ID NO: 6); A light chain complementarity-determining region 2 (CDR-L2) comprising the amino acid sequence: KVSNRFP (SEQ ID NO:7) or KVSNRFS (SEQ ID NO:8); and Light chain complementarity determining region 3 (CDR-L3) containing the amino acid sequence: AQSTHVPRT (SEQ ID NO: 9) or SQSTHFPRT (SEQ ID NO: 10) 4. A pharmaceutical composition for use according to claim 3, comprising:

6. the monoclonal antibody or antigen-binding fragment thereof Amino acid sequence: 【Chemistry 1】 or 【Chemistry 2】 or 【Transformation 3】 and a heavy chain variable region comprising Amino acid sequence: 【Chemistry 4】 or 【Transformation 5】 or 【Transformation 6】 a light chain variable region comprising A pharmaceutical composition for use according to claim 5.

7. the antibody molecule has a binding affinity dissociation constant K of 500 nM, 250 nM, 100 nM, 10 nM, or less for human CCR9 as determined by surface plasmon resonance (SPR). Ｄ 2. The pharmaceutical composition for use according to claim 1, wherein

8. 2. The pharmaceutical composition for use according to claim 1, wherein the antibody molecule exhibits CCR9-specific binding in the presence of human CCL25 at a concentration of 10 μg / mL as measured by flow cytometry.

9. The antibody molecule is CCR9 ＋ 2. The pharmaceutical composition for use according to claim 1, which exhibits lymphocyte depletion.

10. 2. The pharmaceutical composition for use according to claim 1, wherein the chemotherapeutic agent comprises vincristine.

11. 2. The pharmaceutical composition for use according to claim 1, wherein the chemotherapeutic agent comprises vincristine and the method further comprises the simultaneous, sequential or separate administration of dexamethasone.

12. The pharmaceutical composition for use according to claim 1, wherein the treatment method is a treatment method for a hematological tumor.

13. 2. The pharmaceutical composition for use according to claim 1, wherein the method of treatment is a method of treatment for T-cell acute lymphoblastic leukemia (T-ALL), T-cell lymphoma, or acute myeloid leukemia (AML).

14. 1. A chemotherapeutic agent for use in a method for treating cancer in a mammalian subject, wherein said chemotherapeutic agent is administered simultaneously, sequentially, or separately with an anti-CCR9 antibody molecule, said chemotherapeutic agent being selected from the group consisting of vincristine, docetaxel, paclitaxel, nanoparticle albumin-bound paclitaxel, and vinblastine; said chemotherapeutic agent and said anti-CCR9 antibody molecule are not conjugated together; Chemotherapeutic agents.

15. A chemotherapeutic agent for use according to claim 14, wherein the method further comprises administering dexamethasone simultaneously, sequentially or separately.

16. The chemotherapeutic agent for use according to claim 14, wherein said anti-CCR9 antibody molecule is as defined in any one of claims 1 to 9.

17. A chemotherapeutic agent for use according to claim 14 or claim 15, wherein the chemotherapeutic agent is as defined in claim 10 or claim 11.

18. A chemotherapeutic agent for use according to claim 14 or 15, wherein the cancer is as defined in claim 12 or claim 13.

19. 14. Use of an anti-CCR9 antibody molecule in the preparation of a pharmaceutical composition for use in a method for treating cancer in a mammalian subject, said pharmaceutical composition being as defined in any one of claims 1 to 13.

20. Use of a chemotherapeutic agent in the preparation of a pharmaceutical composition for use in a method for treating cancer in a mammalian subject, wherein said chemotherapeutic agent is as defined in claim 14 or 15. ,use.

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