CD123 and CD200 as markers for the diagnosis and immuneradication of leukemia stem cells (LSC)

Abstract

The present invention relates to an antigen-binding molecule that is at least bispecific for and specifically binds to CD200 and CD123 on the cell surface of leukemia stem cells (LSCs). The present invention further relates to a method for identifying such an antigen-binding molecule, and to the use of the antigen-binding molecule for the diagnosis and treatment of leukemia such as AML or CML, particularly their pediatric forms.

Description

Technical Field

[0001] The present invention relates to antigen-binding molecules that bind at least bispecifically and specifically to CD200 and CD123 on the cell surface of leukemia stem cells (LSCs). The present invention further relates to methods for identifying such antigen-binding molecules, and to the use of antigen-binding molecules for the diagnosis and treatment of leukemias such as AML or CML, particularly their pediatric forms.

Background Art

[0002] Cancer is a major lethal disease for humans, and is caused by physiologically uncontrolled cell proliferation that affects the normal physiological symptoms of the human body, often resulting in severe pathological reactions leading to death. Although great efforts have been made in cancer research and treatment, currently cancer remains the main cause of death in humans. There are multiple approaches for treating cancer patients, including surgery, radiotherapy and chemotherapy.

[0003] Despite significant progress in risk-adapted chemotherapy, the treatment of acute myeloid leukemia (AML) remains challenging (Dohner H, Estey E, Grimwade D, Amadori S, Appelbaum FR, Buchner T, et al. Diagnosis and management of AML in adults: 2017 ELN recommendations from an international expert panel. Blood. 2017;129:424-447. The relative 5-year survival probability is only 30% (Acute Myeloid Leukemia - cancer stat facts. [Cited December 6, 2021]. Available from: https: / / seer.cancer.gov / statfacts / html / amyl.html). Dormant "leukemic stem cells" (LSC) can survive cytotoxic therapy and thus cause relapse (Shlush LI, Mitchell A, Heisler L, Abelson S, Ng SWK, Trotman-Grant A, et al. Tracing the origins of relapse in acute myeloid leukaemia to stem cells. Nature. 2017;547:104-108). Targeted immunotherapy could be an effective option for eliminating chemotherapy-resistant leukemic stem cells.

[0004] In acute lymphoblastic B-cell leukemia (B-ALL), so-called "bispecific T-cell engaging antibodies constructs" and chimeric antigen receptor (CAR) T cells have been used with some success (Mohanty R, Chowdhury CR, Arega S, Sen P, Ganguly P, Ganguly N. CAR T cell therapy: A new era for cancer treatment (Review). Oncol Rep. 2019;42:2183-2195; Halford Z, Coalter C, Gresham V, Brown T. A Systematic Review of Blinatumomab in the Treatment of Acute Lymphoblastic Leukemia: Engaging an Old Problem With New Solutions. Ann Pharmacother. 2021;55:1236-1253).

[0005] In the case of CD19, there is a target protein that is strongly overexpressed on leukemic B cells, while healthy non-B cells are negative for the marker (Ghorashian S, Pule M, Amrolia P. CD19 chimeric antigen receptor T cell therapy for haematological malignancies. Br J Haematol. 2015;169:463-478). Since CD19 is also expressed on normal B cells, normal B lineage cells are also eliminated after CD19-CAR-T infusion. This phenomenon is typically called the "on-target off-tumor effect". Subsequent B cell aplasia results in persistent hypogammaglobulinemia, and intermittent immunoglobulin replacement is sometimes required to prevent severe infections.

[0006] Nevertheless, in the case of AML, an “ideal” target does not yet exist. All known targets overexpressed on AML stem cells, such as CD33, CD123, or CLL-1, can also be found on other and healthy cells. Therapeutic approaches to some of these targets have also led to severe off-target toxicity (Kenderian SS, Ruella M, Shestova O, Klichinsky M, Aikawa V, Morrissette JJD, et al. CD33-specific chimeric antigen receptor T cells exhibit potent preclinical activity against human acute myeloid leukemia. Leukemia. 2015;29:1637-1647; Tashiro H, Sauer T, Shum T, Parikh K, Mamonkin M, Omer B, et al. Treatment of Acute Myeloid Leukemia with T Cells Expressing Chimeric Antigen Receptors Directed to C-type Lectin-like Molecule 1. Mol Ther. 2017;25:2202-2213; Gill S, Tasian SK, Ruella M, Shestova O, Li Y, Porter DL, et al. Preclinical targeting of human acute myeloid leukemia and myeloablation using chimeric antigen receptor-modified T cells. Blood. 2014;123:2343-2354).

[0007] Leukemia stem cells (LSCs) in acute myeloid leukemia (AML) play important roles in the initiation and progression of leukemia and were considered to be the origin of chemotherapy drug resistance and disease recurrence. The identification and targeting of LSCs rely on membrane markers such as CD34, CD38, CD123, TIM3, CD25, CD32, and CD96. Furthermore, transcription factors are also therapeutic targets in the eradication of LSCs, such as histone deacetylase (HDAC), NF-κB, HIF-1α, and β-catenin. In addition to membrane markers and transcription factors, intracellular reactive oxygen species (ROS), telomerase, and microRNA have been identified as new targets for removing LSCs in AML (Ding Y, Gao H, Zhang Q. The biomarkers of leukemia stem cells in acute myeloid leukemia. Stem Cell Investig. 2017;4:19. Published 2017 Mar 2. doi:10.21037 / sci.2017.02.10).

[0008] In acute myeloid leukemia (AML), leukemia stem cells (LSCs) lie at the root of death, but their low abundance and high similarity to healthy hematopoietic stem cells (HSCs) make them difficult to isolate (Velten, L., Story, B.A., Hernandez-Malmierca, P. et al. Identification of leukemic and pre-leukemic stem cells by clonal tracking from single-cell transcriptomics. Nat Commun 12, 1366 (2021). https: / / doi.org / 10.1038 / s41467-021-21650-1).

[0009] Haubner, S. et al. (in: Coexpression profile of leukemic stem cell markers for combinatorial targeted therapy in AML. Leukemia 33, 64 - 74 (2019). https: / / doi.org / 10.1038 / s41375 - 018 - 0180 - 3) describe that targeted immunotherapy in acute myeloid leukemia (AML) is challenged by the lack of AML - specific target antigens and clonal heterogeneity, leading to unwanted off - target off - leukemic virus and the risk of relapse from minor clones. They hypothesize that combinatorial targeting of AML cells can enhance therapeutic efficacy without increasing toxicity. To identify combinations of target antigens specific to AML and leukemic stem cells, they generated detailed protein expression profiles based on flow cytometry of primary AML (n = 356) and normal (n = 34) bone marrow samples, and a recently reported integrated normal tissue proteomics dataset. They analyzed antigen expression levels of CD33, CD123, CLL1, TIM3, CD244, and CD7 on AML bulk and leukemic stem cells at initial diagnosis (n = 302) and relapse (n = 54). CD33, CD123, CLL1, TIM3, and CD244 were ubiquitously expressed on AML bulk cells at initial diagnosis and relapse, regardless of genetic characteristics. For each target analyzed, they found further expression in different populations of normal hematopoiesis. Analyzing the co - expression of their six targets in all double combinations (n = 15), they found that CD33 / TIM3 and CLL1 / TIM3 were highly positive in AML compared to normal hematopoietic and non - hematopoietic tissues. They propose that combinatorial targeting of CD33 / TIM3 or CLL1 / TIM3 can enhance therapeutic efficacy in AML immunotherapy without worsening toxicity.

[0010] Targeted identification and elimination of AML stem cells remains an unmet need in the treatment of leukemia. There is also a continuing need in the art to identify agents having therapeutic potential for the treatment of proliferative disorders such as AML and for improved specific diagnostic applications. Identification of these agents is urgently needed to enable further development of therapies from the laboratory to clinical practice. SUMMARY OF THE INVENTION

[0011] Elements of the invention are described below. These elements are listed with specific embodiments, but it should be understood that these elements can be combined in any manner and in any number to create additional embodiments. The various examples and preferred embodiments described should not be construed as limiting the invention to only the explicitly described embodiments. This description is to be understood as supporting and encompassing embodiments that combine two or more of the explicitly described embodiments, or embodiments that combine one or more of the explicitly described embodiments with any number of disclosed and / or preferred elements. Furthermore, any permutation and combination of all elements described in this application should be considered to be disclosed by the description of this application, unless the context indicates otherwise.

[0012] In a first aspect of the invention, the above problem is solved by a method or in vitro method for identifying at least a bispecific antigen-binding molecule that specifically binds to mammalian leukemia stem cells (LSCs), the method comprising: a) providing a first library of candidate antigen-binding molecules specific for protein CD123 or an immunologically recognizable fragment or derivative thereof; a') providing a second library of candidate antigen-binding molecules specific for protein CD200 or an immunologically recognizable fragment or derivative thereof; b) Combining a number of candidate antigen-binding molecules from said first and second libraries to generate a library of candidate molecules that are at least bispecific for CD123 and CD200; c) Providing a suitable screening system comprising said protein CD123 and / or CD200, or an immunologically recognizable fragment or derivative thereof; d) Contacting the library of b) with the screening system of c); e) Identifying from the library of b) binding molecules that specifically bind to said proteins CD123 and CD200, or an immunologically recognizable fragment or derivative thereof. It includes.

[0013] Preferably, the LSC is selected from LSCs in acute myeloid leukemia (AML) or chronic myeloid leukemia (CML).

[0014] The above problem is solved, in a second aspect of the present invention, by at least a bispecific antigen-binding molecule that specifically binds to the proteins CD123 and CD200 of mammalian leukemia stem cells (LSCs), or an immunologically recognizable fragment or derivative thereof, identified by the method according to the present invention.

[0015] In its third aspect, the present invention relates to a pharmaceutical composition comprising at least a bispecific antigen-binding molecule according to the present invention together with a pharmaceutically acceptable carrier and / or adjuvant.

[0016] The above problem is solved, in a fourth aspect of the present invention, by at least a bispecific antigen-binding molecule according to the present invention or a pharmaceutical composition according to the present invention for use in medicine, preferably for the prevention or treatment of cancer such as leukemia, for example AML or CML, and especially its pediatric form, etc. in a patient, and comprises administering to the patient an effective amount of at least a bispecific antigen-binding molecule according to the present invention or a pharmaceutical composition according to the present invention.

[0017] In a fifth aspect of the present invention, the above problem is solved by a method for preventing or treating cancer such as leukemia, for example AML or CML, particularly its pediatric form, etc. in a patient, which comprises administering to the patient an effective amount of at least a bispecific antigen-binding molecule according to the present invention or a pharmaceutical composition according to the present invention.

[0018] In a sixth aspect of the present invention, the above problem is a method for identifying mammalian leukemia stem cells (LSCs) in a biological sample obtained from a mammalian patient suspected of having leukemia, comprising: a) contacting the sample with at least a bispecific antigen-binding molecule of the present invention; b) detecting the binding of at least the bispecific antigen-binding molecule to cells in the sample; and the binding of at least the bispecific antigen-binding molecule to cells, particularly specific binding, identifies leukemia stem cells (LSCs) in the sample, and is solved by the method.

[0019] In a seventh aspect of the present invention, the above problem is a method for diagnosing leukemia in a mammalian patient suspected of having leukemia, the method comprising the method of the present invention and further concluding leukemia in the mammalian patient based on the binding. Preferably, the leukemia is relapsed, malignant and / or metastatic leukemia, AML, particularly its pediatric form, and CML.

[0020] As described above, the present invention in its first aspect relates to an in vitro method for identifying at least a bispecific antigen-binding molecule that specifically binds to mammalian leukemia stem cells (LSCs), the method comprising: a) providing a first library of candidate antigen-binding molecules specific for protein CD123 or an immunologically recognizable fragment or derivative thereof; a') providing a second library of candidate antigen-binding molecules specific for protein CD200 or an immunologically recognizable fragment or derivative thereof; b) Combining a number of candidate antigen-binding molecules from said first and second libraries to generate a library of candidate molecules that are at least bispecific for CD123 and CD200; c) Providing a suitable screening system comprising said protein CD123 and / or CD200, or an immunologically recognizable fragment or derivative thereof; d) Contacting the library of b) with the screening system of c); e) Identifying from the library of b) binding molecules that specifically bind to said protein CD123 and CD200, or an immunologically recognizable fragment or derivative thereof comprising.

[0021] The method according to the invention is preferred when the LSC is selected from LSCs in acute myeloid leukemia (AML) or chronic myeloid leukemia (CML).

[0022] The present invention generally is based on the use of the target proteins differentiation cluster (“CD”) CD123 and CD200 because both markers are co-expressed in leukemia stem cells, particularly AML stem cells.

[0023] CD200 (also known as OX-2) is a membrane protein of the "Ig superfamily" (IgSF) and is expressed in several cell types (Barclay AN, Clark MJ, McCaughan GW. Neuronal / lymphoid membrane glycoprotein MRC OX-2 is a member of the immunoglobulin superfamily with a light-chain-like structure. Biochem Soc Symp. 1986;51:149-157). The CD200 receptor (CD200R) can be found on hematopoietic cells (T, B, and NK cells, as well as myeloid lineage immune cells). Binding of the receptor to CD200 inhibits these immune cells (Wright GJ, Cherwinski H, Foster-Cuevas M, Brooke G, Puklavec MJ, Bigler M, et al. Characterization of the CD200 receptor family in mice and humans and their interactions with CD200. J Immunol. 2003;171:3034-3046).In many AML patients, CD200 is overexpressed on malignant cells and contributes to the "immune evasion" of LSCs (Herbrich S, Baggerly K, Alatrash G, Davis RE, Konopleva MY. CD200 Is a Stem Cell-Specific Immunosuppressive Target in AML. 2018;132:2768-2768; Kawasaki BT, Mistree T, Hurt EM, Kalathur M, Farrar WL. Co-expression of the toleragenic glycoprotein, CD200, with markers for cancer stem cells. Biochem Biophys Res Commun. 2007;364:778-782; Kawasaki BT, Farrar WL. Cancer stem cells, CD200 and immunoevasion. Trends Immunol. 2008;29:464-468; Coles SJ, Wang ECY, Man S, Hills RK, Burnett AK, Tonks A, et al. CD200 expression suppresses natural killer cell function and directly inhibits patient anti-tumor response in acute myeloid leukemia. Leukemia. 2011;25:792-799). A therapeutic approach using CD200 as a checkpoint inhibitor has been argued to be promising (Rastogi N, Baker S, Man S, Uger RA, Wong M, Coles SJ, et al. Use of an anti-CD200-blocking antibody improves immune responses to AML in vitro and in vivo. Br J Haematol. 2020. doi:10.1111 / bjh.17125).A therapeutic antibody against CD200 (samalizumab) is currently being tested in a Phase I clinical trial in patients with chronic lymphocytic leukemia (CLL) and multiple myeloma (MM) (Mahadevan D, Lanasa MC, Farber C, Pandey M, Whelden M, Faas SJ, et al. Phase I study of samalizumab in chronic lymphocytic leukemia and multiple myeloma: blockade of the immune checkpoint CD200. J Immunother Cancer. 2019;7:227). Therefore, CD200 was selected as the first target in the combinatorial approach according to the present invention.

[0024] CD123 (IL3RA) is a surface marker on hematopoietic progenitor cells and is strongly overexpressed in AML-stem cell leukemia (SCL). This correlates with an increase in the proliferation of AML cells (see also below for diagnostic methods). This effect can be explained by IL3-mediated signaling, but the mechanism of the increased resistance of CD123-positive AML cells to chemotherapy is not fully understood (Yan B, Chen Q, Shimada K, Tang M, Li H, Gurumurthy A, et al. Histone deacetylase inhibitor targets CD123 / CD47-positive cells and reverse chemoresistance phenotype in acute myeloid leukemia. Leukemia. 2019;33:931-944; Jordan CT, Upchurch D, Szilvassy SJ, Guzman ML, Howard DS, Pettigrew AL, et al. The interleukin-3 receptor alpha chain is a unique marker for human acute myelogenous leukemia stem cells. Leukemia. 2000;14:1777-1784; Testa U, Pelosi E, Frankel A. CD 123 is a membrane biomarker and a therapeutic target in hematologic malignancies. Biomark Res. 2014;2:4).

[0025] Several clinical trials are investigating the use of various CD123-targeted agents, including chimeric antigen receptor-modified T cells (CD123 expression, monoclonal antibodies, composite CD3-CD123 dual-affinity retargeting antibody therapy, recombinant fusion proteins, and CD123-engager T cells) (El Achi H, Dupont E, Paul S, Khoury JD. CD123 as a Biomarker in Hematolymphoid Malignancies: Principles of Detection and Targeted Therapies. Cancers (Basel). 2020;12(11):3087. Published 2020 Oct 23. doi:10.3390 / cancers12113087). Examples are CD123 neutralizing antibodies, CD3×CD123 bispecific antibodies, dual-affinity retargeting (DART) and CAR-T cells (Shi M, Su RJ, Parmar K-P, Chaudhry R, Sun K, Rao J, et al. CD123: A Novel Biomarker for Diagnosis and Treatment of Leukemia. Cardiovasc Hematol Disord Drug Targets. 2019;19:195-204; Search of: CD123. [cited 6 Dec 2021]. Available: https: / / clinicaltrials.gov / ct2 / results?cond=aml&term=CD123&cntry=&state=&city=&dist=). The experimental antibody-drug conjugate SGN-CD123A targets CD123 as a possible treatment for AML.

[0026] CD123 has been disclosed as a second diagnostic target (Kandeel EZ, Madney Y, Eldin DN, Shafik NF. Overexpression of CD200 and CD123 is a major influential factor in the clinical course of pediatric acute myeloid leukemia. Exp Mol Pathol. 2021;118:104597; Coustan-Smith E, Song G, Shurtleff S, Yeoh AE-J, Chng WJ, Chen SP, et al. Universal monitoring of minimal residual disease in acute myeloid leukemia. JCI Insight. 2018;3. doi:10.1172 / jci.insight.98561).

[0027] Therefore, both target proteins not only show strong overexpression in AML-LSCs, but are also more important for successful immune evasion and increased cancer cell proliferation. Since both target proteins serve as markers and contribute to the malignancy of leukemia such as AML, the possibility of the highly undesirable "loss of target", which is common in other respects, is low.

[0028] Taking the above into consideration, the inventors investigated an advantageous combined approach against the target proteins CD123 and CD200, generating and testing a new recombinant bispecific antigen-binding agent, particularly a trifab-contorsbody.

[0029] Kandeel EZ, et al. (In PMID:33358743, Epub 2020 Dec 23, doi:10.1016 / j.yexmp.2020.104597, Exp Mol Pathol. 2021 Feb;118:104597) disclosed that acute myeloid leukemia (AML) accounts for approximately 20% of all pediatric acute leukemias. The outcome of AML remains unsatisfactory. CD123 and CD200 have been demonstrated to play important roles in hematological malignancies. Both CD200 and CD123 have adverse effects on clinical symptoms and treatment outcomes, and the situation deteriorates significantly when both are overexpressed simultaneously. Therefore, CD200 and CD123 can be used as markers for minimal residual disease in AML. They also speculated about their use as therapeutic targets, but there is no further information on this.

[0030] In the context of the present invention, the term "CD123" generally refers to the interleukin-3 receptor (CD123) protein, subunit α, a molecule found on cells that helps transmit the signal of interleukin-3, a soluble cytokine important in the immune system. This term shall include not only the human form of this protein, but also its homologs, particularly in mice or rats. Information on CD123 can be obtained from the Human Gene Name web page with accession number HGNC:6012 (https: / / www.genenames.org / ). The CD123 protein is accessible at the UniProt web page with accession number P26951, and further, at least the human version as shown in SEQ ID NO:1 (isoform 1). All other isoforms of the protein are also intended to be encompassed by the present invention.

[0031] In the context of the present invention, the term "CD200" generally refers to the CD200 molecular protein, also commonly known as the OX-2 membrane glycoprotein. This term shall include not only the human form of this protein, but also its homologs, particularly in mice or rats. Information regarding CD200 can be obtained from the Human Gene Nomenclature Committee (HGNC) gene name web page with accession number HGNC:7203 (https: / / www.genenames.org / ). The CD200 protein is accessible on the UniProt web page with accession number P41217 (isoform 1), and furthermore, is at least in the human form as shown in SEQ ID NO:2. All other isoforms of the protein are also intended to be encompassed by the present invention.

[0032] Fragments, derivatives, or variants of CD200 and / or CD123 can be readily tested for their ability to bind to the antigen-binding agents according to the present invention. Accordingly, the present invention also discloses a method for determining whether protein derivatives, variants, and / or fragments of CD200 and / or CD123 maintain the ability to interact (bind) with the antigen-binding agents according to the present invention and thus constitute immunologically recognizable fragments. This method comprises contacting candidate derivatives, variants, and / or fragments of CD200 and / or CD123 with the antigen-binding molecules of the present invention and detecting their interaction, i.e., binding. The above method for testing protein variants / derivatives and / or fragments may, in some embodiments, form part of the aforementioned method for identification, preferably when it involves the use of any protein derivatives, variants, and / or fragments of CD123 and / or CD200. These fragments may be included in the screening systems used in the context of the present invention.

[0033] Since the CD123 protein is composed of three extracellular domains (287 amino acids), a single transmembrane domain (30 amino acids), and a short intracellular region (53 amino acids), the preferred fragment for use in the context of the present invention is the extracellular portion, which can be used as a soluble protein and can further be labeled and / or recombinantly produced.

[0034] CD200 is composed of two extracellular (one variable and one constant) immunoglobulin-like domains, a single transmembrane region, and a cytoplasmic tail. Thus, the preferred fragment for use in the context of the present invention is the extracellular portion, which can be used as a soluble protein and can further be labeled and / or recombinantly produced.

[0035] In the context of the present invention, the terms "CD123" and / or "CD200" also include other proteins, their variants or fragments when they have amino acids with at least 50, 60, 70, 80, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity compared to the amino acid sequences of human CD123 or CD200 disclosed herein, for example SEQ ID NO: 1 or 2 respectively. Fragments of CD123 or CD200 or their variants preferably have a length of at least 30, 40, 50, 80, 100, 150, 200, 300 or more amino acids. Variants can include conservative amino acid changes, as known to those skilled in the art.

[0036] As used herein, the term "identical" or "identity" percent, when used in the context of two or more nucleic acid or protein / polypeptide sequences, refers to two or more sequences or subsequences having (or at least having) a specified percentage of amino acid residues or nucleotides that are the same (i.e., when measured using a sequence comparison algorithm or by manual alignment and visual inspection (see, e.g., the NCBI website), are the same or (i.e., when compared over a comparison window or specified region and aligned to maximize identity) are about 60% identical or at least about 60% identical over the specified region, preferably over their full-length sequences, at 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93% or 94% identity, or at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93% or 94% identity, more preferably about 95%, 96%, 97%, 98%, 99% or more identity, or at least about 95%, 96%, 97%, 98%, 99% or more identity). For amino acid identity in particular, use of BLASTP 2.2.28+ with the following parameters is included: Matrix: BLOSUM62; Gap Penalty: existence: 11, extension: 1; Neighborhood Threshold: 11; Window for Multiple Hits: 40.

[0037] In the method according to the invention, any suitable library of candidate antigen-binding molecules can be used that enables the selection of domains involved in antigen recognition for use as binding moieties in the design of bispecific diagnostic and therapeutic agents. Preferably, the library is selected from an antibody library or a phage display library, more preferably a human-based or humanized-based library. Examples of domains involved in antigen recognition include domain antibodies (heavy chain variable domains (V HH:heavy chain-only variable domain)), single-chain Fv fragments (scFv), antigen-binding fragments (Fab), single-chain Fab fragments (scFab), and more recently single-chain Fc fragments (scFc), preferably single-chain variable fragments (scFv) or stabilized forms thereof (e.g., disulfide-stabilized scFv), Fab fragments, Fab' fragments, F(ab') fragments, F(ab')2 fragments, scFab, and heavy chain variable domains (VHH), single-domain antibodies (sdAb), or single-domain antibody fragments such as nanobodies. Further, a TCR recognition domain, i.e., a construct containing the essential CDRs of the TCR that form loops protruding from the core Vα or Vβ domain and mediate specific binding, may be included.

[0038] Once appropriate binding domains / molecules are identified (e.g., by additional antigen-binding assays and / or sequencing of the respective nucleic acid molecules as disclosed herein), these are combined into appropriate bispecific constructs for use in connection with the present invention. The present invention relates to methods according to the present invention where the combination comprises a covalent or non-covalent fusion or combination of candidate antigen-binding molecules from the first and second libraries. The combination may include additional steps of cloning or otherwise inserting the binding domain / molecule into an appropriate scaffold structure (e.g., synthesis of a polypeptide). Each method is described in the literature and well known to those skilled in the art.

[0039] At least bispecific binding agents for CD123 and CD200 can be in any suitable format that enables binding of CD123 and CD200, preferably simultaneous binding, in diagnostic assays, screening assays and / or therapeutic applications as disclosed herein. A library of candidate molecules that are at least bispecific binding agents for CD123 and CD200, i.e., the expected "final product", is a TrioMab, IgG-like, CrossMab, 2:1 CrossMab, 2:2 CrossMab, DuoBody, DVD-Ig, scFv-IgG, IgG-IgG, Fab-scFv-Fc, TF, ADAPTIR, BITE, BITE-Fc, DART, DART-FC, tetravalent DART, TandAb, ImmTAC, TriKE, scFv-scFv-scFv, trispecific nanobody, diabody, triabody, tetrabody or higher-order multimer, and trifab-contobody (see Figures 1 and 2, and Suurs FV, Lub-de Hooge MN, de Vries EGE, de Groot DJA. A review of bispecific antibodies and antibody constructs in oncology and clinical challenges. Pharmacol Ther. 2019 Sep;201:103-119.doi:10.1016 / j.pharmthera.2019.04.006.Epub 2019 Apr 24.PMID:31028837), and a method according to the invention comprising at least one bispecific format selected from the group consisting of is preferred.

[0040] More preferably, the method according to the invention is such that the candidate molecules of the library further comprise at least 1 / 3 antigen-binding domains, such as a CD3-binding domain. This enables additional functionality, especially in immunotherapy or diagnostic applications, but the binder can also be included for the purpose of binding to a matrix or other protein or protein complex. Even more preferred examples are specific binding agents for CD33, TIM3 and / or CLL1, or additional T cell-specific antigens such as CD4, CD8 or CD28.

[0041] A method according to the present invention is more preferred, wherein the candidate molecules of the library as described above further comprise an Fc fragment or a variant or inactivated derivative / version thereof, for example, an Fc fragment lacking cysteine residues and / or cysteine bridges, and / or an Fc fragment comprising a "knob" or "hole" structure.

[0042] Particularly preferred constructs / molecules such as those generated constitute at least bispecific binding agents against CD123 and CD200 in the form of trifab - contosobody (see, for example, Figure 2 and European Patent No. 3704146). The trifab molecule contains two complementary halves and is a T - cell activating antibody format specifically directed against a combination of antigens. Each half of the molecule consists of a light and a heavy antibody chain. Furthermore, the construct contains a so - called "dummy" chain. As in the case of a normal IgG antibody, the light and heavy chains having "variable light" (V L ) and "variable heavy" (V H ) chain domains constitute the antigen - specific binding domains. The Fc fragment (crystallizable fragment) is modified as follows; the cysteine does not bind to another heavy chain, and the CD2 domain is replaced in the first half by the V L of an anti - CD3 antibody and in the second half by the V Hhas been replaced by (Mayer K, Baumann A-L, Grote M, Seeber S, Kettenberger H, Breuer S, et al. TriFabs--Trivalent IgG-Shaped Bispecific Antibody Derivatives: Design, Generation, Characterization and Application for Targeted Payload Delivery. Int J Mol Sci. 2015;16:27497-27507). Furthermore, a knobs-into-holes system has been introduced to avoid dimerization of two identical halves (Ridgway JB, Presta LG, Carter P. "Knobs-into-holes" engineering of antibody CH3 domains for heavy chain heterodimerization. Protein Eng. 1996;9:617-621). Therefore, the halves may be designated as "knob" or "hole", respectively. For the modification, the Fc fragment is inactivated. The "dummy" chains are complementary to their respective Fc fragments but cannot generate an active anti-CD3 domain.

[0043] If only half of the construct binds to a cell expressing only one target, i.e., either CD123 or CD200, the T cell is not activated. Chain exchange thermodynamically promoted by the charge in the Fc fragment occurs only when both target structures are recognized on the cell surface. Reshuffling results in the removal of both "dummy chains", but a functional anti-CD3 domain is formed and thus conditionally ("only in that case") activates the T cell. Therefore, the desired cytotoxic activity is limited and localizes only to cells expressing CD200 and CD123.

[0044] The LSCs can be identified from any mammal having LSCs that express CD200 and CD123 or homologs thereof. Preferred methods according to the invention are those in which the mammal is selected from mice, monkeys, dogs, cats, rats or humans, preferably human children.

[0045] The method according to the invention comprises a suitable system for identifying and / or screening the binding of antigen-binding molecules of a library. The system used according to the invention can be any suitable means, especially for specifically identifying, for identifying the binding of an antigen-binding molecule to either CD123 or CD200 or an antigenic fragment thereof, and can include it. Preferably, such a system comprises cells, such as bacterial or yeast cells or human cells, that express CD123 and CD200 or an antigenic fragment thereof, especially recombinantly. The system can be a cell-free system comprising the antigenic fragment in solution or bound or conjugated to a binder and / or CD123 or CD200 or a matrix. The system can further comprise a suitable reporter moiety or label. Preferred antigenic fragments for use in connection with the present invention are the extracellular portions of CD123 or CD200 (see also above) that can be used as soluble proteins or attached to a matrix, and can further be labeled and / or recombinantly produced. Preferred methods according to the invention are those in which the candidate molecules of the library further comprise at least one marker or label, such as a toxin, an enzyme, or a fluorophore.

[0046] Furthermore, the invention in its second aspect relates to at least a bispecific antigen-binding molecule that specifically binds to the proteins CD123 and CD200 of mammalian leukemia stem cells (LSCs) identified by the method according to the invention or immunologically recognizable fragments or derivatives thereof. The at least bispecific binding agent for CD123 and CD200 can be in any suitable format that enables binding of CD123 and CD200, preferably simultaneous binding, in the diagnostic assays, screening assays and / or therapeutic uses disclosed herein, preferably TrioMab, IgG-like, CrossMab, 2:1 CrossMab, 2:2 CrossMab, DuoBody, DVD-Ig, scFv-IgG, IgG-IgG, Fab-scFv-Fc, TF, ADAPTIR, BITE, BITE-Fc, DART, DART-FC, tetravalent DART, TandAb, ImmTAC, TriKE, scFv-scFv-scFv, trispecific nanobody, diabody, triabody, tetrabody or higher-order multimer, and trifab-contobody (see FIGS. 1 and 2, and Suurs FV, Lub-de Hooge MN, de Vries EGE, de Groot DJA. A review of bispecific antibodies and antibody constructs in oncology and clinical challenges. Pharmacol Ther. 2019 Sep;201:103-119. doi:10.1016 / j.pharmthera.2019.04.006. Epub 2019 Apr 24. PMID:31028837). Furthermore, a construct can be included that contains the TCR recognition domain, i.e., a loop-forming loop protruding from the core Vα or Vβ domain and containing the essential CDRs of the TCR that mediate specific binding.

[0047] Preferred is at least a bispecific antigen-binding molecule according to the invention, wherein its antigen-binding domains are covalently or non-covalently bound.

[0048] More preferably, the at least bispecific antigen-binding molecule according to the invention, wherein the candidate molecule of the library further comprises at least 1 / 3 of the antigen-binding domain, such as the CD3-binding domain. This enables additional functions, particularly in immunotherapy or diagnostic applications, but can also include binding agents for the purpose of binding to a matrix or other protein or protein complex. Even more preferred examples are specific binding agents for CD33, TIM3 and / or CLL1, or additional T cell-specific antigens such as CD4, CD8 or CD28.

[0049] Particularly preferred constructs / molecules such as those produced constitute at least bispecific binding agents for CD123 and CD200 in the form of the trifab - contosobody described above. Another particularly preferred construct / molecule produced constitutes at least a bispecific binding agent for CD123 and CD200 that binds simultaneously to CD123 and CD200 on the surface of LSCs.

[0050] Another particularly preferred embodiment of the construct / molecule produced according to the invention constitutes at least a bispecific binding agent for CD123 and CD200 upon binding and inhibits the biological function, such as its signaling, of CD123, CD200 or both in cells such as LSCs.

[0051] More preferably, the at least bispecific antigen-binding molecule according to the invention, wherein the molecule of the library as described above further comprises an Fc fragment or a mutant or inactivated derivative / version thereof, such as an Fc fragment lacking cysteine residues and / or cysteine bridges, and / or an Fc fragment comprising a "knob" or "hole" structure.

[0052] Preferably, the at least bispecific antigen-binding molecule according to the invention, wherein the LSC is selected from LSCs in acute myeloid leukemia (AML) or chronic myeloid leukemia (CML).

[0053] Preferred are at least bispecific antigen-binding molecules according to the invention, wherein the candidate molecules of the library further comprise at least one marker or label such as a toxin, an enzyme, or a fluorophore.

[0054] Preferred examples are at least bispecific antigen-binding molecules according to the invention, wherein the amino acid sequence of the CD123-binding region is as disclosed in European Patent Application Publication No. 2778175 A1 (incorporated herein by reference in its entirety) or is at least 95% identical to the disclosed sequence, and the amino acid sequence of the CD200-binding region is in accordance with European Patent Application Publication No. 2178561 A1 (incorporated herein by reference in its entirety) or is at least 95% identical to the disclosed sequence.

[0055] Another preferred example is at least a bispecific antigen-binding molecule according to the invention, wherein the amino acid sequence of the light chain variable region (V L ) for binding to CD123 comprises the amino acid sequence according to SEQ ID NO: 3, and the amino acid sequence of the heavy chain variable region (V H ) for binding to CD123 comprises the amino acid sequence according to SEQ ID NO: 4. Optionally, the heavy chain constant region comprises the amino acid substitutions S239D and I332E according to the Kabat EU numbering system or a specific binding sequence that is at least 95% identical to its sequence.

[0056] Yet another preferred example is at least a bispecific antigen-binding molecule according to the invention, wherein the amino acid sequence of the light chain for binding to CD123 comprises the amino acid sequence according to SEQ ID NO: 5, and the amino acid sequence of the heavy chain for binding to CD123 comprises the amino acid sequence according to SEQ ID NO: 6 or a specifically binding sequence that is at least 95% identical to those sequences.

[0057] Yet another preferred example is at least a bispecific antigen-binding molecule according to the invention, wherein the amino acid sequence of its CD200-binding fragment comprises one of the following sets of CDR pairs: (i) HCDR1 containing the amino acid sequence: GFNIKDYYMH (SEQ ID NO: 7); HCDR2 containing the amino acid sequence: WIDPENGDTKYAPKFQG (SEQ ID NO: 8); HCDR3 containing the amino acid sequence: KNYYVSNYNFFDV (SEQ ID NO: 9); LCDR1 containing the amino acid sequence: SASSSVRYMY (SEQ ID NO: 10); LCDR2 containing the amino acid sequence: DTSKLAS (SEQ ID NO: 11); and LCDR3 containing the amino acid sequence: FQGSGYPLT (SEQ ID NO: 12); (ii) HCDR1 containing the amino acid sequence: GFNIKDYYIH (SEQ ID NO: 13); HCDR2 containing the amino acid sequence: WIDPEIGATKYVPKFQG (SEQ ID NO: 14); HCDR3 containing the amino acid sequence: LYGNYDRYYAMDY (SEQ ID NO: 15); LCDR1 containing the amino acid sequence: KASQNVRTAVA (SEQ ID NO: 16); LCDR2 containing the amino acid sequence: LASNRHT (SEQ ID NO: 17); and LCDR3 containing the amino acid sequence: LQHWNYPLT (SEQ ID NO: 18); (iii) HCDR1 containing the amino acid sequence: GYSFTDYIIL (SEQ ID NO: 19); HCDR2 containing the amino acid sequence: HIDPYYGSSNYNLKFKG (SEQ ID NO: 20); HCDR3 containing the amino acid sequence: SKRDYFDY (SEQ ID NO: 21); LCDR1 containing the amino acid sequence: KASQDINSYLS (SEQ ID NO: 22); LCDR2 containing the amino acid sequence: RANRLVD (SEQ ID NO: 23); and LCDR3 containing the amino acid sequence: LQYDEFPYT (SEQ ID NO: 24); (iv) HCDR1 containing the amino acid sequence: GYTFTEYTMH (SEQ ID NO: 25); HCDR2 containing the amino acid sequence: GVNPNNGGALYNQKFKG (SEQ ID NO: 26); HCDR3 containing the amino acid sequence: RSNYRYDDAMDY (SEQ ID NO: 27); LCDR1 containing the amino acid sequence: KSSQSLLDIDEKTYLN (SEQ ID NO: 28); LCDR2 containing the amino acid sequence: LVSKLDS (SEQ ID NO: 29); and LCDR3 containing the amino acid sequence: WQGTHFPQT (SEQ ID NO: 30); or (v) Amino acid sequences: HCDR1 containing the amino acid sequence AFNIKDHYMH (SEQ ID NO: 31); HCDR2 containing the amino acid sequence WIDPESGDTEYAPKFQG (SEQ ID NO: 32); HCDR3 containing the amino acid sequence FNGYQALDQ (SEQ ID NO: 33); LCDR1 containing the amino acid sequence TASSSVSSSYLH (SEQ ID NO: 34); LCDR2 containing the amino acid sequence STSNLAS (SEQ ID NO: 35); and LCDR3 containing the amino acid sequence RQYHRSPPIFT (SEQ ID NO: 36), or sequences that specifically bind and are at least 95% identical to the above sequences, preferably at least 96%, 97%, 98% or 99% identical to the above sequences.

[0058] In the above, HCDR indicates the heavy-chain complementarity-determining region of the antigen-binding molecule binding domain, and LCDR indicates the light-chain complementarity-determining region of the antigen-binding molecule binding domain.

[0059] Combinations of sequences as disclosed above, i.e., sequences selected from SEQ ID NOs: 3 - 6, sequences combined with sequences selected from SEQ ID NOs: 7 - 12, 13 - 18, 19 - 24, 25 - 30 and 31 - 36, or sequences that specifically bind and are at least 95% identical to those sequences, are more preferred for the at least bispecific antigen-binding molecule according to the present invention.

[0060] The at least bispecific antigen-binding molecule according to the present invention is preferred, wherein the sequences used, such as the binding sequences, are selected from mouse sequences, chimeric sequences, humanized sequences and fully human sequences.

[0061] Yet another aspect of the present invention relates to a method for manufacturing a pharmaceutical composition, which includes formulating at least a bispecific antigen-binding molecule according to the present invention, which has been identified, as a pharmaceutical composition together with a pharmaceutically acceptable carrier and / or excipient.

[0062] Yet another aspect of the invention relates to a pharmaceutical composition comprising at least a bispecific antigen-binding molecule according to the invention together with a pharmaceutically acceptable carrier and / or adjuvant. Preferably, the pharmaceutical composition is suitable for immunotherapy of leukemia, particularly AML and its pediatric forms.

[0063] As used herein, the term “pharmaceutically acceptable” carrier, stabilizer or excipient is intended to include any and all solvents, solubilizers, fillers, stabilizers, binders, absorbers, bases, buffers, lubricants, controlled release vehicles, diluents, emulsifiers, humectants, dispersion media, coatings, antibacterial or antifungal agents, isotonic and absorption delaying agents, and the like that are compatible with pharmaceutical administration. The use of such media and agents for pharmaceutical active substances is well known in the art. The use thereof in the present composition is contemplated, except in cases where any conventional media or agent is incompatible with the active compound. Adjuvants can also be incorporated into the composition.

[0064] The pharmaceutical compositions of the invention are formulated to be compatible with their intended route of administration. Examples of routes of administration include parenteral, for example intrathecal, intraarterial, intravenous, intradermal, subcutaneous, oral, transdermal (topical) and transmucosal administration.

[0065] The pharmaceutical composition to be used may contain a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers or excipients include diluents (fillers, extenders, such as lactose, microcrystalline cellulose), disintegrants (such as sodium starch glycolate, croscarmellose sodium), binders (such as PVP, HPMC), lubricants (such as magnesium stearate), glidants (such as colloidal SiO2), solvents / co-solvents (such as aqueous vehicles, propylene glycol, glycerol), buffers (such as citrate, gluconate, lactate), preservatives (such as sodium benzoate, parabens (Me, Pr and Bu), BKC), antioxidants (such as BHT, BHA, ascorbic acid), wetting agents (such as polysorbate, sorbitan esters), thickeners (such as methyl cellulose or hydroxyethyl cellulose), sweeteners (such as sorbitol, saccharin, aspartame, acesulfame), flavoring agents (such as peppermint, lemon oil, butterscotch, etc.), humectants (such as propylene, glycol, glycerol, sorbitol). Other suitable pharmaceutically acceptable excipients include, inter alia, Remington's Pharmaceutical Sciences, 15 th Ed., Mack Publishing Co., New Jersey (1991) and Bauer et al., Pharmazeutische Technologic, 5 th Ed., Govi-Verlag Frankfurt (1997). A person skilled in the art knows the appropriate formulations for each compound, for example for topical use, and can easily select the appropriate pharmaceutically acceptable carrier or excipient according to, for example, the formulation and administration route of the pharmaceutical composition.

[0066] Yet another aspect of the present invention relates to at least a bispecific antigen-binding molecule according to the present invention or a pharmaceutical composition according to the present invention for use in medicine. It comprises administering to a patient in need thereof an effective amount of at least a bispecific antigen-binding molecule according to the present invention or a pharmaceutical composition according to the present invention, wherein the patient is selected from a mouse, a monkey, a dog, a cat, a rat or a human, preferably a human child, and is for the prevention or treatment of cancer in a patient, such as leukemia, for example AML or CML, especially its pediatric form. A bispecific antigen-binding molecule according to the present invention or a pharmaceutical composition according to the present invention is preferred.

[0067] "Treatment" or "treating" means any treatment of a disease or disorder in a mammal, including preventing or defending against a disease or disorder, i.e., not developing the clinical symptoms of the disease; inhibiting the disease, i.e., stopping or suppressing the occurrence of clinical symptoms; and / or alleviating the disease, i.e., causing regression of clinical symptoms.

[0068] A bispecific antigen-binding molecule or pharmaceutical composition for use according to the present invention is preferred, wherein the prevention or treatment of the cancer comprises immunotherapy and / or comprises avoidance of target loss and / or metastasis of the cancer.

[0069] Yet another aspect of the present invention relates to a method for preventing or treating cancer in a patient, such as leukemia, for example AML or CML, especially its pediatric form, comprising administering to the patient an effective amount of at least a bispecific antigen-binding molecule according to the present invention or a pharmaceutical composition according to the present invention. As described above, the patient can be selected from leukemia patients selected from a mouse, a monkey, a dog, a cat, a rat, or a human, preferably a human child.

[0070] A method according to the present invention is preferred, wherein the prevention or treatment of the cancer comprises immunotherapy and / or comprises avoidance of target loss and / or metastasis of the cancer as described herein.

[0071] Methods for preventing or treating diseases using bispecific antigen-binding agents are known to those skilled in the art and are disclosed, for example, in Ma et al. (Bispecific Antibodies: From Research to Clinical Application. Front Immunol. 2021 May 5;12:626616. doi: 10.3389 / fimmu.2021.626616. PMID: 34025638; PMCID: PMC8131538).

[0072] In another important aspect of the present invention, the above object is a method for identifying mammalian leukemia stem cells (LSCs) in a biological sample obtained from a mammalian patient suspected of having leukemia, comprising: a) contacting the sample with at least a bispecific antigen-binding molecule of the present invention; b) detecting the binding of at least a bispecific antigen-binding molecule to cells in the sample; and the binding, particularly specific binding and / or co-binding, of at least a bispecific antigen-binding molecule to CD123 and CD200 on the cells identifies / shows leukemia stem cells (LSCs) in the sample, which is solved by the method.

[0073] In this aspect, at least a bispecific antigen-binding molecule according to the present invention is used to identify leukemia stem cells (LSCs) in a sample. This method can be carried out in vitro, in vivo or ex vivo. The sample can be any suitable sample containing or suspected of containing LSCs, such as a sample containing blood cells or a sample of a hematopoietic organ, such as bone marrow (BM), lymph nodes and spleen.

[0074] A method for identifying mammalian leukemia stem cells (LSCs) in a biological sample according to the present invention is preferred, wherein the at least bispecific antigen-binding molecule comprises at least one detectable marker, enzyme or label such as a toxin, enzyme or fluorophore. These markers, enzymes or labels enable the detection of the antigen-binding molecule when bound to the surface of LSCs, i.e., CD123 and CD200.

[0075] A method for identifying mammalian leukemia stem cells (LSCs) in a biological sample according to the present invention is preferred, wherein the at least bispecific antigen-binding molecule binds simultaneously to CD123 and CD200. The step of identification may further include the identification of additional suitable markers of LSCs as described above.

[0076] A particular preferred embodiment of the method according to the present invention comprises a trifab construct as described above, which can identify the simultaneous binding of LSCs to CD123 and CD200 using CD3 activity (e.g., T cell activation).

[0077] In another important aspect of the present invention, the above object is achieved by a method for diagnosing (LSC-related) leukemia in a mammalian patient suspected of having leukemia, the method according to the present invention, and further concluding leukemia in the mammalian patient based on binding. Preferably, the method according to the present invention further includes diagnosing whether the leukemia is a recurrent, malignant and / or metastatic leukemia, AML, particularly its pediatric form, and CML. The diagnosis may include quantifying LSCs in a sample derived from the patient. Since both CD200 and CD123 have an adverse effect on clinical findings and treatment outcome, and the co-overexpression of both significantly worsens the condition, CD200 and CD123 can therefore be used as prognostic markers for minimal residual disease or the risk of poor disease status and metastasis in AML, and for predicting a worse outcome for cells carrying a greater amount of CD200 and CD123 detected in the patient. The markers CD200 and CD123 can also be used as markers for the stemness of LSCs. The markers CD200 and CD123 can also be used to stratify AML patients.

[0078] The above method can also be carried out in the context of monitoring the prevention or treatment of leukemia in a patient, such as AML or CML, and particularly its pediatric form, etc., a method for identifying or diagnosing a sample derived from the patient as described above during the course of treatment, and comparing the results with a control sample, particularly a sample derived from the patient taken at a time point before the prevention or treatment of leukemia in the patient, such as AML or CML, and particularly its pediatric form, etc. Prevention or treatment preferably includes administering to the patient an effective amount of at least a bispecific antigen-binding molecule according to the present invention or a pharmaceutical composition according to the present invention.

[0079] In yet another aspect of the invention, at least a bispecific antigen-binding molecule according to the invention is used for purifying and / or isolating LSCs expressing CD123 and CD200. Preferably, the at least bispecific antigen-binding molecule simultaneously attaches to CD123 and CD200 and binds to a solid matrix such as a column material, beads and / or a membrane. After contacting the LSCs in a sample with at least a bispecific antigen-binding molecule according to the invention, the LSCs via CD123 and CD200 attach to the at least bispecific antigen-binding molecule and, after a washing and elution step, can be isolated as a fraction enriched in LSCs that can be further used in the establishment or study of cell lines.

[0080] The inventors designed and studied a combined antigen binder for the target structures CD123 and CD200 and thus developed a new method for eliminating leukemia stem cells. CD123 / CD200-dependent and preferably localized activation of T cells is an improved strategy for immunotherapy of leukemia, such as pediatric AML.

[0081] The invention preferably relates to the following items.

[0082] Item 1. A method or in vitro method for identifying at least a bispecific antigen-binding molecule that specifically binds to mammalian leukemia stem cells (LSCs), a) providing a first library of candidate antigen-binding molecules specific for the protein CD123 or an immunologically recognizable fragment or derivative thereof; a') providing a second library of candidate antigen-binding molecules specific for the protein CD200 or an immunologically recognizable fragment or derivative thereof; b) combining a plurality of candidate antigen-binding molecules from the first and second libraries to generate a library of candidate molecules that are at least bispecific for CD123 and CD200; c) providing an appropriate screening system comprising said protein CD123 and / or CD200, or an immunologically recognizable fragment or derivative thereof; d) contacting the library of b) with the screening system of c); e) identifying from the library of b) a binding molecule that specifically binds to said proteins CD123 and CD200, or an immunologically recognizable fragment or derivative thereof A method or in vitro method comprising the steps of.

[0083] Item 2. The method according to item 1, wherein the LSC is selected from LSCs in acute myeloid leukemia (AML) or chronic myeloid leukemia (CML).

[0084] Item 3. The library of candidate antigen-binding molecules is selected from an antibody library or a phage display library, preferably a single-chain variable fragment (scFv) or a stabilized form thereof (e.g., disulfide-stabilized scFv), Fab fragment, Fab' fragment, F(ab') fragment, F(ab')2 fragment, scFab, and a single-domain antibody fragment such as a heavy-chain variable domain (VHH), single-domain antibody (sdAb), or nanobody. The method according to any one of items 1 to 3.

[0085] Item 4. The method according to any one of items 1 to 3, wherein said combining comprises a covalent or non-covalent fusion or combination of the candidate antigen-binding molecules from the first and second libraries.

[0086] The method according to any one of items 1 to 4, wherein said library of candidate molecules that are at least bispecific binding agents for item 5. CD123 and CD200 comprises at least one bispecific format selected from the group consisting of TrioMab, IgG-like, CrossMab, 2:1 CrossMab, 2:2 CrossMab, DuoBody, DVD-Ig, scFv-IgG, IgG-IgG, Fab-scFv-Fc, TF, ADAPTIR, BITE, BITE-Fc, DART, DART-FC, tetravalent DART, TandAb, ImmTAC, TriKE, scFv-scFv-scFv, trispecific nanobody, diabody, tribody, tetrabody or higher order multimer, and trifab-contobody.

[0087] Item 6. The method according to any one of items 1 to 5, wherein said candidate molecule of said library further comprises at least 1 / 3 antigen-binding domains, such as a CD3-binding domain.

[0088] Item 7. The method according to any one of items 1 to 6, wherein said candidate molecule of said library further comprises an Fc fragment or a mutated or inactivated version thereof, such as an Fc fragment lacking cysteine residues and / or cysteine bridges, or an Fc fragment containing a knob or hole structure.

[0089] Item 8. The method according to any one of items 1 to 6, wherein said mammal is selected from mouse, monkey, dog, cat, rat, or human, preferably a human child.

[0090] Item 9. The method according to any one of items 1 to 7, wherein said suitable screening system comprises cells that express CD123 and CD200 or an antigenic fragment thereof, particularly recombinantly express, or is a cell-free system.

[0091] Item 10. The method according to any one of items 1 to 9, wherein said candidate molecule of said library further comprises at least one marker or label.

[0092] Item 11. At least a bispecific antigen-binding molecule that specifically binds to the proteins CD123 and CD200 of mammalian leukemia stem cells (LSCs) or immunologically recognizable fragments or derivatives thereof, identified by the method according to any one of Items 1 to 10.

[0093] Item 12. The at least bispecific antigen-binding molecule according to Item 11, wherein the LSC is selected from LSCs in acute myeloid leukemia (AML) or chronic myeloid leukemia (CML).

[0094] Item 13. The at least bispecific antigen-binding molecule according to Item 11 or 12, wherein the antigen-binding domains thereof are bound by covalent or non-covalent bonds.

[0095] Item 14. The at least bispecific antigen-binding molecule according to any one of Items 11 to 13, selected from at least one bispecific format selected from the group consisting of TrioMab, IgG-like, CrossMab, 2:1 CrossMab, 2:2 CrossMab, DuoBody, DVD-Ig, scFv-IgG, IgG-IgG, Fab-scFv-Fc, TF, ADAPTIR, BITE, BITE-Fc, DART, DART-FC, tetravalent DART, TandAb, ImmTAC, TriKE, scFv-scFv-scFv, trispecific nanobody, diabody, triabody, tetrabody or higher-order multimer, and trifab-contobody.

[0096] Item 15. The at least bispecific antigen-binding molecule according to any one of Items 11 to 14, wherein the candidate molecule of the library further comprises at least 1 / 3 of an antigen-binding domain, for example, a CD3-binding domain.

[0097] Item 16. The at least bispecific antigen-binding molecule according to any one of Items 11 to 15, wherein the molecule further comprises an Fc fragment or a mutant or inactivated version thereof, such as an Fc fragment lacking cysteine residues and / or cysteine bridges, or an Fc fragment containing a knob or hole structure.

[0098] Item 17. The at least bispecific antigen-binding molecule according to any one of Items 11 to 16, wherein the candidate molecule of the library further comprises at least one marker or label.

[0099] Item 18. The amino acid sequence of the CD123 binding region follows European Patent Application Publication No. 2778175 A1, or a specific binding sequence that is at least 95% identical to the said sequence, and the amino acid sequence of the CD200 binding region follows European Patent Application Publication No. 2178561 A1, or a specific binding sequence that is at least 95% identical to the said sequence. The at least bispecific antigen-binding molecule according to any one of Items 11 to 17.

[0100] Item 19. The at least bispecific antigen-binding molecule according to any one of Items 11 to 18, which is murine, chimeric, humanized or fully human.

[0101] Item 20. A pharmaceutical composition comprising the at least bispecific antigen-binding molecule according to any one of Items 11 to 19 together with a pharmaceutically acceptable carrier and / or adjuvant.

[0102] Item 21. Use in medicine, preferably for the prevention or treatment of cancer, such as leukemia, for example AML or CML, and particularly its pediatric form, etc. in a patient, comprising administering to the patient an effective amount of the at least bispecific antigen-binding molecule according to any one of Items 11 to 18 or the pharmaceutical composition according to Item 19. The at least bispecific antigen-binding molecule according to any one of Items 11 to 19 or the pharmaceutical composition according to Item 20.

[0103] Item 22. At least a bispecific antigen-binding molecule or a pharmaceutical composition for use according to item 21, wherein the patient is selected from mouse, monkey, dog, cat, rat or human, preferably a human child.

[0104] Item 23. At least a bispecific antigen-binding molecule or a pharmaceutical composition for use according to item 21 or 22, wherein the prevention or treatment of the cancer involves immunotherapy and / or the avoidance of cancer target loss and / or metastasis.

[0105] Item 24. A method for preventing or treating cancer in a patient, such as leukemia, for example AML or CML, especially its pediatric form, etc., comprising administering to the patient an effective amount of at least a bispecific antigen-binding molecule according to any one of items 11 to 18 or a pharmaceutical composition according to item 19.

[0106] Item 25. The method according to item 24, wherein the patient is selected from mouse, monkey, dog, cat, rat, or human, preferably a human child.

[0107] Item 26. The method according to item 25 or 26, wherein the prevention or treatment of the cancer involves immunotherapy and / or the avoidance of cancer target loss and / or metastasis.

[0108] Item 27. A method for identifying mammalian leukemia stem cells (LSCs) in a biological sample obtained from a mammalian patient suspected of having leukemia, a) contacting the sample with at least a bispecific antigen-binding molecule according to any one of items 11 to 17; and b) detecting the binding of the at least bispecific antigen-binding molecule to cells in the sample, comprising wherein the binding, particularly specific binding, of the at least bispecific antigen-binding molecule to the cells identifies leukemia stem cells (LSCs) in the sample.

[0109] Item 28. A method for identifying mammalian leukemia stem cells (LSCs) in a biological sample according to Item 27, wherein the at least bispecific antigen-binding molecule comprises at least one detectable marker, enzyme, or label.

[0110] Item 29. A method for identifying mammalian leukemia stem cells (LSCs) in a biological sample according to Item 27 or 28, wherein the at least bispecific antigen-binding molecule binds simultaneously to CD123 and CD200.

[0111] Item 30. A method for diagnosing leukemia in a mammalian patient suspected of having leukemia, comprising the method according to any one of Items 27 to 29, and further concluding the leukemia of the mammalian patient based on the binding.

[0112] Item 31. The method according to Item 30, wherein the leukemia is relapsed, malignant, and / or metastatic leukemia, AML, particularly its pediatric form, and CML.

[0113] Item 32. Use of the at least bispecific antigen-binding molecule according to any one of Items 11 to 18 for purifying and / or isolating LSCs.

[0114] The present invention will be further described in the following examples with reference to the accompanying drawings and sequences, but is not limited thereto. For the purposes of the present invention, all references cited herein are incorporated by reference in their entirety.

Brief Description of the Drawings

[0115]

Figure 1

Figure 2

Figure 3

Figure 4

[0116] SEQ ID NO:1 shows the amino acid sequence of human CD123, isoform 1.

[0117] SEQ ID NO:2 shows the amino acid sequence of human CD200, isoform 1.

[0118] SEQ ID NO:3 shows the amino acid sequence of the light chain variable region (V L ) of an exemplary antigen-binding domain that binds to CD123.

[0119] SEQ ID NO:4 shows the amino acid sequence of the heavy chain variable region (V H ) of an exemplary antigen-binding domain that binds to CD123.

[0120] SEQ ID NO:5 shows the amino acid sequence of the light chain of an exemplary antigen-binding domain that binds to CD123.

[0121] SEQ ID NO:6 shows the amino acid sequence of the heavy chain of an exemplary antigen-binding domain that binds to CD123.

[0122] SEQ ID NOs:7-9 show the amino acid sequences of the CDRs of the first antigen-binding domain that binds to CD200.

[0123] SEQ ID NOs:10-12 show the amino acid sequences of the CDRs of the second antigen-binding domain that binds to CD200.

[0124] SEQ ID NOs:13-15 show the amino acid sequences of the CDRs of the third antigen-binding domain that binds to CD200.

[0125] SEQ ID NOs:16-18 show the amino acid sequences of the CDRs of the fourth antigen-binding domain that binds to CD200.

[0126] SEQ ID NOs:19-21 show the amino acid sequences of the CDRs of the fifth antigen-binding domain that binds to CD200.

[0127] SEQ ID NOs:22-24 show the amino acid sequences of the CDRs of the sixth antigen-binding domain that binds to CD200.

[0128] SEQ ID NOs: 25 to 27 show the amino acid sequences of the CDRs of the seventh antigen-binding domain that binds to CD200.

[0129] SEQ ID NOs: 28 to 30 show the amino acid sequences of the CDRs of the eighth antigen-binding domain that binds to CD200.

[0130] SEQ ID NOs: 31 to 33 show the amino acid sequences of the CDRs of the ninth antigen-binding domain that binds to CD200.

[0131] SEQ ID NOs: 34 to 37 show the amino acid sequences of the CDRs of the tenth antigen-binding domain that binds to CD200.

BEST MODE FOR CARRYING OUT THE INVENTION

[0132] Examples The results of in vitro experiments confirmed selective binding to LSCs and controlled and improved, preferably local activation of T cells.

[0133] An exemplary trifab-contobody according to an embodiment of the invention, preferably comprising a combination of antigen-binding domains of the CD123 and CD200 domains disclosed in SEQ ID NOs: 3 to 23 above, against CD123 + / CD200 +Tested in the AML cell line Kasumi-1 (Asou H, Tashiro S, Hamamoto K, Otsuji A, Kita K, Kamada N. Establishment of a human acute myeloid leukemia cell line (Kasumi-1) with 8;21 chromosome translocation. Blood. 1991;77:2031-2036). For this purpose, a combination of anti-CD200-knob (CD200K) and anti-CD123-hole (CD123H) was constructed. Furthermore, individual halves as well as combinations with a scrambled hole (SrcH), which is a non-binding half of CD200K, were used as controls. One CD200K half with a functional anti-CD3 domain instead of a "dummy" was used as a positive control (CD200-act). (Figures 2 and 3). In other constructs, the knob-hole part may be omitted.

[0134] To test how well different combinations / individual halves of the construct can activate T cells, PBMCs were isolated from healthy donors (HDs) and cultured with antibodies and Kasumi-1 cells for 24 hours. The T cells were then examined by flow cytometry for the expression of the activation markers CD25, CD69, and CD137. In the case of PBMCs with Kasumi-1 and CD200K+CD123H, the expression of all three markers increased significantly. In contrast, when PBMCs were co-cultured with Kasumi-1 and individual halves of the antibody, activation was not apparent. The combination of CD200K+ScrH resulted in a slight increase in the CD69 activation marker but was clearly delayed compared to CD200K+CD123H. Increases in the expression of CD25 and CD137 could not be determined with CD200K+ScrH. (Figure 4)

[0135] Next, in the following step, the inventors investigated the cytotoxic activity of T cells dependent on the TriFab exemplary construct with the help of continuous visualization of co-cultures with mCherry-expressing Kasumi-1 cells in vitro. The co-cultures were mixed with defined antibody halves in various combinations.

[0136] Photographs were taken every 2 hours for 72 hours in the bright field and mCherry channels. An increase in mCherry-positive cells was observed for Kasumi-1-mCherry and PBMCs and individual hemibodies. However, for the combination of CD200K+CD123H, a clear eradication of AML cells was found. The combination of CD200K+ScrH showed no difference from the antibody-free control.

[0137] Similar results were obtained with so-called "patient-derived xenograft leukemia cells", which are more heterogeneous than cell lines and are therefore more biologically equivalent to primary leukemia cells [Vick B, Rothenberg M, Sandhofer N, Carlet M, Finkenzeller C, Krupka C, et al. An advanced preclinical mouse model for acute myeloid leukemia using patients' cells of various genetic subgroups and in vivo bioluminescence imaging. PLoS One. 2015;10:e0120925].

[0138] This in vitro experiment shows that the present invention can achieve selective cytotoxicity through spatially and temporally controlled activation of T cells on the surface of CD123+ / CD200+ AML cells.

[0139] Therefore, the combination of the two target structures constitutes a novel strategy for eliminating leukemia stem cells, particularly in the pediatric form of AML. Therefore, CD123 / CD200-dependent and favorably localized activation of T cells is a principle for improving anti-AML immunotherapy.

Claims

1. An in vitro method for identifying at least a bispecific antigen-binding molecule that specifically binds to mammalian leukemia stem cells (LSCs), comprising: a) providing a first library of candidate antigen-binding molecules specific for protein CD123 or an immunologically recognizable fragment or derivative thereof; a') providing a second library of candidate antigen-binding molecules specific for protein CD200 or an immunologically recognizable fragment or derivative thereof; b) combining a plurality of candidate antigen-binding molecules from said first and second libraries to generate a library of candidate molecules that are at least bispecific for CD123 and CD200; c) providing a suitable screening system comprising said protein CD123 and / or CD200, or an immunologically recognizable fragment or derivative thereof; d) contacting the library of b) with the screening system of c); e) identifying, from the library of b), a binding molecule that specifically binds to said protein CD123 and CD200, or an immunologically recognizable fragment or derivative thereof, preferably, said LSCs are selected from LSCs in acute myeloid leukemia (AML) or chronic myeloid leukemia (CML). An in vitro method comprising the above steps.

2. The library of candidate antigen-binding molecules is selected from an antibody library or a phage display library, preferably a single-chain variable fragment (scFv) or a stabilized form thereof (e.g., disulfide-stabilized scFv), Fab fragment, Fab' fragment, F(ab') fragment, F(ab') ２ fragment, scFab, and a heavy chain variable domain (VHH), a single domain antibody (sdAb), or a single domain antibody fragment such as a nanobody, the method according to claim 1.

3. The library of candidate molecules that are at least bispecific binding agents for CD123 and CD200 comprises at least one bispecific format selected from the group consisting of TrioMab, IgG-like, CrossMab, 2:1 CrossMab, 2:2 CrossMab, DuoBody, DVD-Ig, scFv-IgG, IgG-IgG, Fab-scFv-Fc, TF, ADAPTR, BITE, BITE-Fc, DART, DART-FC, tetravalent DART, TandAb, ImmTAC, TriKE, scFv-scFv-scFv, trispecific nanobody, diabody, tribody, tetrabody or higher order multimer, and trifab-contobody. The method according to claim 1 or 2.

4. The method according to any one of claims 1 to 3, wherein the candidate molecules of the library further comprise at least 1 / 3 of an antigen-binding domain, such as a CD3-binding domain.

5. The method according to any one of claims 1 to 4, wherein the suitable screening system comprises cells that express, particularly recombinantly express, CD123 and CD200 or antigenic fragments thereof, or is a cell-free system.

6. At least a bispecific antigen-binding molecule that specifically binds to the proteins CD123 and CD200 or immunologically recognizable fragments or derivatives thereof of mammalian leukemia stem cells (LSCs) identified by the method according to any one of claims 1 to 5, preferably wherein the LSCs are selected from LSCs in acute myeloid leukemia (AML) or chronic myeloid leukemia (CML), at least a bispecific antigen-binding molecule.

7. The amino acid sequence of the CD123-binding region follows the sequence of European Patent Application Publication No. 2778175 A1, or a specific binding sequence that is at least 95% identical to said sequence, and the amino acid sequence of the CD200-binding region follows the sequence of European Patent Application Publication No. 2178561 A1, or a specific binding sequence that is at least 95% identical to said sequence. The at least bispecific antigen-binding molecule according to claim 6.

8. The at least bispecific antigen-binding molecule according to claim 6 or 7, which is murine, chimeric, humanized or fully human.

9. A pharmaceutical composition comprising the at least bispecific antigen-binding molecule according to any one of claims 6 to 8 together with a pharmaceutically acceptable carrier and / or adjuvant.

10. Use in medicine, preferably for the prevention or treatment of cancer such as leukemia, for example AML or CML, and particularly its pediatric form, etc., in a patient, comprising administering to the patient an effective amount of the at least bispecific antigen-binding molecule according to any one of claims 6 to 8 or the pharmaceutical composition according to claim 9. The at least bispecific antigen-binding molecule according to any one of claims 6 to 8 or the pharmaceutical composition according to claim 9.

11. The at least bispecific antigen-binding molecule or pharmaceutical composition for use according to claim 10, wherein the prevention or treatment of the cancer comprises immunotherapy and / or comprises avoidance of target loss and / or metastasis of the cancer.

12. A method for identifying mammalian leukemia stem cells (LSCs) in a biological sample obtained from a mammalian patient suspected of having leukemia, comprising a) contacting the sample with at least a bispecific antigen-binding molecule according to any one of claims 6 to 8; b) detecting the binding of the at least bispecific antigen-binding molecule to cells in the sample; comprising: A method for identifying leukemia stem cells (LSCs) in the sample, wherein the binding, particularly specific binding, of the at least bispecific antigen-binding molecule to the cells identifies leukemia stem cells (LSCs) in the sample.

13. The method for identifying mammalian leukemia stem cells (LSCs) in a biological sample according to claim 12, wherein the at least bispecific antigen-binding molecule binds simultaneously to CD123 and CD200.

14. A method for diagnosing leukemia in a mammalian patient suspected of having leukemia, comprising the method according to claim 12 or 13, and further concluding leukemia in the mammalian patient based on the binding, preferably wherein the leukemia is relapsed, malignant and / or metastatic leukemia, AML, particularly its pediatric form, and CML.

15. Use of at least a bispecific antigen-binding molecule according to any one of claims 6 to 8 for purifying and / or isolating LSCs.

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