CD123 and CD200 as markers for diagnosis and immunoeradication of leukemia stem cells (lsc)

Abstract

The present invention relates to antigen binding molecules that are at least bispecific and specifically bind to CD200 and CD123 on the cell surface of leukemia stem cells (LSCs). The invention further relates to methods for identifying such antigen-binding molecules and to the use of said antigen-binding molecules for the diagnosis and treatment of leukemia, such as AML or CML, and in particular the pediatric form thereof.

Description

[0001] The present invention relates to antigen binding molecules, which are at least bispecific and specifically bind to CD200 and CD123 on the cell surface of leukemia stem cells (LSC). The present invention further relates to methods for identifying such antigen binding molecules and the use of the antigen binding molecules for diagnosing and treating leukemias such as AML or CML and particularly their pediatric forms. Background Art

[0002] Cancer is a major fatal disease in humans and is caused by physiologically uncontrolled cell proliferation, which affects the normal physiological conditions of the human body, resulting in severe pathological reactions and often death. Despite the great efforts made in cancer research and treatment, cancer is still the leading cause of death in humans. There are many ways to treat cancer patients, including surgery, radiotherapy and chemotherapy.

[0003] Despite significant advances in risk-adapted chemotherapy, the treatment of acute myeloid leukemia (AML) remains challenging ( H, Estey E, Grimwade D, Amadori S, Appelbaum FR, Büchner 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 "leukemia stem cells" (LSCs) can survive after cytotoxic therapy and thus become the origin of 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 may be an effective option for eliminating chemotherapy-resistant leukemia stem cells.

[0004] In acute lymphoblastic B-cell leukemia (B-ALL), so-called "bispecific T cell-engaging antibody 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 TA 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] There is a target protein CD19, which is strongly overexpressed on leukemic B cells, while healthy non-B cells are negative for this marker (Ghorashian S, Pule M, Amrolia P. CD19 chimeric antigen receptor T cell therapy for haematological malignancies. Br J Haematol. 2015; 169: 463–478). Because CD19 is also expressed on normal B cells, normal B lineage cells are also eliminated after CD19-CAR-T infusion. This phenomenon is often referred to as the "on-target off-tumor effect". Subsequent B cell aplasia leads to long-term hypogammaglobulinemia, and intermittent immunoglobulin replacement therapy is occasionally required to prevent serious infections.

[0006] However, for AML, there is no "ideal" target yet. All previously known targets that are overexpressed on AML stem cells (such as CD33, CD123 or CLL-1) can also be present on other healthy cells. Treatments targeting some of these targets also result in severe on-target myeloablation (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 T cells. antigen receptor-modified T cells. Blood. 2014;123:2343–2354).

[0007] Leukemia stem cells (LSC) in AML play an important role in the initiation and progression of leukemia and are considered to be the root of chemotherapy resistance and disease relapse. The identification and targeting of LSC depend on membrane markers, such as CD34, CD38, CD123, TIM3, CD25, CD32 and CD96. In addition, transcription factors are also therapeutic targets for eradicating LSC, 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 are identified as new targets for ablating LSC 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 on March 2, 2017, doi: 10.21037 / sci.2017.02.10).

[0008] In acute myeloid leukemia (AML), leukemic stem cells (LSCs) determine mortality, but are difficult to isolate due to their low abundance and high similarity to healthy hematopoietic stem cells (HSCs) (Velten, L., Story, BA, Hernández-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) described that targeted immunotherapy for acute myeloid leukemia (AML) is challenged by the lack of AML-specific target antigens and clonal heterogeneity, resulting in unwanted on-target deleukemia and the risk of relapse from minor clones. They hypothesized that combinatorial targeting of AML cells could enhance therapeutic efficacy without increasing toxicity. To identify combinations of target antigens specific for 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 proteome dataset. They analyzed the antigen expression levels of CD33, CD123, CLL1, TIM3, CD244, and CD7 on AML bulk cells and leukemic stem cells at initial diagnosis (n=302) and at 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 analyzed target, they found additional expression in different populations with normal hematopoiesis. By analyzing the co-expression of our six targets in all double combinations (n=15), they found that CD33 / TIM3 and CLL1 / TIM3 were highly positive in AML compared with normal hematopoietic tissues and non-hematopoietic tissues. They proposed that combined targeting of CD33 / TIM3 or CLL1 / TIM3 may enhance the therapeutic efficacy of immunotherapy for AML without exacerbating toxicity.

[0010] In the treatment of leukemia, targeted identification and elimination of AML stem cells remain unmet needs. There is also a continuing need in the art to identify agents with therapeutic potential for treating proliferative disorders (such as AML) and improved specific diagnostic uses. There is an urgent need to identify these agents so that the therapy can be further developed from the laboratory to clinical practice.

[0011] Hereinafter, the elements of the present invention will be described. These elements are listed together with specific embodiments, however, it should be understood that they can be combined in any way and in any number to produce other embodiments. The examples and preferred embodiments of various descriptions should not be interpreted as limiting the present invention to only the embodiments clearly described. This specification should be understood as supporting and covering the embodiments of combining two or more clearly described embodiments, or the embodiments of combining one or more clearly described embodiments with any number of disclosed and / or preferred elements. In addition, unless the context otherwise indicates, any arrangement and combination of all described elements in this application should be considered to be disclosed by the specification of the application.

[0012] In a first aspect of the present invention, the above problem is solved by a method for identifying at least a bispecific antigen binding molecule that specifically binds to a mammalian leukemia stem cell (LSC), the method comprising the following steps:

[0013] a) providing a first library of candidate antigen-binding molecules specific for protein CD123 or an immunologically recognizable fragment or derivative thereof,

[0014] a') providing a second library of candidate antigen binding molecules specific for protein CD200 or an immunologically recognizable fragment or derivative thereof,

[0015] b) combining a plurality of candidate antigen-binding molecules from the first library and the second library to generate a library of candidate molecules having at least bispecificity for CD123 and CD200,

[0016] c) providing a suitable screening system comprising the proteins CD123 and / or CD200 or immunologically recognizable fragments or derivatives thereof,

[0017] d) contacting the library of b) with the screening system of c), and

[0018] e) identifying from the library of b) binding molecules that specifically bind to proteins CD123 and CD200 or immunologically recognizable fragments or derivatives thereof.

[0019] Preferably, the LSC is selected from LSC in acute myeloid leukemia (AML) or in chronic myeloid leukemia (CML).

[0020] In a second aspect of the present invention, the above-mentioned problem is solved by at least one bispecific antigen binding molecule, which specifically binds to the proteins CD123 and CD200 of mammalian leukemia stem cells (LSC) or their immune-recognizable fragments or derivatives, and the at least bispecific antigen binding molecule is identified by the method according to the present invention.

[0021] 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 and a pharmaceutically acceptable carrier and / or adjuvant.

[0022] In a fourth aspect of the present invention, the above problem is solved by an at least bispecific antigen binding molecule according to the invention or a pharmaceutical composition according to the invention for use in medicine, preferably for the prevention or treatment of cancer, such as leukemia, e.g. AML or CML, and in particular pediatric forms thereof, in a patient, comprising administering to said patient an effective amount of an at least bispecific antigen binding molecule according to the invention or a pharmaceutical composition according to the invention.

[0023] In a fifth aspect of the invention, the above problem is solved by a method for preventing or treating cancer (such as leukemia, e.g. AML or CML, and in particular pediatric forms thereof) in a patient, the method comprising administering to the patient an effective amount of at least a bispecific antigen binding molecule according to the invention or a pharmaceutical composition according to the invention.

[0024] In a sixth aspect of the present invention, the above problems are solved by a method for identifying mammalian leukemia stem cells (LSCs) in a biological sample obtained from a mammalian patient suspected of having leukemia, the method comprising

[0025] a) contacting the sample with at least a bispecific antigen binding molecule according to the invention, and

[0026] b) detecting binding of the at least bispecific antigen binding molecule to cells in said sample, wherein the binding and in particular specific binding of the at least bispecific antigen binding molecule to these cells identifies leukemic stem cells (LSC) in said sample.

[0027] In a seventh aspect of the present invention, the above problem is solved by a method for diagnosing leukemia in a mammalian patient suspected of having leukemia, the method comprising a method according to the present invention, and based on the combination, further concluding that the mammalian patient has leukemia. Preferably, the leukemia is relapsed, malignant and / or metastatic leukemia, AML (particularly its pediatric form) and CML.

[0028] As mentioned 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 leukemic stem cells (LSCs), the method comprising the following steps:

[0029] a) providing a first library of candidate antigen-binding molecules specific for protein CD123 or an immunologically recognizable fragment or derivative thereof,

[0030] a') providing a second library of candidate antigen binding molecules specific for protein CD200 or an immunologically recognizable fragment or derivative thereof,

[0031] b) combining a plurality of said candidate antigen-binding molecules from said first library and said second library to generate a library of candidate molecules having at least bispecificity for CD123 and CD200,

[0032] c) providing a suitable screening system, said screening system comprising said protein CD123 and / or CD200 or an immunologically recognizable fragment or derivative thereof,

[0033] d) contacting the library of b) with the screening system of c), and

[0034] e) identifying from the library of b) binding molecules that specifically bind to proteins CD123 and CD200 or immunologically recognizable fragments or derivatives thereof.

[0035] Preferably, the method according to the invention is wherein said LSC is selected from LSC in acute myeloid leukemia (AML) or in chronic myeloid leukemia (CML).

[0036] The present invention is generally based on the use of the target proteins Cluster of Differentiation ("CD") CD123 and CD200, since these two markers are expressed together in leukemic stem cells, in particular AML stem cells.

[0037] 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 MRCOX-2is a member of the immunoglobulin superfamily with a light-chain-like structure. Biochem Soc Symp. 1986; 51: 149–157). The CD200 receptor (CD200R) is present on hematopoietic cell lines (T cells, B cells and NK cells, as well as immune cells of the myeloid lineage). 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 escape” 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). Therapeutic approaches using CD200 as checkpoint inhibitors are considered 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).Therapeutic antibodies against CD200 (Samalizumab) are currently being tested in patients with chronic lymphocytic leukemia (CLL) and multiple myeloma (MM) in a phase 1 clinical study (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 is selected as the first target in the combination method according to the present invention.

[0038] CD123 (IL3RA) is a surface marker of hematopoietic precursor cells and is strongly overexpressed in AML-stem cell leukemia (SCL). This is associated with an increased proliferation of AML cells (see also diagnostic methods below). Although this effect can be explained by IL3-mediated signaling, the mechanism by which CD123-positive AML cells become more resistant 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 an amembrane biomarker and a therapeutic target in hematologicmalignancies. Biomark Res. 2014; 2:4).

[0039] Several clinical trials are investigating the use of various CD123-targeting agents, including T cells modified with chimeric antigen receptors (expressing CD123, monoclonal antibodies, combined CD3-CD123 dual-affinity redirecting antibody therapy, recombinant fusion proteins, and CD123-engaged 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 on October 23, 2020, doi: 10.3390 / cancers12113087). Examples are CD123 neutralizing antibodies, CD3×CD123 bispecific antibodies, dual affinity redirection (DART), and CAR-T cells (Shi M, Su RJ, Parmar KP, 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: CD123. [cited Dec 6, 2021]. Available from: https: / / clinicaltrials.gov / ct2 / results?cond=aml&term=CD123&cntry=&state=&cit y=&dist=). The experimental antibody-drug conjugate SGN-CD123A targets CD123 as a possible treatment for AML.

[0040] CD123 was 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).

[0041] Therefore, both target proteins not only show strong overexpression in AML-LSCs, but are also important for successful immune evasion and increased cell proliferation of the cancer. Since both target proteins not only represent markers but also contribute to the malignancy of leukemias such as AML, the otherwise common and highly undesirable "target loss" is unlikely to occur.

[0042] In view of the above, the present inventors examined advantageous combinatorial approaches against the target proteins CD123 and CD200, and generated and tested new recombinant bispecific antigen binding agents, in particular trifab-contorsbodies.

[0043] Kandeel EZ et al. (see: Overexpression of CD200 and CD123 is a majorinfluential factor in the clinical course of pediatric acute myeloidleukemia. Exp Mol Pathol. February 2021; 118: 104597. doi: 10.1016 / j.yexmp.2020.104597. Epub December 23, 2020. PMID: 33358743) disclose that acute myeloid leukemia (AML) accounts for about 20% of all childhood acute leukemias. The results of AML are still unsatisfactory. CD123 and CD200 have been shown to play an important role in hematological malignancies. Both CD200 and CD123 have a negative impact on clinical manifestations and treatment outcomes. When both are overexpressed at the same time, clinical manifestations and treatment outcomes are significantly worsened. Therefore, CD200 and CD123 can be used as markers for minimal residual disease in AML. They also speculated on its use as a therapeutic target, but no further information on this was available.

[0044] In the context of the present invention, the term "CD123" generally refers to the interleukin-3 receptor (CD123) protein (alpha subunit), a molecule present on cells that helps transmit signals of interleukin-3, a soluble cytokine important in the immune system. The term should cover the human form of the protein, but also its homologs, particularly those in mice or rats. Information on CD123 can be obtained from the Human Gene Names webpage ( https: / / www.genenames.org / ), whose accession number is HGNC:6012. CD123 protein can be obtained on the UniProt webpage with accession number P26951, and further at least the human form is shown in SEQ ID NO:1 (isoform 1). The present invention should also cover all other isoforms of the protein.

[0045] In the context of the present invention, the term "CD200" generally refers to the CD200 molecule protein, also known as the OX-2 membrane glycoprotein. The term should cover the human form of the protein, but also its homologs, especially those in mouse or rat. Information on CD200 can be obtained from the Human Gene Names webpage ( https: / / www.genenames.org / ), whose accession number is HGNC: 7203. The CD200 protein is available on the UniProt webpage under accession number P41217 (isoform 1), and is at least the human form, shown in SEQ ID NO: 2. The present invention shall also encompass all other isoforms of the protein.

[0046] The fragment or derivative or variant of CD200 and / or CD123 can be easily tested for the ability to bind to the antigen binding agent according to the present invention. Therefore, the present invention also discloses a method for determining whether the protein derivative, variant and / or fragment of CD200 and / or CD123 keep the interaction (binding) ability of binding to the antigen binding agent according to the present invention, and thus constitutes an immunologically recognizable fragment. The method includes contacting the candidate derivative, variant and / or fragment of CD200 and / or CD123 with the antigen binding molecules of the present invention, and detecting the step of their interaction (i.e., binding). In some embodiments, the above-mentioned method for testing protein variants / derivatives and / or fragments can constitute a part of the above-mentioned method for identification, preferably under the condition that the method covers any protein derivative, variant and / or fragment using CD123 and / or CD200. These fragments can also be included in the screening system used in the context of the present invention.

[0047] Since the CD123 protein consists of three extracellular domains (287 amino acids), a single-pass transmembrane domain (30 amino acids) and a short intracellular region (53 amino acids), the preferred fragment used in the context of the present invention is the extracellular part, which can be used as a soluble protein, and which can also be labeled and / or recombinantly produced.

[0048] CD200 consists of two extracellular (one variable and one constant) immunoglobulin-like domains, a single transmembrane region and a cytoplasmic tail. Therefore, the preferred fragment used in the context of the present invention is the extracellular part, which can be used as a soluble protein, which can also be labeled and / or produced recombinantly.

[0049] In the context of the present invention, the term "CD123" and / or "CD200" should also include other proteins, their variants or fragments, provided that their amino acids have a sequence identity of at least 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% compared to the amino acid sequence of human CD123 or CD200, respectively. The fragment of CD123 or CD200 or its variant should preferably have a length of at least 30, 40, 50, 80, 100, 150, 200, 300 or more amino acids. As known to the technician, the variant may include conservative amino acid changes.

[0050] As used herein, the terms "identical" or "percent identity" when used in the context of two or more nucleic acids or protein / polypeptide sequences refer to two or more sequences or subsequences that are identical or have (or have at least) a specified percentage of identical amino acid residues or nucleotides (i.e., equal to or at least about 60% identity, preferably equal to or at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93% or 94% identity, and more preferably equal to or at least about 95%, 96%, 97%, 98%, 99% or more identity (preferably over their full lengths), when compared and aligned over a comparison window or designated region for maximum correspondence, as measured using a sequence comparison algorithm or by manual alignment and visual inspection (see, e.g., NCBI website). In particular, for amino acid identity, use of BLASTP with the following parameters is contemplated. 2.2.28+: Matrix: BLOSUM62; Gap Penalties: Existence: 11, Extension: 1; Neighborhood Word Threshold: 11; Multi-Hit Window: 40.

[0051] In the method according to the present invention, any suitable library of candidate antigen binding molecules can be used, which allows the selection of domains involved in antigen recognition for use as binding moieties in the design of bispecific diagnostic agents and therapeutic agents. Preferably, the library is selected from an antibody library or a phage display library, more preferably a human-based or humanized library. Examples of domains involved in antigen recognition include domain antibodies (only heavy chain variable domains (V HH )) and single-chain Fv fragments (scFv), antigen binding fragments (Fab), single-chain Fab fragments (scFab) and the most recent single-chain Fc fragments (scFc), and preferably include single-chain variable fragments (scFv) or stabilized forms thereof (e.g., scFv stabilized by disulfide bonds), Fab fragments, Fab' fragments, F(ab') fragments, F(ab')2 fragments, scFab and single-domain antibody fragments, such as only the heavy chain variable domain (VHH), single-domain antibodies (sdAb) or nanobodies. In addition, a TCR recognition domain, i.e. a construct comprising the essential CDRs of a TCR, which forms a loop protruding from the core Vα or Vβ domain and mediates specific binding.

[0052] Once suitable binding domains / molecules are identified (e.g., by additional antigen binding tests as disclosed herein and / or sequencing of corresponding nucleic acid molecules), they are combined into suitable bispecific constructs for use in the context of the present invention. The present invention relates to methods according to the present invention, wherein the combination includes covalent or non-covalent fusion or combination of candidate antigen binding molecules from the first library and the second library. The combination may include additional steps of cloning or otherwise inserting (e.g., synthesizing polypeptides) the binding domain / molecule into a suitable scaffold structure. The corresponding methods are described in the literature and are well known to those skilled in the art.

[0053] The at least bispecific binding agent for CD123 and CD200 may have any suitable form that allows for binding, preferably simultaneous binding, of CD123 and CD200 in diagnostic assays, screening assays and / or therapeutic applications as disclosed herein. Preferably, the method according to the invention is characterized in that the library of candidate molecules that are at least bispecific binders to CD123 and CD200 (i.e. the intended "final product") 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 nanobodies, diabodies, triabodies, tetrabodies or higher order multimers, and trifab-contorsbody (see Figure 1 and Figure 2 , and Suurs FV, ​​Lub-de Hooge MN, de Vries EGE, deGroot DJA. A review of bispecific antibodies and antibody constructs inoncology and clinical challenges. Pharmacol Ther. 2019 Sep; 201: 103-119. doi: 10.1016 / j.pharmthera.2019.04.006. Epub April 24, 2019. PMID: 31028837).

[0054] More preferably, the method according to the invention, wherein the candidate molecules in the library further comprise at least one third antigen binding domain, such as a CD3 binding domain. This allows additional functionality, particularly in immunotherapy or diagnostic applications, but may also include a binding agent for binding to a matrix or other protein or protein complex. Further preferred examples are specific binding agents for CD33, TIM3 and / or CLL1, or additional T cell-specific antigens, such as CD4, CD8 or CD28.

[0055] More preferred is the method according to the invention, wherein the candidate molecules in the above library further comprise an Fc fragment or a mutated or inactivated derivative / version thereof, such as, for example, an Fc fragment lacking cysteine ​​residues and / or cysteine ​​bridges and / or comprising a "knob" or "hole" structure.

[0056] Particularly preferred constructs / molecules generated constitute at least bispecific binding agents to CD123 and CD200 in the form of a trifab-contorsbody (see e.g. Figure 2and EP3704146). The trifab molecule is a T cell activating antibody format that consists of two complementary halves and is specific to an antigen combination. Each half of the molecule consists of an antibody light chain and an antibody heavy chain. In addition, the structure also includes a so-called "dummy" chain. Like conventional IgG antibodies, the light and heavy chains with "variable light chain" (VL) and "variable heavy chain (VH) chain domains" constitute the antigen-specific binding domain. The Fc fragment (crystallizable fragment) has been modified as follows: there is no cysteine ​​bonded to the other heavy chain, the CD2 domain is replaced by the VL of the anti-CD3 antibody in the first half and by the VH of the anti-CD3 antibody in the second half (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). In addition, a knob-into-hole system was introduced to avoid dimerization of two identical halves (see Ridgway JB, Presta LG, Carter P. "Knobs-into-holes" engineering of antibody CH3 domains for heavychain heterodimerization. Protein Eng. 1996; 9: 617–621). These halves can therefore be designated as "knob" or "hole", respectively. Due to the modification, the Fc fragment becomes inactive. The "dummy" chain is complementary to the respective Fc fragment but cannot generate an active anti-CD3 domain.

[0057] If only one half of the construct binds to cells expressing only one target (i.e., CD123 or CD200), T cells will not be activated. Chain exchange, which is thermodynamically promoted by the charge in the Fc fragment, will occur only when both target structures are recognized on the cell surface. The reorganization results in the removal of two "dummy chains" while forming a functional anti-CD3 domain, thus activating T cells only conditionally ("only if"). Therefore, the desired cytotoxic activity is limited and confined to cells expressing CD200 and CD123.

[0058] LSCs may be identified from any mammal having LSCs expressing CD200 and CD123 or homologues thereof.Preferred is a method according to the invention wherein the mammal is selected from the group consisting of mouse, monkey, dog, cat, rat or human, and preferably a human child.

[0059] The method according to the present invention relates to a suitable system for identifying and / or screening the combination of the antigen binding molecules of the library. The system to be used according to the present invention can be and include any suitable device for identifying and particularly specifically identifying the combination of antigen binding molecules with CD123 or CD200 or its antigen fragment. Preferably, such a system includes cells expressing and particularly recombinantly expressing CD123 and CD200 or its antigen fragment, such as bacteria or yeast cells or human cells. The system can be a cell-free system, which is in solution or relates to a binding agent and / or CD123 or CD200 or its antigen fragment that is combined or conjugated with a matrix. The system can also include a suitable reporter part or labeling. The preferred antigen fragment used in the context of the present invention is the extracellular part of CD123 or CD200 (also see above), which can be used as a soluble protein or attached to a matrix, and can be labeled and / or recombinantly produced in addition. Preferably, the method according to the present invention, wherein the candidate molecule in the library further includes at least one marker or labeling, such as a toxin, an enzyme or a fluorophore.

[0060] Furthermore, the present invention in its second aspect relates to at least bispecific antigen binding molecules which specifically bind to the proteins CD123 and CD200 of mammalian leukemic stem cells (LSC) or immunologically recognizable fragments or derivatives thereof, identified by the method according to the invention. The at least bispecific binding agent for CD123 and CD200 can have any suitable format that allows CD123 and CD200 to bind, preferably simultaneously, in a diagnostic assay, screening assay, and / or therapeutic application as disclosed herein, and preferably 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 nanobodies, diabodies, triabodies, tetrabodies or higher order multimers, and trifab-contorsbody (see Figure 1 and Figure 2, and Suurs FV, ​​Lub-de Hooge MN, de Vries EGE, de Groot DJA. A review of bispecific antibodies and antibody constructs inoncology and clinical challenges. Pharmacol Ther. 2019 Sep;201:103-119. doi:10.1016 / j.pharmthera.2019.04.006. Epub 2019 Apr 24. PMID:31028837). In addition, a TCR recognition domain may be included, i.e. a construct comprising the basic CDRs of the TCR, which form loops protruding from the core Vα or Vβ domain and mediate specific binding.

[0061] Preferred are at least bispecific antigen binding molecules according to the invention, wherein the antigen binding domains thereof are bound covalently or non-covalently.

[0062] More preferably at least bispecific antigen binding molecules according to the present invention, wherein the candidate molecules in the library also include at least one third antigen binding domain, such as a CD3 binding domain. This allows additional functions, particularly in immunotherapy or diagnostic applications, but may also include binding agents for the purpose of binding to a matrix or other proteins or protein complexes. Further preferred examples are specific binding agents for CD33, TIM3 and / or CLL1, or additional T cell-specific antigens, such as CD4, CD8 or CD28.

[0063] One particularly preferred construct / molecule produced constitutes at least a bispecific binding agent to CD123 and CD200 in the form of a trifab-contorsbody as mentioned above. Another particularly preferred construct / molecule produced constitutes at least a bispecific binding agent to CD123 and CD200, which simultaneously binds to CD123 and CD200 on the surface of LSC.

[0064] Another particularly preferred embodiment of the construct / molecule produced according to the invention constitutes at least a bispecific binding agent to CD123 and CD200 which upon binding inhibits the biological function of CD123, CD200 or both, e.g. their signal transduction in cells such as LSCs.

[0065] More preferred are at least bispecific antigen binding molecules according to the invention, wherein the molecules of the above library further comprise an Fc fragment or a mutated or inactivated derivative / form thereof, such as, for example, an Fc fragment lacking cysteine ​​residues and / or cysteine ​​bridges and / or comprising a "knob" or "hole" structure.

[0066] Preferred is at least a bispecific antigen binding molecule according to the invention, wherein said LSC is selected from LSC in acute myeloid leukemia (AML) or in chronic myeloid leukemia (CML).

[0067] Preferred are at least bispecific antigen binding molecules according to the invention, wherein the candidate molecules in the library further comprise at least one marker or label, such as a toxin, enzyme or fluorophore.

[0068] A preferred example is at least a bispecific antigen binding molecule according to the invention, wherein the amino acid sequence of the CD123 binding region is as disclosed in EP2778175A1 (incorporated by reference in its entirety) or a specific binding sequence having at least 95% identity with the disclosed sequence, and wherein the amino acid sequence of the CD200 binding region is according to EP2178561A1 (incorporated by reference in its entirety) or a specific binding sequence having at least 95% identity with the disclosed sequence.

[0069] Another preferred example is at least a bispecific antigen binding molecule according to the present invention, wherein the light chain variable region (V L ) comprises the amino acid sequence according to SEQ ID NO: 3, for binding to the heavy chain variable region (V H ) comprises the amino acid sequence according to SEQ ID NO: 4, and optionally the heavy chain constant region comprises the amino acid substitutions S239D and I332E according to the EU numbering system of Kabat, or a specific binding sequence that is at least 95% identical to said sequence.

[0070] Another preferred example is the at least 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 specific binding sequence according to SEQ ID NO: 6 or a sequence having at least 95% identity thereto.

[0071] Yet another preferred example is the at least bispecific antigen binding molecule according to the invention, wherein the amino acid sequence of the CD200 binding fragment thereof contains one of the CDRs of the following pair:

[0072] (i) HCDR1 comprising the amino acid sequence: GFNIKDYYMH (SEQ ID NO: 7); HCDR2 comprising the amino acid sequence: WIDPENGDTKYAPKFQG (SEQ ID NO: 8); HCDR3 comprising the amino acid sequence: KNYYVSNYNFFDV (SEQ ID NO: 9); LCDR1 comprising the amino acid sequence: SASSVRYMY (SEQ ID NO: 10); LCDR2 comprising the amino acid sequence: DTSKLAS (SEQ ID NO: 11); and LCDR3 comprising the amino acid sequence: FQGSGYPLT (SEQ ID NO: 12);

[0073] (ii) HCDR1 comprising the amino acid sequence: GFNIKDYYIH (SEQ ID NO: 13); HCDR2 comprising the amino acid sequence: WIDPEIGATKYVPKFQG (SEQ ID NO: 14); HCDR3 comprising the amino acid sequence: LYGNYDRYYAMDY (SEQ ID NO: 15); LCDR1 comprising the amino acid sequence: KASQNVRTAVA (SEQ ID NO: 16); LCDR2 comprising the amino acid sequence: LASNRHT (SEQ ID NO: 17); and LCDR3 comprising the amino acid sequence: LQHWNYPLT (SEQ ID NO: 18);

[0074] (iii) HCDR1 comprising the amino acid sequence: GYSFTDYIIL (SEQ ID NO: 19); HCDR2 comprising the amino acid sequence: HIDPYYGSSNYNLKFKG (SEQ ID NO: 20); HCDR3 comprising the amino acid sequence: SKRDYFDY (SEQ ID NO: 21); LCDR1 comprising the amino acid sequence: KASQDINSYLS (SEQ ID NO: 22); LCDR2 comprising the amino acid sequence: RANRLVD (SEQ ID NO: 23); and LCDR3 comprising the amino acid sequence: LQYDEFPYT (SEQ ID NO: 24);

[0075] (iv) HCDR1 comprising the amino acid sequence: GYTFTEYTMH (SEQ ID NO: 25); HCDR2 comprising the amino acid sequence: GVNPNNGGALYNQKFKG (SEQ ID NO: 26); HCDR3 comprising the amino acid sequence: RSNYRYDDAMDY (SEQ ID NO: 27); LCDR1 comprising the amino acid sequence: KSSQSLLDIDEKTYLN (SEQ ID NO: 28); LCDR2 comprising the amino acid sequence: LVSKLDS (SEQ ID NO: 29); and LCDR3 comprising the amino acid sequence: WQGTHFPQT (SEQ ID NO: 30); or

[0076] (v) HCDR1 comprising the amino acid sequence: AFNIKDHYMH (SEQ ID NO:31); HCDR2 comprising the amino acid sequence: WIDPESGDTEYAPKFQG (SEQ ID NO:32); HCDR3 comprising the amino acid sequence: FNGYQALDQ (SEQ ID NO:33); LCDR1 comprising the amino acid sequence: TASSSVSSSYLH (SEQ ID NO:34); LCDR2 comprising the amino acid sequence: STSNLAS (SEQ ID NO:35); and LCDR3 comprising the amino acid sequence: RQYHRSPPIFT (SEQ ID NO:36), or a specific binding sequence that is at least 95% identical to the above sequences, preferably at least 96%, 97%, 98% or 99% identical to the above sequences.

[0077] In the above, HCDR represents the heavy chain complementarity determining region of the binding domain of an antigen-binding molecule; and LCDR represents the light chain complementarity determining region of the binding domain of an antigen-binding molecule.

[0078] More preferably, it is at least a bispecific antigen binding molecule according to the present invention, which comprises a combination of sequences disclosed above (i.e. a combination of a sequence selected from SEQ ID No: 3 to 6 and a sequence selected from SEQ ID No: 7 to 12, 13 to 18, 19 to 24, 25 to 30 and 31 to 36), or a specific binding sequence having at least 95% identity with said sequences.

[0079] Preferred are at least bispecific antigen binding molecules according to the invention, wherein the sequences used (eg binding sequences) are selected from the group consisting of murine sequences, chimeric sequences, humanized sequences and fully human sequences.

[0080] Yet another aspect of the invention relates to a method for producing a pharmaceutical composition, the method comprising formulating at least a bispecific antigen binding molecule according to the invention, eg, as identified, together with a pharmaceutically acceptable carrier and / or excipient as a pharmaceutical composition.

[0081] Yet another aspect of the invention relates to a pharmaceutical composition comprising at least a bispecific antigen binding molecule according to the invention and a pharmaceutically acceptable carrier and / or adjuvant. Preferably, the pharmaceutical composition is suitable for immunotherapy of leukemia, in particular AML and pediatric forms thereof.

[0082] As used herein, the language "pharmaceutical" carrier, stabilizer or excipient is intended to include any and all solvents, solubilizers, fillers, stabilizers, adhesives, absorbents, substrates, buffers, lubricants, controlled release vehicles, diluents, emulsifiers, wetting agents, dispersion media, coatings, antibacterial or antifungal agents, isotonic agents and absorption delaying agents, etc. that are compatible with drug administration. The use of such media and agents for pharmaceutical active substances is well known in the art. Unless any conventional media or agents are incompatible with the active compound, it is considered to be used in the composition. Supplements may also be incorporated into the composition.

[0083] The pharmaceutical composition of the present invention is formulated to be compatible with its intended route of administration.Examples of routes of administration include parenteral administration, such as intrathecal, intraarterial, intravenous, intradermal, subcutaneous, oral, transdermal (topical), and transmucosal administration.

[0084] The pharmaceutical composition used may include a pharmaceutical carrier. Pharmaceutical carriers or excipients include diluents (fillers, extenders, such as lactose, microcrystalline cellulose), disintegrants (such as sodium starch glycolate, cross-linked sodium carboxymethyl cellulose), binders (such as PVP, HPMC), lubricants (such as magnesium stearate), glidants (such as colloidal SiO2), solvents / cosolvents (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 polysorbates, sorbitan esters), thickeners (such as methylcellulose or hydroxyethylcellulose), sweeteners (such as sorbitol, saccharin, aspartame, acesulfame potassium), flavoring agents (such as mint, lemon oil, butterscotch, etc.), humectants (such as propylene, ethylene glycol, glycerol, sorbitol). Other suitable pharmaceutical excipients are described, inter alia, in Remington's Pharmaceutical Sciences, 15th edition, Mack Publishing Co., New Jersey (1991) and Bauer et al., Pharmazeutische Technologic, 5th edition, Govi-Verlag Frankfurt (1997). The skilled person is aware of suitable formulations, such as topical formulations, for the individual compounds and will be able to readily select suitable pharmaceutical carriers or excipients, depending, for example, on the formulation and route of administration of the pharmaceutical composition.

[0085] Another aspect of the present invention relates to at least a bispecific antigen binding molecule according to the invention or a pharmaceutical composition according to the invention for use in medical treatment. Preferably, at least a bispecific antigen binding molecule according to the invention or a pharmaceutical composition according to the invention is used to prevent or treat cancer in a patient, such as leukemia, e.g., AML or CML and in particular its pediatric form, comprising administering an effective amount of at least a bispecific antigen binding molecule according to the invention or a pharmaceutical composition according to the invention to a patient in need, wherein the patient is selected from a mouse, a monkey, a dog, a cat, a rat or a human, and preferably a human child.

[0086] "Treatment" or "treating" refers to any treatment of a disease or disorder in a mammal, including: preventing or protecting against the disease or disorder, i.e., causing clinical symptoms of the disease not to develop; inhibiting the disease, i.e., suppressing or arresting the development of clinical symptoms; and / or alleviating the disease, i.e., causing regression of clinical symptoms.

[0087] Preferred is at least a bispecific antigen binding molecule or a pharmaceutical composition for use according to the invention, wherein said cancer prevention or treatment comprises immunotherapy and / or comprises avoiding target loss and / or metastasis of said cancer.

[0088] Yet another aspect of the invention relates to a method for preventing or treating a cancer in a patient, such as a leukemia, e.g., AML or CML and in particular a pediatric form thereof, comprising administering to the patient an effective amount of at least a bispecific antigen binding molecule according to the invention or a pharmaceutical composition according to the invention. As described above, the patient may be selected from a leukemia patient selected from a mouse, monkey, dog, cat, rat or human, and preferably a human child.

[0089] Preferred is a method according to the invention, wherein said cancer prevention or treatment comprises immunotherapy and / or comprises avoiding target loss and / or metastasis of said cancer, as described herein.

[0090] Methods for preventing or treating diseases using bispecific antigen binders are known to those skilled in the art and are disclosed in the art, for example, in the following: 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).

[0091] In another important aspect of the present invention, the above object is solved by a method for identifying mammalian leukemia stem cells (LSC) in a biological sample obtained from a mammalian patient suspected of having leukemia, the method comprising

[0092] a) contacting the sample with at least a bispecific antigen binding molecule according to the invention, and

[0093] b) detecting binding of the at least bispecific antigen binding molecule to cells in the sample, wherein binding and in particular specific binding and / or simultaneous binding of the at least bispecific antigen binding molecule to CD123 and CD200 on cells identifies / indicates leukemic stem cells (LSC) in the sample.

[0094] In this aspect, at least a bispecific antigen binding molecule according to the invention is used to identify leukemic stem cells (LSC) in a sample. The method can be performed in vitro, in vivo or ex vivo. The sample can be any suitable sample comprising or suspected of comprising LSC, such as a sample comprising blood cells or a sample of a hematopoietic organ, such as bone marrow (BM), lymph nodes and spleen.

[0095] Preferably, the method for identifying mammalian leukemic stem cells (LSC) in a biological sample according to the invention, 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 allow detection of the antigen binding molecule on the surface of the LSC (i.e. when bound to CD123 and CD200).

[0096] Preferably, the method for identifying mammalian leukemic stem cells (LSC) in a biological sample according to the invention, wherein the at least bispecific antigen binding molecule simultaneously binds to CD123 and CD200. The identification step may also include identifying other suitable markers of LSC as described above.

[0097] A particularly preferred aspect of the method according to the invention relates to a trifab construct as described above, wherein CD3 activity (eg T cell activation) can be used to identify simultaneous binding to CD123 and CD200 of LSCs.

[0098] In another important aspect of the present invention, the above purpose is solved by a method for diagnosing (LSC-related) leukemia in a mammalian patient suspected of having leukemia, the method comprising a method according to the present invention, and based on the combination, it is further concluded that the mammalian patient suffers from leukemia. Preferably, the method according to the present invention also includes diagnosing whether the leukemia is recurrent, malignant and / or metastatic leukemia, AML (particularly its pediatric form) and CML. Diagnosis may include quantifying LSC in a sample derived from a patient. Since both CD200 and CD123 have a negative impact on clinical manifestations and treatment outcomes, when both are overexpressed at the same time, clinical manifestations and treatment outcomes significantly deteriorate, so CD200 and CD123 can be used as minimal residual disease in AML or malignant state of the disease and metastasis risk and markers of disease prognosis, wherein a higher amount of cells carrying CD200 and CD123 detected in the patient predict poor results. Markers CD200 and CD123 can also be used as markers of LSC stemness. Markers CD200 and CD123 can also be used to stratify AML patients.

[0099] The above method can also be performed in the context of monitoring the prevention or treatment of a patient's cancer, such as a leukemia, e.g. AML or CML and in particular its pediatric form, comprising a method of identifying or diagnosing a sample derived from the patient during treatment as described above and comparing the result with a control sample, in particular a sample taken from the patient at a time prior to the prevention or treatment of the patient's cancer, such as a leukemia, e.g. AML or CML and in particular its pediatric form. Preferably, the prevention or treatment comprises administering to the patient an effective amount of at least a bispecific antigen binding molecule according to the invention or a pharmaceutical composition according to the invention.

[0100] In another aspect of the invention, at least a bispecific antigen binding molecule according to the invention is used to purify and / or separate LSC expressing CD123 and CD200. Preferably, the at least bispecific antigen binding molecule simultaneously binds CD123 and CD200 and is attached to a solid matrix, such as a column material, a bead and / or a membrane. After contacting the LSC in the sample with the at least bispecific antigen binding molecule according to the invention, the LSC is attached to the at least bispecific antigen binding molecule via CD123 and CD200 and can be separated into LSC-rich fractions after washing and elution steps, which can be further used for the establishment or study of a cell line.

[0101] The inventors designed and studied a combination antigen binder for the target structures CD123 and CD200, and thus developed a new method for eliminating leukemia stem cells. CD123 / CD200 dependence and favorable local activation of T cells is an improved strategy for immunotherapy of leukemias (such as childhood AML).

[0102] The present invention preferably relates to the following items:

[0103] Item 1. A method or in vitro method for identifying at least a bispecific antigen binding molecule that specifically binds to a mammalian leukemia stem cell (LSC), the method comprising the following steps:

[0104] a) providing a first library of candidate antigen-binding molecules specific for protein CD123 or an immunologically recognizable fragment or derivative thereof,

[0105] a') providing a second library of candidate antigen binding molecules specific for protein CD200 or an immunologically recognizable fragment or derivative thereof,

[0106] b) combining a plurality of said candidate antigen-binding molecules from said first library and said second library to generate a library of candidate molecules having at least bispecificity for CD123 and CD200,

[0107] c) providing a suitable screening system, said screening system comprising said protein CD123 and / or CD200 or an immunologically recognizable fragment or derivative thereof,

[0108] d) contacting the library of b) with the screening system of c), and

[0109] e) identifying from the library of b) binding molecules that specifically bind to proteins CD123 and CD200 or immunologically recognizable fragments or derivatives thereof.

[0110] Item 2. The method according to Item 1, wherein the LSC is selected from LSC in acute myeloid leukemia (AML) or in chronic myeloid leukemia (CML).

[0111] Item 3. A method according to item 1 or 2, wherein the library of candidate antigen-binding molecules is selected from an antibody library or a phage display library, preferably comprising a single-chain variable fragment (scFv) or a stabilized form thereof (e.g., a disulfide-stabilized scFv), a Fab fragment, a Fab' fragment, a F(ab') fragment, a F(ab')2 fragment, a scFab and a single domain antibody fragment, such as only a heavy chain variable domain (VHH), a single domain antibody (sdAb) or a nanobody.

[0112] Item 4. The method according to any one of Items 1 to 3, wherein the combination comprises a covalent or non-covalent fusion or combination of candidate antigen-binding molecules from the first library and the second library.

[0113] Item 5. A method according to any one of items 1 to 4, wherein the library of candidate molecules that are at least bispecific binders to 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 nanobodies, diabodies, triabodies, tetrabodies or higher order multimers, and trifab-contorsbody.

[0114] Item 6. A method according to any one of Items 1 to 5, wherein the candidate molecules in the library further comprise at least one third antigen binding domain, such as a CD3 binding domain.

[0115] Item 7. A method according to any one of Items 1 to 6, wherein the candidate molecules in the library further comprise an Fc fragment or a mutated or inactivated form thereof, such as an Fc fragment lacking cysteine ​​residues and / or cysteine ​​bridges or comprising a knob structure or a hole structure.

[0116] Item 8. The method according to any one of Items 1 to 6, wherein the mammal is selected from a mouse, a monkey, a dog, a cat, a rat or a human, and is preferably a human child.

[0117] Item 9. A method according to any one of Items 1 to 7, wherein the suitable screening system comprises cells expressing and in particular recombinantly expressing CD123 and CD200 or antigenic fragments thereof, or is a cell-free system.

[0118] Item 10. The method according to any one of Items 1 to 9, wherein the candidate molecules in the library further comprise at least one marker or label.

[0119] Item 11. An at least bispecific antigen binding molecule that specifically binds to the proteins CD123 and CD200 of mammalian leukemia stem cells (LSC) or immunologically recognizable fragments or derivatives thereof, identified using a method according to any one of Items 1 to 10.

[0120] Item 12. The at least bispecific antigen binding molecule according to Item 11, wherein the LSC is selected from LSC in acute myeloid leukemia (AML) or in chronic myeloid leukemia (CML).

[0121] Item 13. The at least bispecific antigen binding molecule according to Item 11 or Item 12, wherein the antigen binding domains thereof are covalently or non-covalently bound.

[0122] Item 14. At least a bispecific antigen-binding molecule according to any one of Items 11 to 13, which is selected from at least one bispecific format of the group consisting of the following items: 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 nanobodies, diabodies, triabodies, tetrabodies or higher-order multimers and trifab-contorsbody.

[0123] Item 15. The at least bispecific antigen binding molecule according to any one of Items 11 to 14, wherein the candidate molecules in the library further comprise at least one third antigen binding domain, such as a CD3 binding domain.

[0124] Item 16. An 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 mutated or inactivated form thereof, such as an Fc fragment lacking cysteine ​​residues and / or cysteine ​​bridges or comprising a knob or hole structure.

[0125] Item 17. The at least bispecific antigen binding molecule according to any one of Items 11 to 16, wherein the candidate molecules in the library further comprise at least one marker or label.

[0126] Item 18. An at least bispecific antigen binding molecule according to any one of Items 11 to 17, wherein the amino acid sequence of the CD123 binding region is according to EP2778175A1 or a specific binding sequence having at least 95% identity thereto, and wherein the amino acid sequence of the CD200 binding region is according to EP2178561A1 or a specific binding sequence having at least 95% identity thereto.

[0127] 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.

[0128] Item 20. A pharmaceutical composition comprising at least a bispecific antigen binding molecule according to any one of Items 11 to 19 and a pharmaceutically acceptable carrier and / or adjuvant.

[0129] Item 21. An at least bispecific antigen binding molecule according to any one of items 11 to 19 or a pharmaceutical composition according to item 20 for use in medicine, preferably for the prevention or treatment of cancer, such as leukemia, e.g. AML or CML, and in particular pediatric forms thereof, in a patient, comprising administering to the patient an effective amount of an at least bispecific antigen binding molecule according to any one of items 11 to 18 or a pharmaceutical composition according to item 19.

[0130] Item 22. The at least bispecific antigen binding molecule or pharmaceutical composition for use according to Item 21, wherein the patient is selected from a mouse, a monkey, a dog, a cat, a rat or a human, and preferably a human child.

[0131] Item 23. At least a bispecific antigen binding molecule or a pharmaceutical composition for use according to Item 21 to Item 22, wherein the cancer prevention or treatment comprises immunotherapy and / or comprises avoiding target loss and / or metastasis of the cancer.

[0132] Item 24. A method for preventing or treating cancer in a patient, such as a leukemia, e.g., AML or CML and in particular a pediatric form thereof, the method 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.

[0133] Item 25. The method according to Item 24, wherein the patient is selected from a mouse, a monkey, a dog, a cat, a rat or a human, and preferably a human child.

[0134] Item 26. A method according to Item 25 or Item 26, wherein the cancer prevention or treatment comprises immunotherapy and / or comprises avoiding target loss and / or metastasis of the cancer.

[0135] Item 27. A method for identifying mammalian leukemia stem cells (LSCs) in a biological sample obtained from a mammalian patient suspected of having leukemia, the method comprising

[0136] a) contacting the sample with at least a bispecific antigen binding molecule according to any one of clauses 11 to 17, and

[0137] b) detecting binding of the at least bispecific antigen binding molecule to cells in the sample, wherein the binding and in particular specific binding of the at least bispecific antigen binding molecule to the cells identifies leukemic stem cells (LSC) in the sample.

[0138] 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.

[0139] Item 29. A method for identifying mammalian leukemia stem cells (LSCs) in a biological sample according to Article 27 or Article 28, wherein the at least bispecific antigen binding molecule binds to CD123 and CD200 simultaneously.

[0140] Item 30. A method for diagnosing leukemia in a mammalian patient suspected of having leukemia, comprising a method according to any one of Items 27 to 29, and further concluding based on the combination that the mammalian patient has leukemia.

[0141] Item 31. The method according to Item 30, wherein the leukemia is a relapsed, malignant and / or metastatic leukemia, AML, in particular its pediatric form, and CML.

[0142] 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 LSC.

[0143] The present invention will now be further described in the following examples with reference to the accompanying drawings and sequences, however, the present invention is not limited thereto.For the purposes of the present invention, all references cited herein are incorporated by reference in their entirety.

[0144] Figure 1 : A schematic overview of the structure of bsAb constructs currently being evaluated in clinical trials. IgG antibody constructs consist of a Fab region and an Fc region. The binding portion of the Fab region is called a single-chain variable fragment (scFv). The antibody contains two heavy chains (V H and C H ) and two light chains (V L and C L These chains can be arranged in variable domains (V H and V L ) and constant domain (C H and C L ) segmentation. Adapted from Suurs FV et al., 2019.

[0145] Figure 2 :The structure of trifab-contorsbody according to the embodiment of the present invention.When construct combines CD200 and CD123 simultaneously, the inactive dummy chain is replaced by respective CD3 chain, and forms (three specific) functional CD3 domain, and optionally dissociates the inactive dummy chain at the same time.

[0146] Figure 3 : Flow cytometric analysis of PBMC. PBMC were incubated with Kasumi-1 cells and antibodies on 96-well plates for 24 hours. 30,000 Kasumi-1 cells were seeded per well. The effector to target ratio was 5:1. Stimulation was performed with 10 nM antibody. FACS profiles show the expression of activation markers CD25, CD69 and CD137 on cytotoxic CD8 T cells. The individual halves and the combination of CD200K with the non-binding halves scrambled pit (SrcH) were used as controls.

[0147] Figure 4: Killing curve of Kasumi-1mCherry cells. Kasumi-1-mCherry cells were incubated with PBMCs from different healthy donors (HD1-3) and antibodies in 96-well plates for 72 hours. 30,000 Kasumi-1 cells were seeded per well. The effector to target ratio was 5:1. Stimulation was performed with 10 nM antibody. A picture was taken every 2 hours in bright field and mCherry channel. The curve represents the relative increase / decrease of mCherry-positive cells over time. Each curve consists of three technical replicates. X-axis: t, in [h]; Y-axis: log2 fold change of cell count / FOV.

[0148] SEQ ID NO: 1 shows the amino acid sequence of human CD123, isoform 1.

[0149] SEQ ID NO: 2 shows the amino acid sequence of human CD200, isoform 1.

[0150] SEQ ID NO: 3 shows the light chain variable region (V L )'s amino acid sequence.

[0151] SEQ ID NO: 4 shows the heavy chain variable region (V H )'s amino acid sequence.

[0152] SEQ ID NO: 5 shows the amino acid sequence of the light chain of an exemplary antigen binding domain that binds to CD123.

[0153] SEQ ID NO: 6 shows the amino acid sequence of the heavy chain of an exemplary antigen binding domain that binds to CD123.

[0154] SEQ ID NOs: 7 to 9 show the amino acid sequences of the CDRs of the first antigen-binding domain that binds to CD200.

[0155] SEQ ID NOs: 10 to 12 show the amino acid sequences of the CDRs of the second antigen-binding domain that binds to CD200.

[0156] SEQ ID NOs: 13 to 15 show the amino acid sequences of the CDRs of the third antigen-binding domain that binds to CD200.

[0157] SEQ ID NOs: 16 to 18 show the amino acid sequences of the CDRs of the fourth antigen-binding domain that binds to CD200.

[0158] SEQ ID NOs: 19 to 21 show the amino acid sequences of the CDRs of the fifth antigen-binding domain that binds to CD200.

[0159] SEQ ID NOs: 22 to 24 show the amino acid sequences of the CDRs of the sixth antigen-binding domain that binds to CD200.

[0160] SEQ ID NOs: 25 to 27 show the amino acid sequences of the CDRs of the seventh antigen-binding domain that binds to CD200.

[0161] SEQ ID NOs: 28 to 30 show the amino acid sequences of the CDRs of the eighth antigen-binding domain that binds to CD200.

[0162] SEQ ID NOs: 31 to 33 show the amino acid sequences of the CDRs of the ninth antigen-binding domain that binds to CD200.

[0163] SEQ ID NOs: 34 to 37 show the amino acid sequences of the CDRs of the tenth antigen-binding domain that binds to CD200.

[0164] Examples

[0165] The results of the in vitro experiments demonstrated selective binding to LSCs and a controlled and improved, preferably localized, activation of T cells.

[0166] CD123 + / CD200 + An exemplary trifab-contorsbody according to one embodiment of the present invention (preferably a combination of antigen binding domains comprising the domains of CD123 and CD200 as disclosed in SEQ ID No 3 to 23 above) was tested on 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. In addition, a combination of a single half and a scrambled hole (SrcH) of CD200K and a non-binding half was used as a control. A CD200K half with a functional anti-CD3 domain, rather than a "dummy", was used as a positive control (CD200-act). ( Figure 2and Figure 3 ). In other configurations the pestle-mortar portion may be omitted.

[0167] To test the extent to which different combinations / alone halves of the constructs were able to activate T cells, PBMCs were isolated from healthy donors (HD) and cultured with the antibody and Kasumi-1 cells for 24 hours. The T cells were then examined in a flow cytometer for 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 was significantly increased. In contrast, when PBMCs were co-cultured with the individual halves of Kasumi-1 and the antibody, there was no significant activation. The combination of CD200K+ScrH resulted in a slight increase in the CD69 activation marker, however, it lagged significantly behind the marker CD200K+CD123H. Increased expression of CD25 and CD137 could not be determined with CD200K+ScrH. ( Figure 4 )

[0168] In a next step, the inventors then investigated the cytotoxic activity of T cells depending on the TriFab exemplary constructs by means of sequential visualization of in vitro co-cultures with Kasumi-1 cells expressing mCherry. The co-cultures were mixed with the defined antibody halves in various combinations.

[0169] Pictures were taken every 2 hours in the bright field and mCherry channels for 72 hours. For Kasumi-1-mCherry as well as PBMC and individual halves, an increase in mCherry positive cells was observed. However, for the combination of CD200K+CD123H, AML cells were found to be significantly eradicated. The combination of CD200K+ScrH showed no difference from the control without antibody.

[0170] Similar results have been obtained using so-called “patient-derived xenograft leukemia cells,” which are more heterogeneous than cell lines and are therefore more biologically comparable to primary leukemia cells [Vick B, Rothenberg M, N, Carlet M, Finkenzeller C, Krupka C, et al. An advanced preclinicalmouse model for acute myeloid leukemia using patients' cells of various genetic subgroups and in vivo bioluminescence imaging. PLoS One. 2015; 10:e0120925].

[0171] Current in vitro experiments show that the present invention can achieve selective cytotoxicity by spatially and temporally controlling the activation of T cells on the surface of CD123+ / CD200+ AML cells.

[0172] The combination of these two target structures therefore constitutes a new strategy for eliminating leukemic stem cells, especially in pediatric forms of AML.Thus, CD123 / CD200 dependence and favorable local 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 a mammalian leukemia stem cell (LSC), the method comprising the following steps: 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 said candidate antigen-binding molecules from said first library and said second library to generate a library of candidate molecules having at least bispecificity for CD123 and CD200, c) providing a suitable screening system, said 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), and e) identifying binding molecules that specifically bind to the proteins CD123 and CD200 or immunologically recognizable fragments or derivatives thereof from the library of b), wherein preferably the LSC is selected from LSC in acute myeloid leukemia (AML) or in chronic myeloid leukemia (CML).

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

3. The method of claim 1 or 2, wherein the library of candidate molecules that are at least bispecific binders to 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 nanobodies, diabodies, triabodies, tetrabodies or higher order multimers and trifab-contorsbody.

4. The method according to any one of claims 1 to 3, wherein the candidate molecules in the library further comprise at least one third 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 expressing and in particular recombinantly expressing CD123 and CD200 or antigenic fragments thereof, or is a cell-free system.

6. An at least bispecific antigen binding molecule that specifically binds to the proteins CD123 and CD200 of a mammalian leukemia stem cell (LSC), or an immunologically recognizable fragment or derivative thereof, identified using the method according to any one of claims 1 to 5, wherein preferably the LSC is selected from LSC in acute myeloid leukemia (AML) or in chronic myeloid leukemia (CML).

7. The at least bispecific antigen binding molecule according to claim 6, wherein the amino acid sequence of the CD123 binding region is according to EP2778175A1 or is a specific binding sequence having at least 95% identity thereto, and wherein the amino acid sequence of the CD200 binding region is according to EP2178561A1 or is a specific binding sequence having at least 95% identity thereto.

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 at least a bispecific antigen binding molecule according to any one of claims 6 to 8 and a pharmaceutically acceptable carrier and / or adjuvant.

10. The at least bispecific antigen binding molecule according to any one of claims 6 to 8 or the pharmaceutical composition according to claim 9 for use in medicine, preferably for use in preventing or treating cancer in a patient, such as leukemia, e.g. AML or CML, and in particular pediatric forms thereof, 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.

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 avoiding 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, the method comprising a) contacting the sample with at least a bispecific antigen binding molecule according to any one of claims 6 to 8, and b) detecting binding of said at least bispecific antigen binding molecule to cells in said sample, Wherein the binding and in particular specific binding of the at least bispecific antigen binding molecule to the cell identifies a leukemic stem cell (LSC) in the sample.

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

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

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

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