Method and pharmaceutical composition for the treatment of acute myeloid leukemia by eradicating leukemia stem cells

Abstract

Methods and pharmaceutical compositions for the treatment of acute myeloid leukemia by eradicating leukemic stem cells After intensive chemotherapy, the emergence of drug resistance and / or cells with stem cell characteristics may explain the frequent relapse and poor outcome of acute myeloid leukemia (AML) patients. Herein, we demonstrate that the adrenomedullin receptor CALCRL is expressed in AML patients compared with normal cells, and preferentially in immature CD34 cells. + CD38 - We first demonstrated that ADM-CALCRL is overexpressed in the AML stem cell compartment. Next, they demonstrated its role in maintaining leukemia stem cell function in vivo. Furthermore, CALCRL depletion strongly affected leukemia growth in a xenograft model and sensitized LSCs to the chemotherapy drug cytarabine in vivo. Thus, we demonstrated that the ADM-CALCRL axis drives cell cycle, DNA integrity, and the high OxPHOS state of chemotherapy-resistant AML stem cells in an E2F1- and BCL2-dependent manner. Furthermore, CALCRL depletion sensitized cells to cytarabine, and its expression predicted chemotherapy response in mice in vivo. Furthermore, using a combination of limiting dilution assays and single-cell RNA-seq analysis of primary AML samples at diagnosis and relapse, as well as before and after transplantation into NSG mice, we uncovered the existence of a chemotherapy-resistant leukemia stem cell subpopulation that retained a CALCRL-driven gene signature. Finally, we strongly demonstrated that chemotherapy-resistant LSCs depend on CALCRL. All these data highlight the essential role of CALCRL in stem cell survival, proliferation and metabolism and identify this receptor as a novel marker of chemotherapy-resistant leukemic stem cell populations and as a promising therapeutic target to specifically eradicate them and overcome AML relapse.

Description

[Technical Field]

[0001] Field of Invention The present invention relates to a method and a pharmaceutical composition for treating acute myeloid leukemia (AML) by eradicating leukemia stem cells.

[0002] Background of the invention: Acute myeloid leukemia (AML) arises from self-replicating leukemia stem cells (LSCs), which can be regrown in immunosuppressed mice through assays and xenotransplantation (e.g., PDX; Bonnet and Dick, 1997). Despite the fact that these cells constitute only a small fraction of all leukemia cells, the fact that genetic signatures related to the phenotype or function of stem cells are associated with a poor prognosis in AML strongly supports the hypothesis that their abundance has a real clinical impact (Gentles et al., 2010; Vergez et al., 2011; Eppert et al., 2011; Ng et al., 2016). This clinical relevance is supported by studies showing that relapsed patients exhibit a continuous enrichment of LSC frequencies (Ho et al., 2016) and increased LSC-related genetic signatures (Hackl et al., 2015). While LSCs were initially shown to be immune to chemotherapy (Jordan et al., 2006; Ishikawa et al., 2007), recent studies have shown that cytarabine (AraC) may have a strong impact on the PDX model and the patient LSC pool (Farge et al., 2017; Boyd et al., 2018). These results suggest that there are two distinct LSC populations, some of which are chemotherapy-sensitive (S-LSCs) and thus eradicated by conventional treatment, while others remain chemotherapy-resistant (R-LSCs), regenerating AML and initiating patient relapses. Therefore, better characterizing these R-LSCs phenotypically and functionally is crucial to enabling the development of novel therapeutic strategies that specifically target R-LSCs.

[0003] LSC compartments human AML cells CD34 + CD38 - It was initially proposed that LSCs were limited to subpopulations (Bonnet and Dick, 1997; Ishikawa et al., 2007), but subsequent studies have shown that LSCs are also phenotypic heterogeneous. For example, CD38 from NPM1c mutant specimens. + AML cells or CD34 -The cells can also serially reproduce the disease when assayed in NSG-deficient mice (Taussig et al., 2008; Taussig et al., 2010; Sarry et al., 2011; Quek et al., 2016). This highlights the need for more functional studies to adequately characterize LSCs (Eppert et al., 2011). Eradicating R-LSCs without killing normal hematopoietic stem cells (HSCs) depends on identifying functionally relevant markers that are overexpressed in the AML compartment. In recent years, numerous research efforts have been made to distinguish LSCs from HSCs, enabling the identification of several cell surface markers such as CD47, CD123, CD44, TIM-3, CD25, CD32, and CD93 (Majeti et al., 2009; Jin et al., 2009; Kikushige et al., 2010; Saito et al., 2010; Iwasaki et al., 2015). In addition to these new membrane markers, LSCs also specifically increase BCL2-dependent oxidative phosphorylation (OxPHOS), suggesting a potential Achilles' heel (vulnerability) that could be exploited through treatment with BCL2 inhibitors such as ABT-199 (Lagadinou et al., 2013; Konopleva et al., 2016). This is consistent with several studies, including recent research showing that mitochondrial OxPHOS status contributes to drug resistance in leukemia (Farge et al., 2017; Bosc et al. 2017; Kunst et al. 2017). Taken together, all these results suggest certain characteristics of R-LSCs that leave the door open to targeted therapeutic approaches aimed at eradicating these cells.

[0004] Summary of the invention: The present invention relates to a method and pharmaceutical composition for treating acute myeloid leukemia (AML) by eradicating leukemia stem cells. In particular, the present invention is defined by the claims.

[0005] Detailed description of the invention: Following intensive chemotherapy, the emergence and persistence of AML cells with drug resistance and / or stem cell characteristics may explain the frequent relapses and poor outcomes in patients with acute myeloid leukemia (AML). In this specification, we demonstrate that the adrenomedullin receptor (CALCRL) is more readily available in AML patients compared to normal cells, and preferentially in immature CD34 cells. + CD38 - They first demonstrated that it is overexpressed in the compartment. Next, they demonstrated its role in maintaining leukemia stem cell function in vivo. Furthermore, CALCRL depletion strongly affected leukemia proliferation in xenograft models and sensitized to the chemotherapeutic agent cytarabine in vivo. Thus, the inventors showed that the ADM-CALCRL axis drives the cell cycle, DNA integrity, and high OxPHOS state of chemotherapy-resistant AML stem cells in both E2F1 and BCL2-dependent ways. In addition, CALCRL depletion sensitized cells to cytarabine, and its expression predicted the response to chemotherapy in vivo in mice. Furthermore, using a combination of limiting dilution assays, single-cell RNA-seq analysis of primary AML samples at diagnosis and relapse, as well as before and after transplantation into NSG mice, the inventors revealed the existence of a subpopulation of chemotherapy-resistant leukemia stem cells that possess a CALCRL-driven genetic signature. Finally, the inventors strongly demonstrated that chemotherapy-resistant LSCs are dependent on CALCRL. All of these data highlight the critical role of CALCRL in stem cell survival, proliferation, and metabolism, identifying this receptor as a novel biomarker for the chemotherapy-resistant leukemia stem cell population and a promising therapeutic target for specifically eradicating them and overcoming AML relapses.

[0006] Therefore, the first object of the present invention relates to a method for depleting leukemia stem cells in a subject suffering from AML, comprising administering a therapeutically effective amount of antibody that specifically binds to AMLCL to the subject, thereby depleting the leukemia stem cells.

[0007] A further object of the present invention relates to a method for depleting leukemia stem cells in a subject suffering from AML, comprising administering a therapeutically effective dose of a CALCRL activity or expression inhibitor to the subject, thereby depleting the leukemia stem cells.

[0008] As used herein, the terms “acute myeloid leukemia” or “acute myeloid leukemia” ("AML") refer to cancer of the myeloid lineage of blood cells characterized by the rapid proliferation of abnormal white blood cells accumulating in the bone marrow and interference with the production of normal blood cells.

[0009] As used herein, the term “leukemia stem cells” has its general meaning in the art and refers to pluripotent myeloid stem cells characterized by genetic transformation resulting in disordered cell division. Leukemia stem cells (LSCs) are distinguished from all other AML cells by their self-renewal ability, that is, their ability to produce daughter cells similar to their mother cells. Extensive self-renewal ability is an inherent characteristic of LSCs and has been shown to be essential for the development of leukemia.

[0010] Therefore, the method of the present invention is particularly suitable for the treatment of AML.

[0011] As used herein, the terms “treatment” or “to treat” include both preventive or prophylactic treatment, as well as curative or disease-modifying treatment, including treatment of patients at risk of developing the disease or suspected of having the disease, and of persons diagnosed with the disease or a disease or condition, and including the suppression of clinical relapses. Treatment may be administered to patients with a medical disability or those likely to eventually acquire a disability in order to prevent, cure, delay the onset of, reduce the severity of, or improve one or more symptoms of a disability or recurrent disability, or to extend the patient's survival beyond what would be expected without such treatment. “Treatment regimen” means a pattern of treatment for a disease, e.g., a pattern of medication used during treatment. A treatment regimen may include an induction regimen and a maintenance regimen. The phrase “induction regimen” or “induction period” refers to a treatment regimen (or part of a treatment regimen) used for the initial treatment of a disease. A common goal of an induction regimen is to provide the patient with a high level of medication during the initial period of the treatment regimen. An induction regimen may employ a “loading regimen” (partially or entirely), which may include administering a larger dose of medication than the physician would use during the maintenance regimen, administering medication more frequently than the physician would administer it, or both. The phrase “maintenance regimen” or “maintenance period” refers to a treatment regimen (or part of a treatment regimen) used to maintain a patient’s condition during treatment for a disease, for example, to keep the patient in remission for an extended period (months or years). Maintenance therapy may involve continuous treatment (e.g., administering medication at regular intervals, e.g., weekly, monthly, yearly, etc.) or intermittent treatment (e.g., interrupted treatment, intermittent treatment, treatment at relapse, or treatment at achievement of certain predetermined criteria [e.g., pain, symptoms of the disease, etc.]).

[0012] The method of the present invention is particularly suitable for preventing relapse in patients with AML who have been treated with chemotherapy.

[0013] As used herein, the term “recurrence” refers to the recurrence of cancer after a period of improvement during which cancer could not be detected. Therefore, the method of the present invention is particularly useful in preventing recurrence after treatment with chemotherapy is presumed to have been successful.

[0014] Therefore, a further object of the present invention relates to a method for treating chemotherapy-resistant acute myeloid leukemia (AML) in a patient in need thereof, comprising administering to the patient an antibody that specifically binds to a therapeutically effective amount of CALCRL.

[0015] Therefore, a further object of the present invention relates to a method for treating chemotherapy-resistant acute myeloid leukemia (AML) in a patient in need thereof, comprising administering to the patient an inhibitor of CALCRL activity or expression at a therapeutically effective amount.

[0016] As used herein, the term "chemotherapy-resistant acute myeloid leukemia" refers to the clinical situation of a patient suffering from acute myeloid leukemia, and at an acceptable dose to the patient, the growth of cancer cells cannot be prevented or inhibited by chemotherapeutic agents or combinations of chemotherapeutic agents normally used for the treatment of AML. The leukemia may be inherently resistant prior to chemotherapy. Or, resistance may be acquired during the treatment of leukemia that is initially sensitive to chemotherapy.

[0017] As used herein, the term “chemotherapeutic agent” refers to any chemical agent that has therapeutic utility in the treatment of cancer. As used herein, chemotherapeutic agents encompass both chemical and biological agents. These agents function by inhibiting cellular activity on which cancer cells depend to survive. Categories of chemotherapeutic agents include alkylating agents / alkaloids, antimetabolites, hormones or hormone analogs, and other antitumor agents. Most, if not all, of these agents are directly toxic to cancer cells and do not require immunostimulation. Appropriate chemotherapeutic agents are listed, for example, in Slapak and Kufe, Principles of Cancer Therapy, Chapter 86 in Harrison's Principles of Internal Medicine, 14th edition; Perry et al., Chemotherapeutic, Chapter 17 in Abeloff, Clinical Oncology 2nd ed., 2000 Chrchill Livingstone, Inc.; Baltzer L. and Berkery R. (eds): Oncology Pocket Guide to Chemotherapeutic, 2nd ed. St. Louis, Mosby-Year Book, 1995; and Fischer DS, Knobf MF, Durivage HJ. (eds): The Cancer Chemotherapeutic Handbook, 4th ed. St. Louis, Mosby-Year Handbook.

[0018] In some embodiments, the chemotherapeutic agent is cytarabine (cytarabine, Ara-C, Cytosar-U), quizartinib (AC220), sorafenib (BAY 43-906), lestaurtinib (CEP-701), midostaurin (PKC412), carboplatin, carmustine, chlorambucil, dacarbazine, ifosfamide, lomustine, mechlorethamine, procarbazine, pentostatin, (2’deoxycoformycin), etoposide, teniposide, topotecan, vinblastine, vincristine, paclitaxel, dexamethasone, methylprednisolone, prednisone, all-trans retinoic acid, arsenic trioxide, interferon α, rituximab (Rituxan®), gemtuzumab ozogamicin, imatinib mesylate, (Cytosar-U), melphalan, busulfan (Myleran®), thiotepa, bleomycin, platinum (cisplatin), cyclophosphamide, Cytoxan®), daunorubicin, doxorubicin, idarubicin, mitoxantrone, 5-azacitidine, cladribine, fludarabine, hydroxyurea, 6-mercaptopurine, methotrexate, 6-thioguanine, or any combination thereof. In some embodiments, the leukemia is resistant to daunorubicin, or a combination of idarubicin and cytarabine (AraC).

[0019] In some embodiments, the chemotherapeutic agent is a BCL2 inhibitor. In some embodiments, the Bcl-2 inhibitor comprises 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 (venetoclax, or ABT-199, or GDC-0199, also known as and optionally referred to herein).) or a pharmaceutically acceptable salt thereof.

[0020] In some embodiments, the chemotherapeutic agent is an FLT3 inhibitor. Examples of FLT3 inhibitors include N-(2-diethylaminoethyl)-5-[(Z)-(5-fluoro-2-oxo-1H-indole-3-ylidene)methyl]-2,4-dimethyl-1H-pyrrole-3-carboxamide, also known as sunitinib, SU11248, and marketed as SUTENT (sunitinib malate); and 4-[4-[[4-chloro-3-(trifluoromethyl)phenyl]carbamoylamino]phenoxy]-N-methylpyridine-2-carboxamide, sorafenib (BAY Also known as 43-9006 and marketed as NEXAVAR (sorafenib); (9S,10R,llR,13R)-2,3,10,11,12,13-hexahydro-10-methoxy-9-methyl-11-(methylamino)-9,13-epoxy-lH,9H-diindro[l,2,3-gh:3',2',l'-lm]pyrrolo[3,4-j][l,7]benzodianzonin-l-one, also known as midostaurin or PKC412; (5S,6S,8R)-6-hydroxy-6-(hydroxymethyl)-5-methyl-7,8,14,15-tetrahydro-5H-16-oxa-4b,8a,14-triaza-5,8-methanodibenzo[ This includes [b,h]cycloocta[jkl]cyclopenta[e]-as-indacen-13(6H)-one, also known as restartinib or CEP-701; l-(5-(tert-butyl)isoxazole-3-yl)-3-(4-(7-(2-morpholinoethoxy)benzo[d]imidazo[2,lb]thiazole-2-yl)phenyl)urea, also known as quizartinib or AC220; l-(2-{5-[(3-methyloxetan-3-yl)methoxy]-lH-benzimidazole-l-yl}quinoline-8-yl)piperidine-4-amine, also known as crenolanib or CP-868,596-26; see, for example, Wander SA, TherAdv Hematol. 5: 65-77 (2014).Other FLT3 inhibitors include pexidartinib (PLX-3397), Tap et al, N Engl J Med, 373:428-437 (2015); gilteritinib (ASP2215), Smith et al., Blood: 126 (23) (2015); FLX-925, also known as AMG-925, Li et al. Mol. Cancer Ther. 14: 375-83 (2015); and G-749, Lee et al, Blood. 123: 2209-2219 (2014).

[0021] In some embodiments, the chemotherapeutic agent is an IDH (isocitrate dehydrogenase) inhibitor. In some embodiments, the IDH inhibitor is a member of the oxazolidinone (3-pyrimidinyl-4-yl-oxazolidine-2-one) family, a specific inhibitor of the novel morphological activity of the IDH1 mutant, and has the chemical name (S)-4-isopropyl-3-(2-((S)-1-(4-phenoxyphenyl)ethyl)amino)pyrimidin-4-yl)oxazolidine-2-one.

[0022] As used herein, the term "CALCRL" has its general meaning in the art and refers to the calcitonin receptor (gene ID: 10203), which is a receptor-like structure. CALCRL is also called CRLR or CGRPR. CALCRL is linked to one of three single transmembrane domain receptor-modified proteins (RAMPs) essential for its functional activity. The association of CALCRL with different RAMP proteins generates different receptors: i) association with RAMP1: generation of the CGRP receptor; ii) association with RAMP2: generation of the adrenomedullin (AM) receptor called AM1; iii) association with RAMP3: generation of the receptor dual CGRP / AM called AM2. These receptors are linked to the G protein G, which activates adenylyl cyclase, and activation generates intracellular cyclic adenosine monophosphate (cAMP). An exemplary amino acid sequence of CALCRL is represented by Sequence ID No. 1.

[0023] [Table 1]

[0024] As used herein, the term “depleted” with respect to leukemia stem cells refers to a measurable decrease in the number of leukemia stem cells in a subject. The decrease may be at least about 10%, for example, at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or more. In some embodiments, the term refers to a decrease in the number of leukemia stem cells in a subject or sample to a level below a detectable limit.

[0025] According to the present invention, the antibody specifically mediates the depletion of a subset population of leukemia stem cells, but does not mediate the depletion of a population of hematopoietic cells.

[0026] As used herein, the term “antibody” has the general meaning in the art and includes monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies formed from at least two intact antibodies), human antibodies, humanized antibodies, camelized antibodies, chimeric antibodies, single-chain Fvs(scFv), single-chain antibodies, single-domain antibodies, domain antibodies, Fab fragments, F(ab')2 fragments, antibody fragments exhibiting desired biological activity, disulfide-linked Fvs(sdFv), and anti-idiotype (anti-Id) antibodies (e.g., anti-Id antibodies against the antibodies of the present invention), intrabodies, and any of the epitope-binding fragments described above. In particular, antibodies include immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, i.e., molecules containing antigen-binding sites. Immunoglobulin molecules can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), a class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or a subclass.

[0027] According to the present invention, the antibody binds to at least one extracellular domain of CALCRL.

[0028] In this specification, the term "bound" indicates that the antibody has affinity for a surface molecule. Furthermore, the term "affinity" refers to the strength of the antibody's binding to an epitope. Antibody affinity is given by the dissociation constant Kd and is defined as [Ab] × [Ag] / [Ab-Ag], where [Ab-Ag] is the molar concentration of the antibody-antigen complex, [Ab] is the molar concentration of the unbound antibody, and [Ag] is the molar concentration of the unbound antigen. The affinity constant Ka is defined as 1 / Kd. Preferred methods for determining the affinity of mAbs include Harlow, et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1988), Coligan et al., eds., Current Protocols in Immunology, Greene Publishing Assoc. and Wiley Interscience, NY, (1992, 1993), and Muller, Meth. Enzymol. 92:589-601 (1983), which are fully incorporated herein by reference. One of the preferred standard methods well known in the art for determining the affinity of mAbs is the use of a Biacore instrument.

[0029] In some embodiments, the antibodies of the present invention are monoclonal antibodies. As used herein, the term “monoclonal antibody” means an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies constituting the population are identical except for any spontaneously occurring variations that may be present in trace amounts. Monoclonal antibodies act specifically on a single antigenic site. Furthermore, while conventional polyclonal antibodies contain antibodies against different determinants (epitopes), monoclonal antibodies are antibodies against a single determinant on an antigen. In addition to their specificity, monoclonal antibodies have the advantage of being synthesized by hybridoma cells that are not contaminated with other immunoglobulin-producing cells. For example, monoclonal antibodies can be produced by cells that have been stably or transiently transfected with the heavy and light chain genes encoding the monoclonal antibody.

[0030] Monoclonal antibodies can be produced using the method of Kohler and Milstein (Nature, 256:495, 1975). To prepare monoclonal antibodies useful for the present invention, mice or other suitable host animals are immunized with a suitable antigen form (i.e., the polypeptide of the present invention) at appropriate intervals (e.g., twice a week, once a week, twice a month, or once a month). The animals may receive a final "boost" of the antigen within one week of sacrificing. It is often desirable to use an immunological adjuvant during immunization. Suitable immunological adjuvants include Freund's complete adjuvant, Freund's incomplete adjuvant, alum, Ribi adjuvant, Hunter's Titermax, saponin adjuvants such as QS21 and Quil A, or CpG-containing immunostimulatory oligonucleotides. Other suitable adjuvants are well known in the field. Animals may be immunized via routes such as subcutaneous, intraperitoneal, intramuscular, intravenous, or intranasal. Some animals may be immunized via multiple routes with multiple forms of antigens. Briefly, the recombinant polypeptides of the present invention may be provided by expression using recombinant cell lines. Recombinant forms of polypeptides may be provided using any of the previously described methods. Following the immunization regimen, lymphocytes are isolated from the spleen, lymph nodes, or other organs of an animal and fused with a suitable myeloma cell line using a drug such as polyethylene glycol to form hydridomas. After fusion, the cells are placed in a medium that allows the growth of the hybridomas using a standard method, but does not allow the growth of the fusion partner. After culturing the hybridomas, the cell supernatant is analyzed to confirm the presence of antibodies with the desired specificity, i.e., antibodies that selectively bind to the antigen. Analytical techniques include ELISA, flow cytometry, immunoprecipitation, and Western blotting. Other screening techniques are well known in the field. Preferred techniques include non-denaturing ELISA, flow cytometry, and immunoprecipitation, which confirm the binding of antibodies to conformationally intact, natively folded antigens.

[0031] In some embodiments, the monoclonal antibody of the present invention is a chimeric antibody, particularly a chimeric mouse / human antibody. In this specification, the term "chimeric antibody" refers to an antibody comprising the VH and VL domains of a non-human antibody and the CH and CL domains of a human antibody. In some embodiments, the human chimeric antibody of the present invention can be produced by obtaining nucleic acid sequences encoding the VL and VH domains as described above, inserting them into an expression vector for animal cells containing genes encoding human antibody CH and human antibody CL to construct a human chimeric antibody expression vector, and then introducing the expression vector into animal cells to express the coding sequences. The CH domain of the human chimeric antibody can be any region belonging to human immunoglobulin, but IgG class domains are preferred, and any subclass belonging to the IgG class, such as IgG1, IgG2, IgG3, or IgG4, can also be used. Furthermore, the CL of the human chimeric antibody can be any region belonging to Ig, and kappa class or lambda class domains can be used. Conventional methods for producing chimeric antibodies using recombinant DNA or gene transfer techniques are well known in this industry (see Morrison SL. et al. (1984) and patent documents US5,202,238; and US5,204,244).

[0032] In some embodiments, the monoclonal antibody of the present invention is a humanized antibody. In particular, in the humanized antibody, the variable domain consists of a human acceptor framework region, a human constant domain if optionally present, and a non-human donor CDR such as a mouse CDR. According to the present invention, the term "humanized antibody" refers to an antibody that has the variable region framework and constant region of a human antibody but retains the CDR of a previous non-human antibody. The humanized antibody of the present invention can be produced by obtaining a nucleic acid sequence encoding the CDR domain as described above, inserting it into an expression vector for animal cells having (i) a gene encoding the same heavy chain constant region as the human antibody, and (ii) a gene encoding the same light chain constant region as the human antibody to construct a humanized antibody expression vector, and then introducing the expression vector into animal cells to express the gene. The humanized antibody expression vector may be of the type in which the gene encoding the antibody heavy chain and the gene encoding the antibody light chain are on separate vectors, or of the type in which both genes are on the same vector (tandem type). From the viewpoint of ease of construction, ease of introduction into animal cells, and balance of antibody H and L chain expression levels in animal cells, tandem humanized antibody expression vectors are preferred. Examples of tandem humanized antibody expression vectors include pKANTEX93 (WO 97 / 10354) and pEE18. Methods for producing humanized antibodies based on conventional recombinant DNA and gene transfer technologies are well known in the industry (see, for example, Riechmann L. et al. 1988; Neuberger MS. et al. 1985). Antibodies can be humanized using various techniques known in the art, such as CDR grafting (EP 239,400; PCT Publication WO91 / 09967; U.S. Patents 5,225,539; 5,225,539; 5,530,101; and 5,585,089).Various techniques known in the art are used, including veneering or resurfacing (EP 592,106; EP 519,596; Padlan EA (1991); Studnicka GM et al. (1994); Roguska MA. et al. (1994)); and chain shuffling (U.S. Patent No. 5,565,332). Common recombinant DNA techniques for preparing such antibodies are also known (see European Patent Application EP 125023 and International Patent Application WO 96 / 02576).

[0033] In some embodiments, the antibodies of the present invention are human antibodies. As used herein, the term “human antibody” is intended to include antibodies having variable and constant regions derived from human immunoglobulin sequences. The human antibodies of the present invention may include amino acid residues not encoded by human immunoglobulin sequences (e.g., mutations introduced by in vitro random mutation or site-directed mutagenesis, or by in vivo somatic mutation). However, as used herein, the term “human antibody” is not intended to include antibodies in which CDR sequences derived from germ cells of another mammalian species, such as mouse, are grafted onto a human framework sequence. Human antibodies can be manufactured using a variety of techniques known in the art. Human antibodies are generally described in van Dijk and van de Winkel, cur. Opin. Pharmacol. 5; 368-74 (2001) and lonberg, cur. Opin.Immunol. 20; 450-459 (2008). Human antibodies can be prepared by administering immunogens to transgenic animals modified to produce intact human antibodies or intact antibodies containing human variable regions in response to antigen challenge. Such animals typically contain all or part of the human immunoglobulin loci, or have them randomly integrated into the animal's chromosomes, either extrachromosomally or in a random manner. In such transgenic mice, endogenous immunoglobulin loci are generally inactivated. For methods of obtaining human antibodies from transgenic animals, see Lonberg, Nat. Biotech. 23;1117-1125 (2005). See also, for example, U.S. Patent Nos. 6,075,181 and 6,150,584 describing the XENOMOUSE™ technology, U.S. Patent No. 5,770,429 describing the HUMAB® technology, U.S. Patent No. 7,041,870 describing the KM MOUSE® technology, and U.S. Patent Application Publication No. US 2007 / 0061900 describing the VELOCIMOUSE® technology.Human variable regions from intact antibodies produced by such animals can be further modified, for example, by combining them with different human constant regions. Human antibodies can also be produced by hybridoma-based methods. Human myeloma cell lines and mouse-human heterozygous myeloma cell lines for producing human monoclonal antibodies have been described (see, for example, Kozbor J. Immunol., 13: 3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987); and Boerner et al., J. Immunol., 147: 86 (1991)). Human antibodies produced using human B-cell hybridoma technology are described in Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006). Additional methods include those described in, for example, U.S. Patent No. 7,189,826 (describes the production of monoclonal human IgM antibodies from hybridoma cell lines) and Ni, Xiandai Mianyixue, 26(4):265-268 (2006) (describes human-human hybridomas). Human hybridoma technology (trioma technology) is also described in Vollmers and Brandlein, Histology and Histopathology, 20(3):927-937 (2005) and Vollmers and Brandlein, Methods and Findings in Experimental and Clinical Pharmacology, 27(3):185-91 (2005). Complete human antibodies can also be derived from phage display libraries (disclosed in Hoogenboom et al., 1991, J. Mol. Biol. 227:381; and Marks et al., 1991, J. Mol. Biol. 222:581).Phage display technology mimics immunoselection by displaying an antibody repertoire on the surface of filamentous bacteriophages and then selecting phages by binding to selected antigens. One such technology is described in PCT Publication No. WO 99 / 10494. The human antibodies described herein can also be prepared using SCID mice in which human immune cells are reconstituted to produce a human antibody response during immunization. Such mice are described, for example, in U.S. Patents No. 5,476,996 and No. 5,698,767 to Wilson et al.

[0034] In some embodiments, the antibodies of the present invention mediate antibody-dependent cell-mediated cytotoxicity. As used herein, “antibody-dependent cell-mediated cytotoxicity” or “ADCC” refers to a cell-mediated response in which nonspecific cytotoxic cells (e.g., natural killer (NK) cells, neutrophils, and macrophages) recognize bound antibodies on target cells, subsequently causing lysis of the target cells. While not intended to be limited to a specific mechanism of action, these cytotoxic cells mediating ADCC generally express Fc receptors (FcRs).

[0035] As used herein, the “Fc region” includes polypeptides that constitute the stereotactic region of an antibody, excluding the first-order region immunoglobulin domains. Thus, Fc refers to the last two typical region immunoglobulin domains of IgA, IgD, and IgG, the last three typical region immunoglobulin domains of IgE and IgM, and the flexible hinges at the N-terminus of these domains. In the case of IgA and IgM, Fc may include a J chain. In the case of IgG, Fc consists of the immunoglobulin domains Cgamma2 and Cgamma3 (Cγ2 and Cγ3) and the hinge between Cgamma1 (Cγ1) and Cgamma2 (Cγ2). While the boundaries of the Fc region vary, the human IgG heavy chain Fc region is typically defined as encompassing residues C226 or P230 to its carboxyl terminus, with numbering here following the EU index described by Kabat et al. (1991, NIH Publication 91-3242, National Technical Information Service, Springfield, Va.). The "EU index described by Kabat" refers to the residue numbering of the human IgG1 EU antibody described by Kabat et al. Fc may refer to this region alone, or to this region in the context of an antibody, antibody fragment, or Fc fusion protein. An Fc variant protein may be an antibody, an Fc fusion, or any protein or protein domain constituting the Fc region. Particularly preferred are proteins consisting of variant Fc regions, which are non-naturally occurring variants of the Fc region. The amino acid sequence of a non-naturally occurring Fc region (also referred to herein as the "variant Fc region") consists of at least one amino acid substitution, insertion, and / or deletion compared to the wild-type amino acid sequence. New amino acid residues appearing in the variant Fc region sequence as a result of insertion or substitution are sometimes called non-native amino acid residues. Note: Polymorphisms have been observed at many Fc positions, including but not limited to Kabat 270, 272, 312, 315, 356, and 358, and therefore slight differences may exist between the presented sequence and the prior art sequence.

[0036] The terms "Fc receptor" or "FcR" are used to describe receptors that bind to the Fc region of antibodies. NK cells, the primary cells of ADCC, express FcγRIII, while monocytes express FcγRI, FcγRII, FcγRIII, and / or FcγRIV. FcR expression in hematopoietic cells is summarized in Ravetch and Kinet, Annu. Rev. Immunol., 9:457-92 (1991). To evaluate the ADCC activity of the molecule, an in vitro ADCC assay, such as that described in U.S. Patent No. 5,500,362, can be performed. In vitro ADCC assays such as those described in Patent No. 5,500,362 or No. 5,821,337 can be performed. Useful effector cells for such assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells. Alternatively, or additionally, the ADCC activity of the molecule in question may be evaluated in vivo, for example, in an animal model such as that disclosed in Clynes et al., Proc. Natl. Acad. Sci. (USA), 95:652-656 (1998). In this specification, the term “effector cell” refers to a leukocyte that expresses one or more FcRs and performs effector function. This cell expresses at least FcγRI, FCγRII, FcγRIII, and / or FcγRIV and performs 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.

[0037] In some embodiments, the antibody of the present invention is a full-length antibody. In some embodiments, the full-length antibody is an IgG1 antibody. In some embodiments, the full-length antibody is an IgG3 antibody.

[0038] In some embodiments, the antibody of the present invention includes a variant Fc region with increased affinity for FcγRIA, FcγRIIA, FcγRIIB, FcγRIIIA, FcγRIIIB, and FcγRIV. In some embodiments, the antibody of the present invention includes a variant Fc region comprising at least one amino acid substitution, insertion, or deletion, the substitution, insertion, or deletion of the at least one amino acid residue resulting in increased affinity for FcγRIA, FcγRIIA. In some embodiments, the antibody of the present invention includes a variant Fc region comprising at least one amino acid substitution, insertion, or deletion, the at least one amino acid residue is selected from the group consisting of residues 239, 330, and 332, where the amino acid residues are numbered following the EU index. In some embodiments, the antibody of the present invention comprises a variant Fc region comprising at least one amino acid substitution, where the at least one amino acid substitution is selected from the group consisting of S239D, A330L, A330Y, and 1332E, where the amino acid residue is numbered according to the EU index.

[0039] In some embodiments, the glycosylation of an antibody is modified. For example, an aglycosylated antibody can be produced (i.e., the antibody lacks glycosylation). Glycosylation can be modified, for example, to increase the antibody's affinity for an antigen. Such glycosylation modifications can be achieved, for example, by altering one or more glycosylation sites in the antibody sequence. For example, glycosylation can be eliminated at a site by making one or more amino acid substitutions that result in the elimination of a glycosylation site in one or more variable region frameworks. Such aglycosylation can increase the antibody's affinity for an antigen. Such approaches are described in more detail in U.S. Patents 5,714,350 and 6,350,861 by Co et al. Furthermore, or alternatively, antibodies with altered glycosylation types can be produced, for example, low-fucosylated or non-fucosylated antibodies with a reduced amount of fucosyl residues or no fucosyl residues, or antibodies with increased bifid GlcNac structures. Such alterations of glycosylation patterns have been demonstrated to enhance the antibody's ADCC capability. Such glycosylation modifications can be achieved, for example, by expressing antibodies in host cells with altered glycosylation mechanisms. Cells with altered glycosylation mechanisms have been described in the Art and can be used as host cells for expressing the recombinant antibodies of the present invention to produce antibodies with altered glycosylation. For example, Hang et al. EP 1,176,195 describes a cell line in which the FUT8 gene encoding fucosyltransferase is functionally disrupted, and antibodies expressed in such cell lines exhibit hypofucosylation or lack fucosyl residues. Therefore, in some embodiments, the human monoclonal antibodies of the present invention can be produced by recombinant expression in cell lines exhibiting hypofucosylation or nonfucosylation patterns, such as mammalian cell lines lacking expression of the FUT8 gene encoding fucosyltransferase.Presta's PCT publication WO 03 / 035835 describes Lecl3 cells, a mutant CHO cell line with reduced ability to attach fucose to Asn(297)-binding carbohydrates, resulting in decreased fucosylation of antibodies expressed in its host cells (see also Shields, RL et al, 2002 J. Biol. Chem. 277:26733-26740). Umana et al.'s PCT publication WO99 / 54342 describes a cell line engineered to express glycoprotein-modified glycosyltransferases (e.g., β(l,4)-N-acetylglucosaminyltransferase III (GnTIII)), and antibodies expressed in these engineered cell lines show increased bifurcating GlcNac structures that enhance the antibody's ADCC activity (see also Umana et al, 1999 Nat. Biotech. 17: 176-180). Eureka Therapeutics further describes genetically modified CHO mammalian cells capable of producing antibodies with altered mammalian glycosylation patterns that do not contain fucosyl residues (http: / / www.eurekainc.com / a&boutus / companyoverview.html). Alternatively, the human monoclonal antibodies of the present invention can be produced in yeast or filamentous fungi that are designed to produce mammalian-like glycosylation patterns and can produce antibodies lacking fucose as part of the glycosylation pattern (see, for example, EP1297172B1).

[0040] In some embodiments, the antibodies of the present invention mediate complement-dependent cytotoxicity. "Complement-dependent cytotoxicity" or "CDC" refers to the ability of a molecule to initiate complement activation and lyse a target in the presence of complement. The complement activation pathway is initiated when the first component of the complement system (C1q) binds to a molecule (e.g., an antibody) conjugated with an homologous antigen. To evaluate complement activation, a CDC assay can be performed, for example, the CDC assay described in Gazzano-Santaro et al., J. Immunol. Methods, 202:163 (1996).

[0041] In some embodiments, the antibodies of the present invention mediate antibody-dependent phagocytosis. Hereinafter, the terms “antibody-dependent phagocytosis” or “opsonization” refer to a cell-mediated response in which nonspecific cytotoxic cells expressing FcγR recognize bound antibodies on target cells, subsequently causing phagocytosis of the target cells.

[0042] In some embodiments, the antibody of the present invention is a polyspecific antibody comprising a first antigen-binding site directed to CALCRL and at least one second antigen-binding site directed to effector cells, as described above. In these embodiments, the second antigen-binding site is used to recruit a killing mechanism, such as binding an antigen to human effector cells. In some embodiments, effector cells can induce ADCCs such as natural killer cells. For example, monocytes and macrophages expressing FcR are involved in the specific killing of target cells or the presentation of antigens to other components of the immune system. In some embodiments, effector cells can phagocytose target antigens or target cells. The expression of specific FcRs on effector cells may be regulated by humoral factors such as cytokines. Effector cells can phagocytose or lyse target antigens or target cells. Suitable cytotoxic agents and second therapeutic agents are exemplified below and include toxins (such as radiolabeled peptides), chemotherapeutic agents, and prodrugs. In some embodiments, the second binding site binds to the Fc receptor defined above. In some embodiments, the second binding site can bind to surface molecules of NK cells and activate the cells.

[0043] Exemplary formats of the multispecific antibody molecule of the present invention include, but are not limited to, the following: (i) two antibodies crosslinked by chemical heteroconjugation, one specific to a specific surface molecule of leukemia stem cells and the other specific to a second antigen; (ii) a single antibody comprising two different antigen-binding regions; (iii) a single-chain antibody comprising two different antigen-binding regions, e.g., a single-chain antibody comprising two different antigen-binding regions, e.g., two scFvs linked in tandem by an extra peptide linker; (iv) a dual variable domain antibody (DVD-Ig) comprising two variable domains, each light chain and heavy chain linked in tandem via short peptide bonds (Wu et al., Generation and Characterization of a Dual Variable Domain Immunoglobulin (DVD-Ig) TM) Molecule, In: Antibody Engineering, Springer Berlin Heidelberg (2010)) (v) Chemically bound bispecific (Fab')2 fragments, (vi) Tandab, a quadrivalent bispecific antibody formed by fusing two single-stranded diabodies to have two binding sites for each target antigen, and (vii) flexibody, a polyvalent molecule formed by combining scFvs and diabodies. viii) A so-called "dock and lock" molecule that utilizes the "dimerization and docking domain" of protein kinase A, which, when applied to Fabs, yields a trivalent bispecific binding protein in which two identical Fab fragments are bound to different Fab fragments. (ix) For example, a so-called scorpion molecule containing two scFvs fused to both ends of a human Fab arm, and (x) a diabody. Another example of a bispecific antibody is an IgG-like molecule with a complementary CH3 domain to force heterodimerization. Such molecules can be prepared using known techniques, such as those known as Triomab / Quadroma (Trion Pharma / Fresenius Biotech), Knob-into-Hole (Genentech), CrossMAb (Roche) and electrostatic-matched (Amgen), LUZ-Y (Genentech), Strand Exchange Engineered Domain body (SEEDbody) (EMD Serono), Biclonic (Merus), and DuoBody (Genmab A / S).

[0044] In some embodiments, bispecific antibodies are obtained, or can be obtained, via controlled Fab-arm exchange, typically using DuoBody technology. In vitro methods for producing bispecific antibodies by controlled Fab-arm exchange are described in WO2008119353 and WO2011131746 (both by Genmab A / S). In one exemplary method described in WO2008119353, a bispecific antibody is formed by “Fab-arm” or “half-arm” exchange (exchange of the heavy chain and attached light chain) between two monospecific antibodies, both consisting of IgG4-like CH3 regions, during incubation under reducing conditions. The result is a bispecific antibody having two Fab arms that may contain different sequences. In another exemplary method described in WO 2011131746, the bispecific antibody of the present invention is prepared by a method comprising the following steps: a) providing a first antibody comprising an Fc region of an immunoglobulin, wherein the Fc region comprises a first CH3 region; b) providing a second antibody comprising an Fc region of an immunoglobulin, wherein the Fc region comprises a second CH3 region, wherein the sequences of the first and second CH3 regions are different and the heterodimer interaction of the first and second CH3 regions is stronger than the homodimer interaction of each of the first and second CH3 regions; c) incubating the first antibody together with the second antibody under reducing conditions; and d) obtaining the bispecific antibody wherein the first antibody is the human monoclonal antibody of the present invention, and the second antibody has a different binding specificity or vice versa. Reducing conditions may be provided, for example, by adding a reducing agent selected from 2-mercaptoethylamine, dithiothreitol, and tris(2-carboxyethyl)phosphine. Step d) may further include returning to a non-reducing or low-reducing state by removing the reducing agent, for example, by desalting.Preferably, the sequences of the first and second CH3 regions are different, consisting of only a few fairly conservative asymmetric mutations, such that the heterodimer interaction between the first and second CH3 regions is stronger than the homodimer interaction between each of the first and second CH3 regions. Details of these interactions and how they are realized are described in WO 2011131746, which is incorporated herein by reference in whole. The following are exemplary embodiments of such asymmetric mutation combinations, in which one or both Fc regions are of the IgGl isotype. In some embodiments, the first Fc region has an amino acid substitution at a position selected from the group consisting of 366, 368, 370, 399, 405, 407, and 409. The second Fc region has an amino acid substitution at a position selected from the group consisting of 366, 368, 370, 399, 405, 407, and 409. The first and second Fc regions are characterized by having an amino acid substitution at a position selected from the group consisting of 366, 368, 370, 399, 405, 407, and 409, and the first and second Fc regions are not substituted at the same position. In some embodiments, the first Fc region has an amino acid substitution at position 405, and the second Fc region has an amino acid substitution at a position selected from the group consisting of: 366, 368, 370, 399, 407, and 409, optionally 409. In some embodiments, the first Fc region has an amino acid substitution at position 409, and the second Fc region has an amino acid substitution at a position selected from the group consisting of: 366, 368, 370, 399, 405, and 407, optionally 405 or 368. In some embodiments, both the first and second Fc regions are IgGl isotypes, the first Fc region has Leu at position 405, and the second Fc region has Arg at position 409.

[0045] In some embodiments, the antibody of the present invention is conjugated with a therapeutic moiety, i.e., a drug. The therapeutic site may be, for example, a cytotoxin, a chemotherapeutic agent, a cytokine, an immunosuppressant, an immunostimulant, a lysed peptide, or a radioisotope. Such conjugates are referred to herein as “antibody-drug conjugates” or “ADCs.”

[0046] In some embodiments, the antibodies of the present invention are conjugated to cytotoxic sites. These cytotoxic sites include, for example, taxol; cytochalasin B; gramicidin D; ethidium bromide; emetine; mitomycin; etoposide; tenoposide; vincristine; vinblastine; cortisine; doxorubicin; daunorubicin; dihydroxyanthracine dione; tubulin inhibitors such as maytansine or its analogs / derivatives; antimitomitotic agents such as monomethyl auristatin E or F or its analogs / derivatives; and drastatin 10 or 15 or its analogs. Irinotecan or its analogues; mitoxantrone; mitramycin; actinomycin D; 1-dehydrotestosterone; antimetabolites such as glucocorticoids, procaine, tetracaine, lidocaine, propranolol, promycin, kalichamycin or its derivatives, methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, fludarabine, 5-fluorouracil, decarbazine, hydroxyurea, asparaginase, gemcitabine, and cladribine.Alkylating agents such as mechloretamine, thioepa, chlorambucil, melphalan, carmustine (BSNU), lomustine (CCNU), cyclophosphamide, busulfan, dibromomanitol, streptozotocin, dacarbazine (DTIC), procarbazine, and mitomycin C; platinum derivatives such as cisplatin and carboplatin; duocalmycin A, duocalmycin SA, rashelmycin (CC-1065), or their analogs or derivatives; antibiotics such as dactinomycin, bleomycin, daunorubicin, doxorubicin, idarubicin, mitramycin, mitomycin, mitoxantrone, plicamycin, and anthramycin (AMC); pyrrolo[2,lc][l,4]-benzodiazepine (PDB); related molecules such as diphtheria toxin and its active fragments and hybrid molecules; lysine toxins such as lysine A and deglycosylated lysine A chain toxin; cholera toxin, SLT I, SLT The group of toxins that can be selected from includes Shiga-like toxins such as II and SLT IIV, LT toxin, C3 toxin, Shiga toxin, pertussis toxin, tetanus toxin, soybean Bowman-Birk protease inhibitors, Pseudomonas endotoxins, alorin, saporin, modeccin, geranin, abrin A chain, modesin A chain, α-sacrin, Aleurites fordii protein, dianthin protein, Phytolacca americana proteins such as PAPI, PAPII, PAP-S, PAPII, PAP-S, momordica charantia inhibitors, curcin, crotin, sapaonaria officinalis inhibitors, geronin, mitogenin, restrictocin, phenomycin and enomycin toxins; ribonuclease (RNase), DNase I, Staphylococcus enterotoxin A, porkweed antiviral protein, diphtherin toxin, and Pseudomonas endotoxin.

[0047] In some embodiments, the antibodies of the present invention are conjugated with auristatin or its peptide analog, derivative, or prodrug. Auristatin has been shown to interfere with microtubule dynamics, GTP hydrolysis, and nuclear and cell division (Woyke et al (2001) Antimicrob. Agents and Chemother. 45(12): 3580-3584), and possesses anticancer (US5663149) and antifungal activity (Pettit et al, (1998) Antimicrob. Agents and Chemother. 42: 2961-2965. For example, auristatin E can be reacted with paraacetylbenzoic acid or benzoylvaleric acid to produce AEB and AEVB, respectively. Other representative auristatin derivatives include AFP, MMAF (monomethyl auristatin F), and MMAE (monomethyl auristatin E). Suitable auristatin, auristatin analogs, derivatives, prodrugs, and auristatin conjugates to Abs. Suitable linkers are described, for example, in U.S. Patents Nos. 5,635,483, 5,780,588, 6,214,345, and International Patent Application Publications WO02088172, WO2004010957, WO2005081711, WO2005084390, WO2006132670, WO03026577, WO200700860, WO207011968, and WO205082023.

[0048] In some embodiments, the antibodies of the present invention are conjugated with pyrrolo[2,lc][l,4]-benzodiazepine (PDB) or its analogues, derivatives, or prodrugs. Suitable PDBs and PDB derivatives, as well as related techniques, are described, for example, in Hartley JA et al., Cancer Res 2010; 70(17): 6849-6858; Antonow D. et al., Cancer J 2008; 14(3): 154-169; Howard PW et al., Bioorg Med Chem Lett 2009; 19: 6463-6466; and Sagnou et al., Bioorg Med Chem Lett 2000; 10(18): 2083-2086.

[0049] In some embodiments, the antibodies of the present invention are conjugated to a cytotoxic site selected from the group consisting of anthracyclines, maytansine, calicheamicin, duocalmycin, rashelmycin (CC-1065), drastatin 10, drastatin 15, irinotecan, monomethyl auristatin E, monomethyl auristatin F, PDB, or any analog, derivative, or prodrug thereof.

[0050] In some embodiments, the antibody of the present invention is conjugated to an anthracycline or its analog, derivative, or prodrug. In some embodiments, the antibody is conjugated to meitansine or its analog, derivative, or prodrug. In some embodiments, the antibody is conjugated to kalichamycin or its analog, derivative, or prodrug. In some embodiments, the antibody is conjugated to duocalmycin or its analog, derivative, or prodrug. In some embodiments, the antibody is conjugated to rakelmycin (CC-1065) or its analog, derivative, or prodrug. In some embodiments, the antibody is conjugated to drastatin 10 or its analog, derivative, or prodrug. In some embodiments, the antibody is conjugated to drastatin 15 or its analog, derivative, or prodrug. In some embodiments, the antibody is conjugated to monomethyl auristatin E or its analog, derivative, or prodrug. In some embodiments, the antibody is conjugated to monomethyl auristatin F or its analog, derivative, or prodrug. In some embodiments, the antibody is conjugated to pyrrolo[2,lc][l,4]-benzodiazepine or its analog, derivative, or prodrug. In some embodiments, the antibody is conjugated to irinotecan or its analogues, derivatives, or prodrugs.

[0051] The technique of binding molecules to antibodies is well known in this field (see, for example, Arnon et al., "Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy," in Monoclonal Antibodies And Cancer Therapy (Reisfeld et al. eds., Alan Riss, Inc., 1985); Hellstrom et al., "Antibodies For Drug Delivery," in Controlled Drug Delivery (Robinson et al., Marcel Deiker, Inc., 2nd edition)); Hellstrom et al., "Antibodies For Drug Delivery," Controlled Drug Delivery (Robinson et al. eds., Marcel Deiker, Inc., 2nd edition, 1987); Thorpe, "Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review," in Monoclonal Antibodies '84: Biological And Clinical Applications (Pinchera et al. eds., 1985); "Analysis, Results, and Future" See "Prospective of the Therapeutic Use of Radiolabeled Antibody In Cancer Therapy," in Monoclonal Antibodies For Cancer Detection And Therapy (Baldwin et al. eds., Academic Press, 1985); and Thorpe et al., 1982, Immunol. Rev. 62:119-58. Also, see, for example, PCT Publication WO 89 / 12624). Typically, nucleic acid molecules are covalently bound to lysine or cysteine ​​on the antibody via N-hydroxysuccinimide ester or maleimide functionalization, respectively.Methods have been reported to improve the homogeneity of conjugates by using artificial cysteine ​​or incorporating unnatural amino acids (Axup, JY, Bajjuri, KM, Ritland, M., Hutchins, BM, Kim, CH, Kazane, SA, Halder, R., Forsyth, JS, Santidrian, AF, Stafin, K., et al. (2012). Synthesis of site-specific antibody-drug conjugates using unnatural amino acids. Proc. Natl. Acad. Sci. USA 109, 16101-16106.; Junutula, JR, Flagella, KM, Graham, RA, Parsons, KL, Ha, E., Raab, H., Bhakta, S., Nguyen, T., Dugger, DL, Li, G., et al.). (2010), an artificial thio-trastuzumab-DM1 conjugate improved the therapeutic index targeting human epidermal growth factor receptor 2-positive breast cancer. (Clin. Cancer Res. 16, 4769-4778.) Junutula et al. (2008) developed "THIOMABs" (TDCs), a cysteine-based site-specific conjugation, and claim that it shows an improved therapeutic index compared to conventional conjugation methods. Conjugation to non-natural amino acids incorporated into antibodies has also been investigated with ADCs, but the generality of this approach has not yet been established (Axup et al., 2012). In particular, those skilled in the art can envision that acyl-donor glutamine-containing tags (e.g., Gin-containing peptide tags or Q-tags) or Fc-containing polypeptides engineered with endogenous glutamine may be made reactive by polypeptide engineering (e.g., via deletion, insertion, substitution, or mutation of amino acids on the polypeptide).Next, transglutaminase can covalently bind to an amine donor (e.g., a small molecule consisting of or bound to a reactive amine) to form a stable and homogeneous population of engineered Fc-containing polypeptide conjugates, in which the amine donor is site-specifically bound to the Fc-containing polypeptide via an acyl-donor glutamine-containing tag or accessible / exposed / reactive endogenous glutamine (WO 2012059882).

[0052] In some embodiments, the inhibitor is a compound (e.g., an antibody) that inhibits the binding of CALCRL to RAMP1 and / or RAMP2 and / or RAMP3. In some embodiments, the inhibitor (e.g., an antibody) inhibits the binding of CALCRL to one of the ligands, such as adrenomedullin.

[0053] In some embodiments, the inhibitor is an inhibitor of the expression of CALCRL, RAMP1, RAMP2, or RAMP3. “Inhibitor of expression” means a natural or synthetic compound that has a biological effect of inhibiting gene expression. In preferred embodiments of the present invention, the gene expression inhibitor is an siRNA, antisense oligonucleotide, or ribozyme. For example, antisense oligonucleotides, including antisense RNA molecules and antisense DNA molecules, act to directly block the translation of CALCRL, RAMP1, RAMP2, or RAMP3 by binding to the mRNA of CALCRL, RAMP1, RAMP2, or RAMP3 and interfering with protein translation or increasing mRNA degradation, thereby reducing the level and activity of CALCRL, RAMP1, RAMP2, or RAMP3 in the cell. For example, an antisense oligonucleotide of at least about 15 nucleotides that is complementary to a specific region of the mRNA transcription sequence encoding CALCRL, RAMP1, RAMP2, or RAMP3 can be synthesized, for example, by conventional phosphodiester techniques. Methods using antisense techniques to specifically inhibit the expression of genes with known sequences are well known in the art (see, for example, U.S. Patent Nos. 6,566,135, 6,566,131, 6,365,354, 6,410,323, 6,107,091, 6,046,321, and 5,981,732). Small inhibitory RNAs (siRNAs) can also function as expression inhibitors for use in the present invention. The expression of CALCRL, RAMP1, RAMP2, or RAMP3 genes can be reduced by contacting a subject or cell with small double-stranded RNA (dsRNA), or a vector or construct that induces the production of small double-stranded RNA (i.e., RNA interference or RNAi), so that the expression of CALCRL, RAMP1, RAMP2, or RAMP3 genes is specifically inhibited. The antisense oligonucleotides, siRNAs, shRNAs, and ribozymes of the present invention may be delivered in vivo, either alone or in association with a vector.In a broad sense, a “vector” is any vehicle that can facilitate the delivery of antisense oligonucleotides, siRNAs, shRNAs, or ribozyme nucleic acids to cells, typically cells expressing CALCRL, RAMP1, RAMP2, or RAMP3. Typically, a vector delivers nucleic acids to cells with reduced degradation compared to the degree of degradation that would occur in the absence of the vector. Generally, vectors useful in the present invention include, but are not limited to, plasmids, phagemids, viruses, and other vehicles derived from viral or bacterial sources that have been manipulated by the insertion or incorporation of antisense oligonucleotides, siRNAs, shRNAs, or ribozyme nucleic acid sequences. Viral vectors are a preferred type of vector and include, but are not limited to, nucleic acid sequences from the following viruses: RNA viruses such as retroviruses, for example, Moloney's mouse leukemia virus, Harvey's mouse sarcoma virus, mouse mammary tumor virus, and Rose's sarcoma virus, adenoviruses, adeno-associated viruses, SV40 virus, polyomavirus, Epstein-Barr virus, papillomavirus, herpesvirus, vaccinia virus, poliovirus, and retroviruses. In addition, other vectors known in the art, although not named, can be readily employed.

[0054] "Therapeutally effective dose" means a sufficient amount of antibody or inhibitor with a reasonable benefit-to-risk ratio applicable to medical practice. It is understood that the total daily dose of the compounds and compositions of the present invention should be determined by the attending physician within the bounds of sound medical judgment. A specific therapeutically effective dose level for a particular subject depends on a variety of factors, including the disease being treated and its severity, the activity of the specific compound being employed, the specific composition being employed, the subject's age, weight, general health status, sex, and diet, the timing of administration, route of administration, and excretion rate of the specific compound being employed, the duration of treatment, any drugs used in combination with or concurrently with the specific polypeptide being employed, and similar factors well known in medical technology. For example, it is easy for those skilled in the art to start administration of a compound at a lower dose than necessary to achieve the desired therapeutic effect and gradually increase the dose until the desired effect is obtained. However, the daily dose of the product can vary widely from 0.01 to 1,000 mg per adult per day. Preferably, the composition contains 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 250, and 500 mg of the active ingredient to symptomatically adjust the dosage for the target to be treated. The drug typically contains about 0.01 mg to about 500 mg of the active ingredient, preferably 1 mg to about 100 mg of the active ingredient. An effective dose of the drug is usually supplied in doses of 0.0002 mg / kg to about 20 mg / kg of body weight per day, particularly about 0.001 mg / kg to 7 mg / kg of body weight.

[0055] Typically, the antibodies or inhibitors of the present invention are combined with pharmaceutically acceptable excipients and optionally with a sustained-release matrix such as a biodegradable polymer to form a pharmaceutical composition. "Pharmaceutically" or "pharmaceutically acceptable" means molecular entities and compositions that, when administered to mammals, particularly humans, do not, as appropriate, cause adverse reactions, allergic reactions, or other undesirable reactions. Pharmaceutically acceptable carriers or excipients refer to non-toxic solid, semi-solid, or liquid fillers, diluents, encapsulating materials, or any type of formulation aid. Generally, pharmaceutical compositions include a pharmaceutically acceptable vehicle as an injectable formulation. These are particularly isotonic sterile salines (monosodium phosphate, disodium phosphate, sodium chloride, potassium, calcium, magnesium, etc., or mixtures of these salts), or dry, particularly lyophilized compositions, which may optionally be combined with sterile water or saline to form an injectable solution. Pharmaceutical forms suitable for injection include sterile aqueous solutions or dispersions, formulations containing sesame oil, peanut oil, or aqueous propylene glycol, and sterile powders for the immediate preparation of sterile injection solutions or dispersions. In all cases, the solution must be sterile and fluid enough to be easily injected. It must also be stable under manufacturing and storage conditions and protected from contamination by microorganisms such as bacteria and fungi. Sterile injectable solutions are prepared by placing the required amount of active ingredient in a suitable solvent, incorporating some of the other components listed above as needed, and then sterilizing by filtration. Generally, dispersions are prepared by incorporating various sterile active ingredients into a sterile vehicle containing a basic dispersion medium and other necessary components. For sterile powders for preparing sterile injectable solutions, preferred preparation methods are vacuum drying and freeze-drying techniques, where the powder of the active ingredient and any additional desired components is obtained from a previously sterile filtered solution.

[0056] A further object of the present invention relates to a method for identifying leukemia stem cells in a sample obtained from a subject with AML, comprising identifying and selecting a population of cells expressing CALCRL and at least one stem cell marker.

[0057] The samples used in the diagnostic method of the present invention can be obtained from various sources, particularly blood, but in some cases, samples such as bone marrow, lymph, cerebrospinal fluid, synovial fluid, etc. may also be used. Such samples can be separated by centrifugation, elution, density gradient separation, apheresis, affinity selection, panning, FACS, centrifugation with Hypaque, etc. before analysis. Once the sample is obtained, it can be used as it is, frozen, or maintained in an appropriate medium for a short period. Various media can be employed to maintain the cells. The sample can be obtained by any convenient method such as blood collection, venipuncture, biopsy, etc. Usually, the sample contains at least about 10 2 cells, more usually at least about 10 3 cells, preferably 10 4 cells, 10 5 cells or more. Appropriate solutions can be used for dispersing or suspending the cell sample. Such solutions are generally balanced salt solutions, such as normal saline, PBS, Hank's balanced salt solution, etc., conveniently supplemented with fetal calf serum or other naturally occurring factors and used in combination with a tolerable buffer at a low concentration, generally 5 - 25 mM. Convenient buffers include HEPES, phosphate buffer, lactate buffer, etc.

[0058] Leukemia stem cells can be predictively separated or identified from primary tumor samples. In particular, leukemia stem cells have unique characteristics of cancer stem cells in functional assays for self-renewal and differentiation of cancer stem cells. Methods for separating leukemia cells and leukemia stem cells are well known in the art and typically involve the presence or absence of specific cell surface markers. For example, leukemia stem cells and normal corresponding human hematopoietic stem cells (HSCs), which have the phenotype Lin - CD34 + CD38 - CD90 + ; or the phenotype Lin - CD34 + CD38 - CD90+ CD45RA - Cells possessing this feature are included without limitation, including human hematopoietic pluripotent progenitor cells (MPPs), which include phenotypic Lin - CD34 + CD38 - CD90 - ; or phenotype Lin - CD34 + CD38 - CD90 - CD45RA - A comparison can be made between cells that have [specific characteristic] and those that do not limit the range of cells that possess [specific characteristic].

[0059] In some embodiments, a panel of binding partners specific to the target cell surface marker is used to determine the presence or absence of the cell surface marker. These binding partners include, but are not limited to, antibodies, aptamers, and peptides. By using binding partners, cell populations expressing the marker can be screened. Various techniques can be used to screen cell populations expressing the target cell surface marker, typically including magnetic separation using antibody-coated magnetic beads, "panning" using antibodies attached to a solid matrix (i.e., a plate), and flow cytometry (see, e.g., U.S. Patent No. 5,985,660 and Morrison et al. Cell, 96:737-49 (1999)).

[0060] In some embodiments, the binding partner is a polyclonal or monoclonal antibody, preferably monoclonal, specifically directed to a single cell surface marker. The polyclonal antibody or fragment of the present invention can be elevated according to known methods by administering a suitable antigen or epitope to a selected host animal, such as a pig, cattle, horse, rabbit, goat, sheep, and mouse. Antibody production can be enhanced using various adjuvants known in the art. While the antibody useful for carrying out the present invention may be polyclonal, monoclonal antibodies are preferred. The monoclonal antibody or fragment of the present invention can be prepared and isolated using any technique that provides antibody molecule production by a serial cell line in culture. Techniques for production and isolation include, but are not limited to, hybridoma techniques; human B-cell hybridoma techniques; and EBV-hybridoma techniques.

[0061] In some embodiments, the binding partner panel is specific to at least one cell surface marker selected from the group consisting of CD33, CD34, CD36, CD38, CD39, CD45, CD81, CD90, and CD123, and therefore includes at least one binding partner specific to CALCRL.

[0062] Generally, binding partners are bound to labels for use in separation. Labels include magnetic beads that enable direct separation, biotin that can be removed with avidin or streptavidin bound to a support, and fluorescent dyes that can be used in fluorescence-activated cell sorters, facilitating the separation of specific cell types. Fluorescent dyes include phycobiliproteins such as phycoerythrin and allophycocyanin, fluorescein, and Texas Red. Typically, each antibody is labeled with a different fluorescent dye, allowing for independent sorting by marker. Suitable fluorescent detection elements include, but are not limited to, fluorescein, rhodamine, tetramethylrhodamine, eosin, erythrosine, coumarin, methylcoumarin, pyrene, malachite green, stilbene, Lucifer Yellow, Cascade Blue®, Texas Red, IAEDANS, DANS, BODIPY FL, LC Red 640, Cy 5, Cy 5.5, LC Red 705, and Oregon Green. The appropriate optical dyes are listed in Richard P. Haugland's "1996 Molecular Probes Handbook," which are explicitly incorporated here by reference.Suitable fluorescent labels also include green fluorescent protein (GFP; Chalfie et al., Science 263(5148):802-805 (February 11, 1994); and EGFP; Clontech-Genbank Accession Number U55762), blue fluorescent protein (BFP; 1. Quantum Biotechnologies, Inc. 1801 de Maisonneuve Blvd. West, 8th Floor, Montreal (Quebec) Canada H3H 1J9; 2. Stauber, RH Biotechniques 24(3):462-471 (1998); 3. Heim, R. and Tsien, RY Curr. Biol. 6:178-182 (1996)), and enhanced yellow fluorescent protein (EYFP; 1. Clontech Laboratories, Inc., 1020 East Meadow Circle, Palo Alto, Calif. 94303), luciferase (Ichiki, et al., J. Immunol. 150(12):5408-5417(1993)), β-galactosidase (Nolan, et al., Proc Natl Acad Sci USA 85(8):2603-2607(April 1988)), and lenira WO 92 / 15673; WO 95 / 07463; WO 98 / 14605; WO 98 / 26277; WO 99 / 49019; US Pat. (as described in Patent Nos. 5,292,658; 5,418,155; 5,683,888; 5,741,668; 5,777,079; 5,804,387; 5,874,304; 5,876,995; and 5,925,558).) All of the above cited references are expressly incorporated herein by reference. In some embodiments, the detection elements for use in the present invention include:This is an exemplary list including Alexa-Fluor dyes (Alexa Fluor® 350, Alexa Fluor® 405, Alexa Fluor® 430, Alexa Fluor® 488, Alexa Fluor® 500, Alexa Fluor® 514, Alexa Fluor® 532, Alexa Fluor® 546, Alexa Fluor® 555, Alexa Fluor® 568, Alexa Fluor® 594, Alexa Fluor® 610, Alexa Fluor® 633, Alexa Fluor® 647, Alexa Fluor® 660, Alexa Fluor® 680, Alexa Fluor® 700, Alexa Fluor® 750), Cascade Blue, Cascade Yellow, R-phycoerythrin (PE) (Molecular Probes) (Eugene, Oreg.), FITC, Rhodamine, Texas Red (Pierce, Rockford, These include Cy5, Cy5.5, and Cy7 (Amersham Life Science, Pittsburgh, Pa.). Tandem conjugate protocols for Cy5PE, Cy5.5PE, Cy7PE, Cy5.5APC, and Cy7APC are known in the industry. Fluorescent dyes bound to antibodies or other binding elements are activated by a laser and re-emit light of different wavelengths. The amount of light detected from the phosphor is related to the number of binding element targets associated with the cells passing through the beam. Any particular set of detection elements in any embodiment, e.g., fluorescently tagged antibodies, may depend on the type of cells under study and the presence of activatable elements within those cells. Since multiple detection elements, e.g., fluorescently labeled antibodies, can be used simultaneously, the measurements performed as a single cell passes through the laser beam consist of scattered light intensity and light intensity from each fluorescent label. Thus, characterization of a single cell consists of a series of measured light intensities, represented as coordinate positions in multidimensional space.Considering only the light from the phosphor, there is one coordinate axis corresponding to each detection element, such as a fluorescently tagged antibody. The number of coordinate axes (spatial dimension) is the number of fluorescent dyes used. Modern flow cytometers can measure thousands of cells per second, with multiple colors associated with different fluorescent dyes. Thus, data from a single subject can be described by a collection of measurements related to the number of antigens for each of thousands of individual cells (typically). See Krutzik et al., High-content single-cell drug screening with phosphospecific flow cytometry. Nature Chemical Biology, Vol.4 No.2, Pgs. 132-42, February 2008. Such methods can optionally use barcodes to improve throughput and reduce consumable consumption. See Krutzik, P. and Nolan, G., Fluorescent cell barcoding in flow cytometry allows high-throughput drug screening and signaling profiling. Nature Methods, Vol.3 No.5, Pgs. 361-68, May 2006.

[0063] In some embodiments, the binding partner is a metallic chemical element such as a lanthanide. Lanthanides offer several advantages over other labels, including being stable isotopes, having up to 100 or more different labels available, being relatively stable, having high detectability when detected using mass spectrometry, and being easily decomposed between detection channels. Lanthanide labels also feature a wide detection dynamic range. Because lanthanides are highly sensitive and unaffected by light and time, they are very flexible and robust and can be used in a variety of applications. Lanthanides are a group of 15 metallic chemical elements with atomic numbers 57 to 71. They are also called rare earth elements. Lanthanides can be detected using CyTOF technology. CyTOF is an inductively coupled plasma time-of-flight mass spectrometer (ICP-MS). A CyTOF instrument can analyze up to 1000 cells per second for the same number of parameters as the number of available stable isotope tags.

[0064] Typically, the binding partner is added to the cell suspension and incubated for a sufficient time to allow the available cell surface antigens to bind. Incubation is usually at least about 5 minutes, and typically less than 30 minutes. It is desirable to have a sufficient concentration of the binding partner in the reaction mixture so that the efficiency of separation is not limited by a shortage of the binding partner. The appropriate concentration is determined by titration. The medium used to separate the cells can be any medium that maintains cell viability. A preferred medium is phosphate-buffered saline containing 0.1 to 0.5% BSA. Mediums are commercially available and, depending on the nature of the cells, can be used, such as Dulbecco's modified Eagle medium (dMEM), Hanks' basic salt solution (HBSS), Dulbecco's phosphate-buffered saline (dPBS), RPMI, Isobe medium, or PBS containing 5 mM EDTA, and can be frequently supplemented with fetal calf serum, BSA, HSA, etc.

[0065] The diagnostic method of the present invention is particularly suitable for determining whether a subject is at risk of relapse, where the presence of leukemia stem cells indicates that the subject is at risk of relapse. Furthermore, in some embodiments, the diagnostic method of the present invention is particularly suitable for determining the survival period of a subject, where the presence of leukemia stem cells indicates that the subject has a short survival period.

[0066] The present invention is further illustrated by the following figures and examples. However, these embodiments and figures should not be construed in any way as limiting the scope of the present invention. [Brief explanation of the drawing]

[0067] [Figure 1A] Downregulation of CALCRL impairs leukemia growth in vivo. Experimental design to evaluate the frequency of leukemia stem cells: Cells were ex vivo treated with siCTR or siCALCRL, and then injected into the tail vein of mice with reduced cell concentrations (500,000; 100,000; 10,000; 1,000) (n=4 in each group). After 12 weeks, the mice were dissected, and the engraftment of human cells in the mouse bone marrow was evaluated. A percentage of human cells greater than 0.1% was considered positive engraftment in the bone marrow. [Figure 1B] The effect of CALCRL depletion on the frequency of LIC. [Figure 1C] Investigation of the role of CALCRL in leukemia cell growth in vivo. 2.106 MOLM-14 or OCI-AML3 cells expressing doxycycline-inducible shCTR, shCAL#1, or shCAL#2 were injected into the tail vein of NSG mice. On the day of injection, 0.2 mg / ml of doxycycline was added to drinking water containing 1% sucrose to induce shRNA expression until the end of the experiment. After 25 days, some mice were sacrificed to measure cell engraftment (total cellular tumor burden = number of blasts in bone marrow + spleen) (5-6 mice per group), and survival rates were tracked for another group of mice (7-9 mice per group). [Figure 1D]The tumor burden of all cells was measured using the mCD45.1- / hCD45+ / hCD33+ / AnnV- markers. Error bars represent Mean ± SEM, and each group was compared using an unpaired two-sided t-test with Welch correction. [Figure 1E] Mouse survival was observed. Comparisons were made using the log-rank (Mantel-Cox) test. *p<0.05; **p<0.01; ***p<0.001; no significant difference in ns. [Figure 2A] Depletion of CALCRL sensitizes cells to chemotherapy. This experimental design evaluates the effect of CALCRL depletion on the in vivo chemotherapy response. 2.106MOLM-14 expressing the specified inducible shRNA was injected into the tail vein of NSG mice. Ten days later, once the disease state was established, the mice were administered 30 mg / kg / d of cytarabine for 5 days. [Figure 2B] The tumor burden of all cells was measured using the mCD45.1- / hCD45+ / hCD33+ / AnnV- markers. [Figure 2C] Mouse survival was observed. Group comparisons were performed using the log-rank (Mantel-Cox) test. *p<0.05;**p<0.01;***p<0.001;ns, no significant difference. [Figure 3A] CALCRL expression levels predict the response to chemotherapy. Schematic diagram of chemotherapy regimens and schedules used to treat an NSG-based PDX model with AraC. Peripheral blood graft survival rates were assessed between 8 and 18 weeks, and mice were assigned to experimental groups of 4–10 mice, with similar mean graft survival rates in each group. Mice were administered either a vehicle (PBS) or 60 mg / kg / day of AraC by daily intraperitoneal injection for 5 days. Post-treatment mice were sacrificed on day 8, and surviving residual AML cells were characterized. [Figure 3B]The total number of viable human AML cells expressing hCD45, hCD33, and / or hCD44 was analyzed using flow cytometry and quantified compared to bone marrow from PBS-treated AML-xenografted mice. For each AML patient sample, the fold reduction in total tumor burden in AraC-treated mice compared to control mice was calculated individually. Patients were then divided into two categories: low-responders (FC>10) and high-responders (FC>10). [Figure 3C] Graph showing the proportion of CALCRL-positive cells in the low-response and high-response groups (cell surface expression was determined by flow cytometry analysis of vehicle-treated cells). [Figure 3D] Correlation between the decrease in the multiplier and the proportion of CALCR-positive cells. Linear regression was performed, and R² and p-value were calculated. *p<0.05;**p<0.01;***p<0.001;ns, no significant difference. [Figure 4A] Targeting CALCRL eradicates chemotherapy-resistant leukemia stem cells. The role of CALCRL in the LSC-positive chemotherapy-resistant population. Primary AML samples were injected into the bloodstream of mice, followed by daily intraperitoneal injection of either a vehicle (PBS) or 60 mg / kg / day of AraC for 5 days. On day 8, mice were sacrificed, and human cells treated with PBS and AraC were transfected ex vivo with siRNA. Subsequent cells were injected into the tail vein of mice at reduced concentrations (n=4 per group). After 12 weeks, mice were dissected, and the engraftment of human cells in the mouse bone marrow was evaluated using the mCD45.1- / hCD45+ / AnnV- markers. A percentage of human cells greater than 0.5% was considered positive for bone marrow or spleen transplantation. [Figure 4B] Graph showing the frequency of LIC (Low-Invasive Leukocyte Transfusion) to the bone marrow and spleen. Frequency and statistical analysis were performed using L-calc software (Stemcell Technologies).

[0068] Examples: Materials and methods. Human trials Samples from newly diagnosed AML patients were obtained from Toulouse University Hospital (TUH), Toulouse, France. Frozen samples were obtained from patients diagnosed with AML at TUH after obtaining informed consent signed in accordance with the Declaration of Helsinki, and stored in the HIMIP collection (BB-0033-00060). In accordance with French law, the HIMIP biobank collection was declared to the Ministry of Higher Education and Research (DC2008-307, Collection 1), approved by the Ethics Committee (Comite de Protection des Personnes Sud-Ouest et Outremer II), and a transfer agreement (AC2008-129) was obtained. Clinical and biological annotations of the samples were declared to the CNIL (Comite National Informatique et Libertes ie Data processing and Liberties National Committee). For age, sex, cytogenetic, and mutation information of the human samples used in this study, please refer to Table S3.

[0069] In vivo animal experiments NSG (NOD.Cg-Prkdcscid Il2rgtm1WjI / SzJ) mice (manufactured by Charles River Laboratories) were used for transplantation of AML cell lines or primary AML samples. Male or female mice aged 6 to 9 weeks were used in the experiments, and mice were randomly assigned to experimental groups before cell injection or drug treatment. Mice were housed in sterile conditions using micro-isolators with HEPA filters and fed irradiated feed and sterile water at the Animal Core Facility of the Cancer Research Center in Toulouse, France. All animals were used in accordance with protocols reviewed and approved by the Institutional Animal Care and Use Committee of Region Midi-Pyrenees, France.

[0070] Cell lines and primary cultures For primary human AML cells, peripheral blood or bone marrow samples were frozen in FCS containing 10% DMSO and stored in liquid nitrogen. The percentage of blast cells was determined by flow cytometry and morphological characteristics before purification. The cells were thawed in a 37°C water bath and washed with a thawing medium consisting of IMDM and 20% FBS. Subsequently, in all experiments, the cells were maintained in IMDM, 20% FBS, and 1% Pen / Strep (GIBCO).

[0071] Cell lines and culture conditions Human AML cell lines were cultured at 37°C and 5% CO2 in RPMI media (Gibco) supplemented with 10% FBS (Invitrogen), 100 U / mL penicillin, and 100 μg / mL streptomycin. The cultured cells were divided every 2-3 days and maintained in an exponential growth phase. All AML cell lines were purchased from DSMZ or ATCC, and liquid nitrogen stocks were renewed every two years. These cell lines were regularly tested for mycoplasma contamination in the laboratory. U937 cells were obtained from DSMZ in February 2012 and from ATCC in January 2014. MV4-11 and HL-60 cells were obtained from DSMZ in February 2012 and 2016. KG1 cells were obtained from DSMZ in February 2012 and from ATCC in March 2013. KG1a cells were obtained from DSMZ in February 2016. MOLM14 cells were obtained from Pr., specifically from Martin Carroll (University of Pennsylvania, Philadelphia, PA) in 2011.

[0072] Mouse Xenograph Model NSG mice were produced on the Genotoul Anexplo platform in Toulouse, France, using breeders obtained from Charles River Laboratories. The transplanted mice were treated with antibiotics (Baytril) throughout the experimental period. In experiments evaluating the response of the PDX model to chemotherapy, mice (6-9 weeks old) were administered busulfan (30 mg / kg) subpleurally 24 hours before infusion of leukemia cells. Leukemia samples were thawed in a 37°C water bath, washed with IMDM 20% FBS, and then treated with Hanks equilibrium salt solution (1-10 × 10⁶ 6 The cells were suspended at a final concentration of cells / 200 μL and injected into the tail vein of NSG mice. Eight to eighteen weeks after AML cell transplantation, when the mice had engrafted (examined by flow cytometry of peripheral blood or bone marrow aspirate), NSG mice were intraperitoneally administered 60 mg / kg of AraC or vehicle (PBS) daily for five days. AraC was supplied from the TUH pharmacy. On day eight, the mice were sacrificed and human leukemia cells were collected from the mouse bone marrow. For the AML cell line, the mice were treated with busulfan (20 mg / kg) 24 hours before injecting the leukemia cells. Subsequently, the cells were thawed and washed using the method described above, and 2 × 10⁶ cells per 200 μL were used. 6 After being suspended in HBSS at the final concentration, the cells were injected into the bloodstream of NSG mice. In experiments using inducible shRNA, doxycycline (0.2 mg / ml + 1% sucrose) was added to the drinking water from the day of cell injection or 10 days later until the end of the experiment. Mice were treated for 5 days with daily intraperitoneal injections of 30 mg / kg of AraC and sacrificed on the 8th day. Mice were monitored daily for disease symptoms (disheveled coat, hunchback, weakness, decreased motor skills), and the timing of euthanasia of injected animals showing signs of distress was determined.

[0073] Evaluation of leukocyte transfer After the experiment, NSG mice were humanely killed in accordance with European ethical guidelines. Bone marrow (a mixture from the tibia and femur) and spleen were dissected and washed with HBSS containing 1% FBS. MNCs from the bone marrow and spleen were labeled with anti-hCD33, anti-mCD45.1, anti-hCD45, anti-hCD3, and / or anti-hCD44 antibodies (all from BD), and the percentage of viable human blast cells (hCD3-hCD45+mCD45.1-hCD33+hCD44+AnnV- cells) was measured using flow cytometry. In some experiments, anti-CALCRL, anti-CD34, and anti-CD38 were also added to characterize AML stem cells. Monoclonal antibodies recognizing the extracellular domain of CALCRL were produced in-house with the cooperation of Biotem (France). The antibodies were then labeled with R-Phycoerythrin using the Lightning-Link kit (Expedeon). All antibodies were used at concentrations ranging from 1 / 50 to 1 / 200 depending on specificity and cell density. Analysis was performed using an LSRFortessa flow cytometer and DIVA software (BD Biosciences) or a CytoFLEX flow cytometer and CytoExpert software (Beckman Coulter). AML cell counts / μL in peripheral blood and AML cell counts in total tumor volume (bone marrow and spleen) were measured using CountBright beads (Invitrogen) according to the described protocol.

[0074] In the LDA experiment, a human transplant was considered positive if at least 0.1% of the cells in the mouse bone marrow were hCD45+mCD45.1-hCD33+. In AML#31, transplantation was measured based solely on hCD45+mCD45.1-, so the cutoff value increased to 0.5% or higher. Limiting dilution analysis was performed using ELDA software.

[0075] Western blot analysis Proteins were degraded using Bis-Tris gels for 4-12% polyacrylamide gel electrophoresis (Life Technology, Carlsbad, CA) and electrophoresed on nitrocellulose membranes. After blocking with Tris-buffered saline (TBS) 0.1%, Tween 20%, and 5% bovine serum albumin, immunostaining was performed overnight with an appropriate primary antibody, followed by incubation with a secondary antibody conjugated to HRP. Immunoreaction bands were visualized using enhanced chemiluminescence with a Syngene camera (ECL Supersignal West Pico; Thermo Fisher Scientific). Chemiluminescence signals were quantified using Syngene's GeneTools software.

[0076] Cell death assay After processing, 5.10 5 Individual cells were washed with PBS and resuspended in 200 μL of Annexin-V binding buffer (BD Biosciences). 2 μL of Annexin-V-FITC (BD Biosciences) and 7-amino-actinomycin D (7-AAD; Sigma Aldrich) were added, and the cells were treated at room temperature in the dark for 15 minutes. All samples were analyzed using an LSRFortessa or CytoFLEX flow cytometer.

[0077] Cell cycle analysis Cells were harvested, washed with PBS, and fixed at -20°C using ice-cold 70% ethanol. The cells were then permeabilized with 1×PBS containing 0.25% Triton X-100, resuspended in 1×PBS containing 10 μg / ml propidium iodide and 1 μg / ml RNase, and incubated at 37°C for 30 minutes. Data were collected using a CytoFLEX flow cytometer.

[0078] Chronogenic assay Primary cells from AML patients were thawed and resuspended in 100 μl of Nucleofector Kit V (Amaxa, Cologne, Germany). The cells were then nucleofected with 200 nM siRNA scramble (ON-TARGETplus Non-targeting siRNA #2, Dharmacon) or anti-CALCRL (SMARTpool ON-TARGETplus CALCRL siRNA, Dharmacon) according to the manufacturer's instructions (Program U-001 Amaxa, Cologne, Germany). The cells were then incubated in H4230 methylcellulose medium (STEMCELL Technologies) supplemented with 10% 5637-CM as a stimulant, reaching a final concentration of 1 × 10⁶. 5 After adjusting the cell / ml concentration, the cells were plated two per 35mm petri dish and grown in a humidified CO2 incubator (5% CO2, 37°C) for 7 days. On day 7, leukemia colonies (5 or more cells) were scored.

[0079] shRNA, lentivirus production and introduction into leukemia cells The shRNA sequence was constructed in pLKO-TET-ON or cloned into a pLKO vector. Each construct (6 μg) was co-transmitted into 293T cells using 20 μL of lipofectamine 2000 in a 10 cm dish, along with psPax2 (4 μg, providing the packaging protein) and pMD2.G (2 μg, providing the VSV-g envelope protein) plasmids, to generate lentiviral particles. 24 hours after cell transfection, the medium was removed and 10 ml of opti-MEM + 1% Pen / Strep was added. Approximately 72 hours after transfection, the 293T culture supernatant containing the lentiviral particles was collected, filtered, aliquoted, and stored in a -80°C freezer for future use. On the day of transduction, 2.106 cells were mixed with 2 ml of freshly thawed lentivirus, and the cells were infected with polyblen at a final concentration of 8 ug / ml. On the third day after infection, the introduced cells were selected using 1 μg / ml puromycin.

[0080] EC50 experiment The day before the experiment, the cells were 3 x 10 5 The final concentration of cells / ml was adjusted and plated into a 96-well plate (final volume: 100 μl). To measure the maximum inhibitory concentration (EC50), increased concentrations of AraC or idarubicin were added to the culture medium. After 2 days, 20 μl of MTS solution (Promega) was added per well, and after 2 hours, the absorbance at 490 nm was recorded using a 96-well plate reader. The dose (EC50) that reduces cell viability to 50% was analyzed using GraphPad Prism software with Nonlinear regression log [inhibitor] vs. normalized response-variable slope.

[0081] Measurement of oxygen consumption of AML cultured cells using the seahorse assay All XF assays were performed using the XFp Extracellular Flux Analyser (Seahorse Bioscience, North Billerica, MA). The day before the assay, the sensor cartridge was placed in the calibration buffer provided by Seahorse Bioscience and hydrated overnight. 50 μl of Cell-Tak (Corning, Cat#354240) solution at a concentration of 22.4 μg / ml was coated onto the wells of a Seahorse XFp microplate and stored overnight at 4°C. Subsequently, the Cell-Tak coated wells of the Seahorse microplate were washed with distilled water, and AML cells were plated at a density of 10⁵ cells per well using XF-based minimal DMEM medium containing 11 mM glucose, 1 mM pyruvate, and 2 mM glutamine. Then, 180 μl of XF-based minimal DMEM medium was added to each well, and the microplates were obtained by centrifugation at 80 g for 5 minutes. After incubation at 37°C for 1 hour in CO2-free air, the basal oxygen consumption rate (OCR, as an indicator of mitochondrial respiration) and extracellular acidification rate (ECAR, as an indicator of glycolysis) were measured using an XFp analyzer.

[0082] RNA microarray and bioinformatics analysis For primary AML samples, human CD45+CD33+ was isolated from engrafted BM mice (3 cases) treated with PBS or AraC using a cell sorter cytometer. RNA from AML cells was extracted using Trizol (Invitrogen) or RNeasy (Qiagen). For the MOLM-14 AML cell line, mRNA was extracted from 2,106 cells using RNeasy (Qiagen). RNA purity was monitored with a NanoDrop 1ND-1000 spectrophotometer, and RNA quality was evaluated with an Agilent 2100 Bionalyzer and RNA 6000 Nano assay kit. No RNA degradation or contamination was detected (RIN > 9). 100 ng of total RNA was extracted using an Affymetrix GeneChip. (c) On the Human Gene 2.0 ST Array, Affymetrix GeneChip (c)Analysis was performed using the WT Plus Reagent Kit, following the manufacturer's instructions (Manual Target Preparation for GeneChip® Whole Transcript (WT) Expression Arrays P / N 703174 Rev. 2). Arrays were washed and scanned, and the raw files generated by the scanner were transferred to R software for preprocessing (using the RMA function in the Oligo package), quality control (box plots, clustering, PCA), and differential expression analysis (using the eBayes function in the LIMMA package). Before expression analysis, all clusters of transcripts unrelated to the gene were removed. Transcription cluster-gene mapping was performed using annotations provided by Affymetrix (HuGene-2_0-st-v1.na36.hg19.transcript.csv) and the R / Bioconductor package hugene20sttranscriptcluster.db. The p-values ​​generated by the eBayes function were adjusted to suppress false discoveries using the Benajmin and Hochberg procedure. [RMA] Irizarry et al., Biostatistics, 2003; [Oligo package] Carvalho and Irizarry, Bioinformatics, 2010; [LIMMA reference] Ritchie et al., Nucleic Acids Research, 2015; hugene20sttranscriptcluster.db: MacDonald JW 2017, Affymetrix hugene20 annotation data (chip hugene20sttranscriptcluster); [FDR]: Benjamini et al., Journal of the Royal Statistical Society, 1995.

[0083] GSEA analysis GSEA analysis was performed using GSEA version 3.0 (Broad Institute). The gene signatures used in this study were obtained from the Broad Institute database, literature, or constructed in-house. The following parameters were used: number of permutations = 1000, permutation type = gene_set. Other parameters were left at their default values.

[0084] Quantification and statistical analysis Statistical analysis of the differences between the two sets of data was performed using a two-tailed (non-directional) Student's t-test with Welch's correction. The Log-rank (Mantel-Cox) test was used for survival analysis. In the Limit Dilution Assay experiment, frequency and statistical analysis were performed using L-calc software (Stemcell Technologies). A p-value less than 0.05 indicates significance. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ns, not significant. Detailed information on each test is provided in the legend of the figure.

[0085] result Leukemia stem cells have high expression of CALCRL, which is necessary for their maintenance. Using clinically relevant chemotherapy models, we previously showed that LSCs are not necessarily enriched in AraC-residual AML, suggesting that these cells are also targets for chemotherapy and that both chemosensible and chemosensible subpopulations exist (Farge et al.). To identify drug-resistant LSCs, we analyzed transcriptome data from three different studies: i) we identified 134 genes overexpressed in functionally defined LSCs compared to their normal hematopoietic stem cell counterparts (Eppert et al.); ii) a study that identified 114 genes associated with poor prognosis when overexpressed in AML (the Cancer Genome Atlas, AML cohort, 2013); and iii) a study that selected 536 genes overexpressed at relapse compared to pairwise-matched diagnoses after intensive chemotherapy (Hackl et al.). Surprisingly, we found one unique gene common to these three transcriptome databases: CALCRL, which encodes a G protein-coupled receptor and has not yet been described in cancer or AML. Using three independently published cohorts of AML patients (TCGA, AML cohort, GSE12417, GSE14468), we demonstrated that patients with high CALCRL expression had significantly lower overall survival and event-free survival. Furthermore, CALCRL gene expression was higher in AML cells from relapsed patients compared to cells matched at diagnosis, and even higher expression was observed in functionally and phenotypically defined LSC populations compared to normal in the leukemia compartment. Flow cytometry analysis using a monoclonal antibody developed by our laboratory revealed that CALCRL cell surface expression was significantly higher between leukemia bulk (n=37) and normal bulk (n=9) cells (Fold change, FC = 2.3), and that it was concentrated in the immature CD34+CD38- compartment of AML patients (FC AML CD34+CD38- vs AML bulk = 1.4, and FC AML CD34+CD38- vs normal CD34+CD38- = 2.6, respectively).Interestingly, the proportion of cells positive for this receptor was similar across all surveyed populations, indicating that CALCRL is overexpressed in a small population of AML cells. Next, a similar approach was taken to evaluate the expression of ADM, a previously reported ligand for CALCRL, in several cancer models. The results showed that the ADM gene was overexpressed in AML cells compared to normal cells, and that its gene expression remained unchanged even during relapse after chemotherapy in AML patients. Furthermore, using techniques such as RNA microarrays, confocal microscopy, and Western blotting, CALCRL, its three co-receptors RAMP1, RAMP2, RAMP3, and ADM (excluding another ligand, CGRP), were expressed in all cell lines tested, and that the CALCRL receptor was well present on the cell membrane of these cells. These observations were also confirmed in primary AML patient samples. Thus, intracellular expression of CALCRL in immature AML cells serves as a novel marker for LSCs and suggests a role for this receptor in LSC biology.

[0086] Next, we examined the impact of CALCRL and ADM protein levels on patient outcomes. IHC analysis confirmed that elevated CALCRL and ADM protein levels were associated with decreased complete remission rates, 5-year overall survival rates, and event-free survival rates in a cohort of 198 AML patients. Furthermore, when patients were classified into four groups based on their CALCRL and ADM expression levels (- / - vs - / + vs + / - vs + / +, data not shown), overall survival was significantly shortened in the CALCRL high / ADM high group, and high expression of either CALCRL or ADM alone dramatically reduced EFS and complete remission rates (data not shown). These data support the hypothesis that the ADM-CALCRL axis is activated autocrinely in AML and is associated with poor prognosis.

[0087] CALCRL is necessary for maintaining leukemia stem cells. Using the Gene Sheet Enrichment Analysis (GSEA) approach, we first confirmed that several LSC-related gene signatures (Eppert et al., 2011; Gentles, 2010; Ng et al., 2016) were enriched in AML patients with the highest CALCRL expression compared to AML patients with the lowest CALCRL expression. To specifically investigate the role of CALCRL in maintaining LSC function, we first performed ex vivo assays by knocking down CALCRL in primary AML samples and then injecting NSG immunodeficient mice with different doses of cells (Figure 1A). After 12 weeks, the mice were sacrificed, and the percentage of human cells in the animal bone marrow was evaluated. As a result, inactivating CALCRL significantly reduced the frequency of in vivo LSCs in secondary transplants (1 / 62,444 for siCTR vs. 1 / 525,000 for siCALCRL, Figure 1B), demonstrating its necessity in maintaining LSC function.

[0088] result CALCRL is essential for cell growth and survival both in vitro and in vivo. Since several GPCRs identified in AML, such as CXCR4 and GPR56, are involved in cell survival and proliferation (Chen et al. 2013), we investigated whether the ADM-CALCRL axis influences these characteristics. CALCRL was knocked down using shRNA in MOLM-14 and OCI-AML3 cell lines. The results showed that CALCRL deficiency suppressed blast cell proliferation, increased cell death, and cleaved caspase-3 and PARP, which are pro-apoptotic markers. Furthermore, shRNA targeting adrenomedullin phenocopyed the effects of shCALCRL on cell proliferation and apoptosis in MOLM-14 and OCI-AML3 cells. To confirm these results in vivo and maintain target inactivation over time, we developed a tetracycline-inducible shRNA model. First, we confirmed that CALCRL depletion, similar to the constitutive shRNA approach, is accompanied by decreased cell proliferation and increased apoptosis. After injecting AML cells into mice, RNA depletion was activated from day one by doxycycline (Figure 1C). On day 25 post-transplant, engraftment of human leukemia cells from the mouse bone marrow and spleen was evaluated using mCD45.1-hCD45+hCD33+AnV markers (Figure 1C). As a result, in mice injected with shCAL#1 and shCAL#2, the number of AML blasts was significantly lower than that of shCTR in both MOLM-14 (shCTR=13.9M vs shCAL#1=0.3M vs shCAL#2=0.1M, Figure 1D) and OCI-AML3 cells (shCTR=17.2M vs shCAL#1=2.0M vs shCAL#2=1.7M, Figure 1D). Thus, the mouse bone marrow was maintained by reducing the tumor burden of cells. Finally, knockdown of CALCRL significantly extended the survival time of mice (Figure 1E). To maximize the potential of this induceable model and enhance its clinical relevance, we evaluated the impact of CALCRL depletion on pre-existing diseases. shRNA expression was induced 10 days post-transplantation of shCTR or shCAL MOLM14 cells, after confirming that engraftment levels were comparable in both groups and after group randomization.In this established disease model, mice xenotransplanted with shCAL AML cells showed a significant reduction in myeloblasts compared to a cohort of shCTR mice with advanced disease. Furthermore, downregulating CALCRL greatly improved mouse survival. These results demonstrate that CALCRL is necessary for the proliferation and maintenance of AML cells in vivo.

[0089] Next, we examined the impact of CALCRL and ADM protein levels on patient outcomes. IHC analysis confirmed that elevated CALCRL and ADM protein levels were associated with decreased complete remission rates, 5-year overall survival rates, and event-free survival rates in a cohort of 198 AML patients. Furthermore, when patients were classified into four groups based on their CALCRL and ADM expression levels (- / - vs - / + vs + / - vs + / +, data not shown), overall survival was significantly shortened in the CALCRL high / ADM high group, and high expression of either CALCRL or ADM alone dramatically reduced EFS and complete remission rates (data not shown). These data support the hypothesis that the ADM-CALCRL axis is activated autocrinely in AML and is associated with poor prognosis.

[0090] CALCRL is necessary for maintaining leukemia stem cells. Using the Gene Sheet Enrichment Analysis (GSEA) approach, we first confirmed that several LSC-related gene signatures (Eppert et al., 2011; Gentles, 2010; Ng et al., 2016) were enriched in AML patients with the highest CALCRL expression compared to AML patients with the lowest CALCRL expression. To specifically investigate the role of CALCRL in maintaining LSC function, we first performed ex vivo assays by knocking down CALCRL in primary AML samples and then injecting NSG immunodeficient mice with different doses of cells (Figure 1A). After 12 weeks, the mice were sacrificed, and the percentage of human cells in the animal bone marrow was evaluated. As a result, inactivating CALCRL significantly reduced the frequency of in vivo LSCs in secondary transplants (1 / 62,444 for siCTR vs. 1 / 525,000 for siCALCRL, Figure 1B), demonstrating its necessity in maintaining LSC function.

[0091] CALCRL is essential for cell growth and survival both in vitro and in vivo. Since several GPCRs identified in AML, such as CXCR4 and GPR56, are involved in cell survival and proliferation (Chen et al. 2013), we investigated whether the ADM-CALCRL axis influences these characteristics. CALCRL was knocked down using shRNA in MOLM-14 and OCI-AML3 cell lines. The results showed that CALCRL deficiency suppressed blast cell proliferation, increased cell death, and cleaved caspase-3 and PARP, which are pro-apoptotic markers. Furthermore, shRNA targeting adrenomedullin phenocopyed the effects of shCALCRL on cell proliferation and apoptosis in MOLM-14 and OCI-AML3 cells. To confirm these results in vivo and maintain target inactivation over time, we developed a tetracycline-inducible shRNA model. First, we confirmed that CALCRL depletion, similar to the constitutive shRNA approach, is accompanied by decreased cell proliferation and increased apoptosis. After injecting AML cells into mice, doxycycline activated RNA depletion from day one (Figure 1C). On day 25 post-transplant, engraftment of human leukemia cells from the mouse bone marrow and spleen was evaluated using mCD45.1-hCD45+hCD33+AnV markers (Figure 1C). As a result, in mice injected with shCAL#1 and shCAL#2, the number of AML blasts was significantly lower than that of shCTR in both MOLM-14 (shCTR=13.9M vs shCAL#1=0.3M vs shCAL#2=0.1M, Figure 1D) and OCI-AML3 cells (shCTR=17.2M vs shCAL#1=2.0M vs shCAL#2=1.7M, Figure 1D). Thus, the mouse bone marrow was maintained by reducing the tumor burden of cells. Finally, knockdown of CALCRL significantly extended the survival time of mice (Figure 1E). To maximize the potential of this induceable model and enhance its clinical relevance, we evaluated the impact of CALCRL depletion on pre-existing diseases. shRNA expression was induced 10 days post-transplantation of shCTR or shCAL MOLM14 cells, after confirming that engraftment levels were comparable in both groups and after group randomization.In this established disease model, mice xenotransplanted with shCAL AML cells showed a significant reduction in myeloblasts compared to a cohort of shCTR mice with advanced disease. Furthermore, downregulating CALCRL greatly improved mouse survival. These results demonstrate that CALCRL is necessary for the proliferation and maintenance of AML cells in vivo.

[0092] CALCRL depletion leads to a decrease in cellular energy state, BCL2, cell cycle, and DNA repair pathways in AML. To elucidate the regulatory pathway downstream of CALCRL, we generated shCTR vs shCALCRL MOLM-14 cells and performed comparative transcriptome and functional assays. Interestingly, knockdown of CALCRL significantly decreased the expression of 623 genes and increased the expression of 278 genes (FDR < 0.05, p < 0.05). Since mitochondrial metabolism has emerged as a crucial regulator of cell proliferation and survival in AML (Scotland et al., 2013; Skrtic et al., 2015; Molina et al., 2018), we first analyzed the impact of CALCRL depletion on this pathway. GSEA results showed that gene signatures related to mitochondrial oxidative metabolism were significantly depleted in shCALCRL MOLM-14 cells. Furthermore, when OCR was measured, basal OCR decreased, but maximal respiration was maintained and reserve capacity increased. This suggests that cells may be able to promote mitochondrial utilization as needed. Furthermore, a significant decrease in mitochondrial ATP production and downregulation of the mitochondrial transcription factor TFAM were observed. Finally, in the absence of CALCRL, the basal energy state of cells was reduced, suggesting that shCALCRL cells transitioned from a proliferative state to a quiescent state. Consistent with this hypothesis, data mining analysis showed that genes involved in the cell cycle and DNA integrity pathways were significantly enriched in shCTR cells. Western blotting confirmed that CALCRL depletion affected RAD51 expression and the protein levels of CHEK1 and BCL2. This was associated with the accumulation of G0 / G1 phase cells. Interestingly, enrichment analysis revealed that CALCRL deficiency affected the genetic information of several transcription factors considered important cell cycle regulators, including E2F1, P53, and FOXM1.

[0093] Next, we focused on the E2F1 transcription factor, whose biological importance in LSCs derived from chronic myeloid leukemia has recently been discussed (Pellicano et al. 2018). We first confirmed that CALCRL depletion is closely associated with a significant decrease in E2F1 activity. We then demonstrated that E2F1 knockdown affects the protein expression of RAD51 and CHK1 in both MOLM-14 and OCI-AML3, inhibiting cell proliferation and cell cycle progression, and inducing high levels of cell death. Furthermore, E2F1 depletion affected the energy state of cells and mitochondria. These results strongly suggest that E2F1 is downstream of CALCRL and governs cell proliferation, survival, and metabolism. Next, we investigated whether CALCRL controls the proliferation of primary AML cells. Interestingly, we first confirmed that CALCRL protein levels are positively correlated with clonal ability in methylcellulose. As expected, deficiency of CALCRL reduced the number of colonies and decreased the protein levels of BCL2 and RAD51. These results suggest that CALCRL is involved in the proliferation of AML blast cells and controls important pathways involved in the DNA repair process.

[0094] CALCRL downregulation sensitizes leukemia cells to the chemotherapy drugs cytarabine and idarubicin. Considering proteins positively regulated by CALCRL, such as BCL2, CHK1, or FOXM1 (David et al., 2016; Khan et al., 2017; Konopleva et al., 2016), we hypothesized that CALCRL is involved in the chemotherapy resistance process. Therefore, CALCRL depletion sensitized MOLM-14 and OCI-AML3 cells to cytarabine and idarubicin, resulting in increased cell viability, induction of cell death, and increased cleavage of apoptotic proteins CASPASE-3 and PARP. Furthermore, we demonstrated that cells were still sensitized to the compounds even after depletion of ADM and E2F1. This indicates that the ADM-CALCRL-E2F1 axis is involved in in vitro drug resistance. To confirm these results in vivo, we used a model of inducible shRNA targeting CALCRL. After verifying in vitro that these inducible shRNAs closely replicated the sensitization observed with constitutive shRNAs, MOLM-14 cells expressing shCTR or shCAL#2 were injected into the blood of NSG mice. Ten days later, shRNA expression was activated, and the mice were administered 30 mg / kg / day of cytarabine for five days (Figure 2A). As a result, the combination of AraC and shCALCRL significantly reduced the total blast count compared to shCTR+ / AraC and shCALCRL alone (Figure 2B), induced cell death at a higher rate, and extended the survival time of the mice (Figure 2C). Furthermore, MOLM-14 cells expressing shCTR and treated with either the vehicle or AraC were sorted by flow cytometry and plated in vitro for further experiments. Interestingly, after one week of culture, cells from AraC-treated mice showed greater resistance to AraC (EC50: 2.238 μM in the vehicle group vs. 6.712 μM in the AraC-treated group) and idarubicin (EC50: 28.64 nM in the vehicle group vs. 60.55 nM in the AraC-treated group). These results strongly suggest that a resistance mechanism common to both drugs persists over time. Next, we showed that in vivo, cells treated with AraC not only had elevated levels of CALCRL protein expression, but also slightly increased RAD51 and BCL2, while CHK1 remained similar to that of untreated cells. To evaluate the role of CALCRL in this in vivo acquired chemotherapy resistance, we attempted to deplete CALCRL in these cells. As a result, knockdown of CALCRL with two types of shRNA sensitized both vehicle-treated and AraC-treated cells to AraC and idarubicin. Surprisingly, the EC50 levels of AraC and idarubicin when shCALCRL #1 and #2 were introduced into AraC-treated cells were similar to those observed when shCTR was introduced into MOLM-14 cells from vehicle-treated mice. This suggests that the increase in CALCRL and its downstream signaling pathways can partially, if not fully, explain the chemotherapy resistance of cells. These results clearly demonstrate that CALCRL promotes chemotherapy resistance in AML cells.

[0095] CALCRL-dependent BCL2 expression is necessary to maintain a high oxo state and resistance to chemotherapeutic agents. We previously suggested that after AraC (cytarabine) treatment, residual cells exhibited impaired oxidative metabolism, and that targeting mitochondria in combination with conventional chemotherapy could be a groundbreaking treatment for AML (Farge et al.). Since CALCRL depletion in our cells reduced oxidative metabolism, we evaluated the cellular energy state in relation to AraC. As a result, knockdown of CALCRL significantly counteracted the increase in basal respiratory volume, maximal respiratory volume, or reserve capacity caused by AraC. Furthermore, we confirmed that mitochondrial ATP production decreased in response to AraC, but ECAR was unaffected. Based on these results, we focused on BCL2. BCL2 has already been reported to be involved in the regulation of drug resistance and the oxidative state of AML cells. First, we showed that overexpression of BCL2 in MOLM-14 did not alter OCR, mitochondrial ATP production, or ECAR, suggesting that basal levels of BCL2 do not need to be involved in the regulation of mitochondrial energy state. However, when BCL2 was overexpressed in response to AraC treatment, it was confirmed that it was sufficient to rescue reserve capacity similar to maximal respiratory volume, but basal respiratory volume was not rescued. From this, it is thought that the CALCRL-BCL2 axis plays a role in maintaining the mitochondrial response capacity to AraC. Furthermore, mitochondrial ATP production and ECAR were unaffected, indicating that it was unrelated to energy production. Finally, BCL2 overexpression completely inhibited basal apoptosis induced by CALCRL depletion and concomitant use with AraC or idarubicin.

[0096] Chemotherapy is selected for CALCRL-positive leukemia stem cells. We addressed the role of CALCRL in chemotherapy for primary AML samples using a clinically appropriate PDX model of AraC-treated mice (Farge et al., 2017). After injecting primary cells into the blood of NSG mice and confirming engraftment, the mice were treated with 60 mg / kg / day of AraC for 5 days and sacrificed on day 8 to examine minimal residual disease (Figure 3A). Ten PDXs were tested and ranked as low response (FC Vehicle / AraC <10) or high response (FC>10) according to their responsiveness to AraC (Figure 3B). The percentage of CALCRL-positive cells was twice as high in the low response group (3.578% vs. 7.786%), and there was an inverse linear correlation between the percentage of positive cells and tumor reduction rate (R2=0.418) (Figures 3C and 3D). Furthermore, the proportion of CALCRL-positive blasts was significantly increased in cells with minimal residual disease (5.6% vs. 23%), and this was observed in all subgroups examined. On the other hand, the average fluorescence intensity of CALCRL-positive cells remained largely constant. These results suggest that chemotherapy selectively targets CALCRL-positive cells, and that the CALCRL expression level in blast cells actually increased. To further investigate the role of CALCRL in chemotherapy from the perspective of LSCs, we used an approach that combined single-cell RNA-seq (scRNA-seq) assays at diagnosis, relapse, and after injection into immunodeficient mice, with measurements of stem cell frequencies of CALCRL+ and CALCRL- cells. First, scRNA-seq analysis at diagnosis detected two cell clusters with different gene expression signatures. Since only cluster 1 was present after injection into mice, it means that only cells with this gene signature could be transplanted into NSG mice. Furthermore, GSEA analysis showed that this cluster was rich in the CALCRL_UP (as previously defined in our transcriptome) and LSC+_UP (as functionally defined by Eppert et al.) genes. At the time of relapse, only cluster 1' (associated with cluster 1) was present and enriched in the CALCRL_UP, LSC+_UP, and Relapse_UP (Hackl et al.) genes. Finally, in an LDA study conducted by sorting CALCRL- and CALCRL+ cell populations at diagnosis and relapse and then injecting them into mice, it was shown that the CALCRL+ cell population was significantly richer in stem cells at diagnosis compared to the CALCRL- cell population. At the time of relapse, an overall increase in the frequency of LSCs was observed in both the CALCRL- and CALCRL+ cell populations, suggesting that both populations acquired the stem cell phenotype.

[0097] Recently, Shlush et al. proposed an elegant model of relapse consisting of two scenarios. The first model is called “relapse origin-primitive” (ROp), where relapse arises from rare LSC clones detected only after HSPC or xenotransplantation. The second model is called “relapse origin-committed” (ROc), where relapsed clones arise from immunophenotypically committed leukemia cells, and bulk cells retain a stem-like transcriptional profile (Shlush et al.). We analyzed this transcriptome database and confirmed that at diagnosis, CALCRL expression was higher in ROc-phenotypic blasts than in ROp-phenotypic blasts, consistent with CALCRL expression in cells possessing stem cell characteristics (data not shown). Interestingly, CALCRL was strongly elevated at relapse in ROp patients, which correlated with the emergence of stem cell-characterized clones at this stage of the disease (data not shown). These observations supported our hypothesis that there is a pre-existing population of rare (ROp) or abundant (ROc) relapse-associated LSCs expressing high levels of CALCRL.

[0098] Another challenge was to clarify the role of CALCRL in the chemotherapy resistance of LSCs. Primary AML cells were injected into NSG mice, engrafted, treated with AraC, and then human cells were selected. Before reinjection into mice, the cells were transfected with siCTR or siCALCRL, and the frequency of stem cells was examined (Figure 4A). As a result, it was confirmed that the frequency of stem cells was significantly reduced with siCALCRL compared to siCTR in both bone marrow and spleen (Figure 4B). Furthermore, a significant reduction was observed in the AraC-administered condition compared to the vehicle-administered condition, suggesting that the remaining chemoresistant stem cells are highly dependent on CALCRL. These results strongly support the hypothesis that chemotherapy selects a cell population rich in LSCs and positive for CALCRL.

[0099] Consideration Because LSC populations exhibit high plasticity and heterogeneity not only in phenotype (Taussig et al., 2010; Eppert et al., 2011; Sarry et al., 2011) but also in drug sensitivity (Farge et al., 2017; Boyd et al., 2018), the clinical efficacy of LSC-selective targeted therapy has not been demonstrated in the treatment of AML. However, basic research focusing on the essential characteristics of this cell population, such as resistance to chemotherapy, is critically necessary for the development of improved and more specific therapies in AML.

[0100] This study provides important insights into the biology and drug resistance of hematopoietic stem cells and reveals that the ADM receptor CALCRL is a master regulator of hematopoietic stem cells. Building on Eppert's research functionally characterizing LSCs, this study is the first to reveal that the CALCRL gene is overexpressed in the leukemia compartment compared to normal cells. CALCRL may be specifically upregulated by LSC-related transcription factors such as HIF1α and ATF4 (Wang et al., 2011; van Galen et al., 2018). In fact, both ADM and CALCRL have a consensus hypoxia-response element (HRE) in their 5' flanking region and are genes regulated by HIF1α (Nikitenko et al., 2003). More recently, it has been revealed that the integrated stress response and the transcription factor ATF4 are involved in the proliferation of AML cells and are specifically activated in HSCs and LSCs (van Galen et al., 2018; Heydt et al., 2018). Interestingly, the maintenance of HSCs in mice under proliferative stress, rather than in a steady state, is dependent on CALCRL signaling (Suekane et al., 2019). Therefore, CALCRL may support hematopoiesis in leukemia and overcome the stress induced by the high proliferation rate of AML cells.

[0101] The results of this study clearly demonstrate that targeting CALCRL expression affects genes related to clonal capacity, cell cycle progression, and DNA repair and genomic stability. While cancer stem cells and LSCs are primarily conserved from chemotherapy by being quiescent, recent studies have suggested that LSCs also exhibit a more active circulating phenotype (Iwasaki et al., 2015; Pei et al., 2018). The C-type lectin CD93 is expressed in a subset of actively circulating, non-quiescent AML cells rich in LSC activity (Iwasaki et al., 2015). Recently, Pei et al. showed that targeting the AMPK-FIS1 axis inhibits mitophagy, induces cell cycle arrest in AML, and depletes the potential of LSCs in primary AML. These results are consistent with the existence of subpopulations of LSCs with different proliferation states. Furthermore, removal of FIS1 induces decreased expression of several genes (CCND2, CDC25A, PLK1, CENPO, AURKB, etc.) as well as the E2F1 gene signature, which was also observed after CALCRL knockdown. Recently, it has been proposed that E2F1 plays a crucial role in regulating the proliferation and survival status of CML stem / progenitor cells (Pellicano et al., 2018). Several signaling pathways, such as MAPK, CDK / cyclin, or PI3K / AKT, have been described as being stimulated by the ADM / CALCRL axis and may regulate the activity of the pRB / E2F1 complex (Hallstrom et al., 2008; Wang et al., 1998). Other signal mediators activated by LSCs, such as c-Myc and CEBPα, regulate E2F1 transcription and enable interaction between the E2F1 protein and the E2F gene promoter, activating genes essential for DNA replication, cell proliferation, and survival in G1 / S AML (Leung et al., 2008; O'Donnell et al., 2005; Rishi et al., 2014). Thus, analyzing intracellular signaling downstream of CALCRL has revealed novel pathways important for LSC maintenance and chemotherapy resistance.

[0102] Identifying the characteristics of R-LSCs (reactive low-scaling cells) that are present at relapse and have disappeared due to chemotherapy is necessary to develop new therapeutic strategies to eradicate R-LSCs. Boyd et al. proposed the existence of Leukemic Regenerating Cells (LRCs), a transient state during the immediate and acute response to AraC (Boyd et al., 2018), which are responsible for disease regeneration by delaying the recovery of the LSC pool. In this intriguing model and the dynamics of MRD after chemotherapy, CALCRL-positive AML cells are part of this LRC subpopulation, and CALCRL is essential for maintaining the LSC potential of chemotherapy-resistant primary AML. It is interesting to clarify whether only CALCRL-positive cells are saved by chemotherapy, or whether it induces an adaptive response to stress that increases CALCRL expression. It is necessary to identify transcription factors that are activated in response to chemotherapy and deepen our understanding of the acute response to chemotherapy. Therefore, CALCRL-targeted therapies should be clinically investigated to specifically eradicate MRD in AML and prevent relapse. Finally, since several molecules that prevent the binding of the neuropeptide CGRP to CALCRL are being developed for the treatment of other diseases (Hutchings et al., 2017; Schuster and Rapoport, 2017), this will facilitate future pharmacological approaches to antagonize the ADM-CALCRL axis in AML.

[0103] Based on the above, our data clearly demonstrate that CALCRL is a novel stem cell actor necessary for maintaining AML development in vivo. This receptor regulates genes involved in chemoresistance mechanisms, and deficiency of this receptor leads to sensitization of AML cells to both cytarabine and anthracyclines in vitro and in vivo. This suggests that LSCs resistant to these drugs share a common activation pathway involved in these resistance mechanisms. These results strongly suggest that CALCRL is a novel and promising therapeutic target candidate for anti-LSC therapy.

[0104] References Throughout this application, various references describe the state of the art to which the present invention relates. The disclosures of these documents are incorporated into this disclosure by reference.

[0105] [Table 2] TIFF0007897700000003.tif230165

[0106] [Table 3]

[0107] [Table 4] TIFF0007897700000006.tif205165

Claims

1. A pharmaceutical composition for use in a method for treating chemotherapy-resistant acute myeloid leukemia (AML) in patients in need thereof, comprising administering to the patient a therapeutically effective dose of an antibody that specifically binds to CALCRL, the pharmaceutical composition comprising an antibody that specifically binds to CALCRL.

2. The pharmaceutical composition according to claim 1, wherein the antibody is a chimeric antibody, a humanized antibody, or a human antibody.

3. The pharmaceutical composition according to claim 1, wherein the antibody mediates antibody-dependent cell-mediated cytotoxicity.

4. The pharmaceutical composition according to claim 3, wherein the antibody is an IgG1 antibody.

5. The pharmaceutical composition according to claim 1, wherein the antibody mediates complement-dependent cytotoxicity.

6. The pharmaceutical composition according to claim 1, wherein the antibody mediates antibody-dependent phagocytosis.

7. The pharmaceutical composition according to claim 1, wherein the antibody is a multispecific antibody comprising a first antigen-binding site directed toward CALCRL and at least one second antigen-binding site directed toward effector cells.

8. The pharmaceutical composition according to claim 1, wherein the antibody is conjugated to the cytotoxic portion.

9. A pharmaceutical composition for use in a method for treating chemotherapy-resistant acute myeloid leukemia (AML) in a patient in need thereof, comprising administering a therapeutically effective amount of a CALCRL activity or expression inhibitor to the patient, wherein the pharmaceutical composition comprises a CALCRL activity or expression inhibitor, wherein the CALCRL activity or expression inhibitor is an antibody against CALCRL, RAMP2, RAMP3 or adrenomedullin, or an siRNA, antisense oligonucleotide or ribozyme that inhibits the expression of CALCRL, RAMP2 or RAMP3.

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