Antibodies that bind to CD123 and the gamma delta T cell receptor

Abstract

The present invention relates to an antibody capable of binding to human CD123 and to the V52 chain of the human Vy9V52 T cell receptor. The present invention further relates to a pharmaceutical composition comprising the antibody of the invention and to the use of the antibody of the invention for medical treatment.

Description

[Technical field]

[0001] The present invention relates to novel multispecific antibodies capable of binding to human CD123 and to the V52 chain of the human Vy9V52 T cell receptor. The present invention further relates to pharmaceutical compositions comprising the antibodies of the invention and to the use of the antibodies of the invention for medical treatment. [Background technology]

[0002] CD123, or the interleukin-3 (IL3) receptor α chain, is a membrane protein that transduces signaling by IL3, a cytokine involved in blood cell production. CD123 forms a heterodimer with the common β chain CD131. CD123 is normally expressed by several types of blood cells, such as plasmacytoid dendritic cells or monocytes, and by a subset of normal myeloid progenitor cells. However, CD123 is strongly overexpressed on leukemic stem cells in patients with acute myeloid leukemia. Thus, CD123 is a potential therapeutic target in several hematological malignancies, including acute myeloid leukemia.

[0003] Several bispecific CD123-CD3 T cell-engaging antibodies have been described (Kuo et al. (2012) Protein Eng Des Set 10:561; Al-Hussaini et al. (2016) Blood 127:122). Bispecific T cell-engaging antibodies have tumor target binding specificity and T cell binding specificity, thus enhancing efficacy by redirecting T cell cytotoxicity to malignant cells, see, for example, Huehls et al. (2015) Immunol Cell Biol 93:290; Ellerman (2019) Methods, 154:102; de Bruin et al. (2017) Oncoimmunology 7(1):e1375641 and WO2015156673. However, the results are very different. For example, in one study combining a CD3-binding moiety with binding moieties for eight different B cell targets (CD20, CD22, CD24, CD37, CD70, CD79b, CD138, and HLA-DR), bispecific antibodies targeting different tumor targets showed strong variation in their ability to induce target cell cytotoxicity and cytotoxicity. It was found that efficacy did not correlate with antigen expression levels. For example, CD3-based bispecific antibodies targeting HLA-DR or CD138 failed to induce cytotoxicity despite intermediate to high HLA-DR and CD138 expression levels (Engelberts et al. (2020) Ebiomedicine 52:102625). Most of the T cell redirection therapies have not reached late-stage clinical development, likely due to significant toxicity, manufacturing issues, immunogenicity, narrow therapeutic window and low response. In particular, toxicity may occur when the T cell engager contains a CD3-binding arm, resulting in uncontrolled, exaggerated, immune activation and cytokine release.

[0004] Thus, although significant progress has been made, there remains a need for novel CD123-targeting antibodies that are therapeutically effective but have acceptable toxicity, as well as stability and manufacturability. Summary of the Invention

[0005] The present invention provides novel antibodies for CD123-based therapy. A bispecific antibody was constructed that combines a single domain CD123 binding region with a binding region that can bind to the V52 chain of the human Vy9V52 T cell receptor and thus engage γδ T cells. Surprisingly, the bispecific antibody was highly potent in mediating the activation of Vy9V52 T cells and inducing the killing of CD123-expressing cell lines as well as patient-derived tumor cells in the presence of Vy9V52 T cells.

[0006] Thus, in a first aspect, the invention provides a multispecific antibody comprising a first antigen-binding region capable of binding to human CD123 and a second antigen-binding region capable of binding to the V52 chain of the human Vy9V52 T cell receptor.

[0007] In a further main aspect, the present invention provides an antibody comprising a first antigen-binding region capable of binding to human CD123, said first antigen-binding region being a single domain antibody, (i) comprising a VH CDR1 sequence as set forth in SEQ ID NO: 2, a VH CDR2 sequence as set forth in SEQ ID NO: 3, and a VH CDR3 sequence as set forth in SEQ ID NO: 4, wherein preferably the first antigen-binding region comprises or consists of a sequence selected from the group of sequences set forth in SEQ ID NOs: 1, 25 to 34, or a sequence having at least 90%, such as at least 92%, for example at least 94%, for example at least 96%, for example at least 98% sequence identity to a sequence selected from the group of sequences set forth in SEQ ID NOs: 1, 25 to 34, or (ii) An antibody comprising a VH CDR1 sequence as set forth in SEQ ID NO: 10, a VH CDR2 sequence as set forth in SEQ ID NO: 11 and a VH CDR3 sequence as set forth in SEQ ID NO: 12, wherein preferably said first antigen-binding region comprises or consists of a sequence as set forth in SEQ ID NO: 9 or a sequence having at least 90%, such as at least 92%, for example at least 94%, such as at least 96%, for example at least 98% sequence identity to said sequence as set forth in SEQ ID NO: 9.

[0008] Further aspects and embodiments of the invention are described below. [Brief description of the drawings]

[0009] [Figure 1] ELISA showing binding of all different bispecific VHHs to CD123, Vy9V52 (GDT) TCR and BSA (as negative control). OD values ​​are shown, values ​​are the mean of duplicate measurements and error bars indicate standard error of the mean. A monovalent anti-V52 VHH was used as a control for TCR staining ("TCR control") and a commercially available anti-CD123 antibody ("anti-CD123") was used as a control for CD123 antigen coating. "No AB" indicates a negative control without primary antibody. [Diagram 2] Figure 2 shows the specificity of binding of bispecific VHHs using flow cytometry. The geometric mean of the fluorescent signal is plotted as a function of the antibody and cell type used. A VHH recognizing EGFR endogenously expressed by 293F cells was included as a positive control. A monovalent anti-V52 VHH was used as a negative control VHH. [Diagram 3] Figure 1 shows the apparent affinity of 1D2-5C8var1 for binding to CD123 determined by flow cytometry. A dilution series of purified 1D2-5C8var1 was tested for binding to 293F cells transiently expressing CD123 or CD131, or both CD123 and -131. The geometric mean of fluorescence intensity is plotted as a function of the antibody concentration used. EC50 values ​​were determined by curve fitting. [Figure 4] Representative BLI analysis of 1D2-5C8var1 binding to CD123. The shift in light reflectance (measured in nm), representative of protein mass binding, is plotted as a function of time. 0-300 sec: association phase, 300-900 sec: dissociation phase. [Diagram 5] Figure 2 shows C1R-neo target cell-dependent, 1D2-5C8var1-mediated Vγ9Vδ2 T cell activation. The percentage of CD3+-Vγ9+ T cells showing CD107A expression (degranulation) is plotted as a function of the concentration of antibody used. Experiments using two different donors are depicted. [Figure 6] Figure 1 shows 1D2-5C8var1 induced, Vy9V52 T cell mediated C1R-neo target cell cytotoxicity. The percentage of live C1R-neo target cells is plotted as a function of the concentration of bispecific VHH used. Data obtained with two different donors of Vy9V52 T cells are shown. [Figure 7] Figure 1 shows bispecific VHH-induced, Vy9V52 T cell-mediated THP-1 target cell cytotoxicity. The percentage of viable THP-1 target cells is plotted as a function of the concentration bispecific VHH used. [Figure 8] Figure 1 shows bispecific VHH-mediated Vy9V52 T cell activation and bispecific VHH-mediated T cell-induced lysis of a primary AML sample from a patient. The top panel shows T cell activation as measured by CD107A expression. The bottom panel shows lysis of AML blasts by T cells in conjunction with bispecific VHH. [Figure 9] Figure 2 shows the HP-SEC profile of purified anti-CD123xVy9V52 TCR bispecific antibody 1D2-5C8var1(Y105F)-Fc. [Figure 10] Figure 1 shows that 1D2-5C8var1(Y105F)-Fc induces Vγ9Vδ2 T cell activation. A typical experiment is shown. The percentage of CD107a- (lysosomal-associated protein-1, or LAMP-1) positive Vγ9Vδ2 cells is shown as a function of the concentration of compound used. EC50 values ​​(in pM, determined by curve fitting) are shown below the graph. Data points are the average of triplicate determinations. Error bars represent standard deviation. [Figure 11] Figure 1 shows 1D2-5C8var1(Y105F)-Fc-induced T cell-mediated target cell lysis. A typical experiment is shown. The graph shows the percentage of target cells killed after 24 hours of co-culture as a function of the concentration of compound used. EC50 values ​​(in pM, determined by curve fitting) are shown below the graph. Data points are the average of triplicate determinations. Error bars represent standard deviation. [Figure 12]CD123 expression levels on plasmacytoid dendritic cells (top panel) and THP-1 cell line (bottom panel). Representative staining is shown. Histograms showing unstained cells, isotype control staining (left overlapping histogram), and staining for CD123 (right peak) are shown. Number of events (Y-axis) is shown as a function of fluorescence intensity (X-axis). [Figure 13] 1D2-5C8var1(Y105F)-Fc induces preferential killing of THP-1 cells compared to pDC. Representative results are shown. The percentage of killed target cells is shown as a function of the concentration of compound used per target cell population (i.e., THP-1 or pDC). EC50 values ​​(in pM, determined by curve fitting) are shown below the graph. Data points are the average of triplicate determinations. Error bars represent standard deviation. [Figure 14] The primary amino acid sequence of full-length human CD123 (GenBank Accession No. NM_002183.4) (SEQ ID NO: 23) is shown. Residues found to be cross-linked by the 1D2 antibody are in bold and underlined. Residues in italics (adjacent to the reactive residues found) may also be part of the recognized epitope. [Figure 15] Cα trace model of CD123 (IL-3 receptor α chain: Broughton et al., 2018 2018 Nat Commun. 9:386), showing residues found to be cross-linked by the antibody. The transmembrane helix is ​​located on the left side of the diagram. [Figure 16A-1] Stress-induced changes as determined by size exclusion chromatography, and thus measured aggregates and fragments, under denaturing (SDS) conditions (CE-SDS) and after reduction and detection by UV absorption (SEC-UV). [Figure 16A-2] Stress-induced changes as determined by size exclusion chromatography, and thus measured aggregates and fragments, under denaturing (SDS) conditions (CE-SDS) and after reduction and detection by UV absorption (SEC-UV). [Figure 16A-3]Stress-induced changes as determined by size exclusion chromatography, and thus measured aggregates and fragments, under denaturing (SDS) conditions (CE-SDS) and after reduction and detection by UV absorption (SEC-UV). [Figure 16A-4] Stress-induced changes as determined by size exclusion chromatography, and thus measured aggregates and fragments, under denaturing (SDS) conditions (CE-SDS) and after reduction and detection by UV absorption (SEC-UV). [Figure 16A-5] Stress-induced changes as determined by size exclusion chromatography, and thus measured aggregates and fragments, under denaturing (SDS) conditions (CE-SDS) and after reduction and detection by UV absorption (SEC-UV). [Figure 16B-1] Stress-induced changes as determined by aggregates and fragments measured by capillary gel electrophoresis under denaturing (SDS) conditions (CE-SDS) and after reduction are shown. [Figure 16B-2] Stress-induced changes as determined by aggregates and fragments measured by capillary gel electrophoresis under denaturing (SDS) conditions (CE-SDS) and after reduction are shown. [Figure 16B-3] Stress-induced changes as determined by aggregates and fragments measured by capillary gel electrophoresis under denaturing (SDS) conditions (CE-SDS) and after reduction are shown. [Figure 16B-4] Stress-induced changes as determined by aggregates and fragments measured by capillary gel electrophoresis under denaturing (SDS) conditions (CE-SDS) and after reduction are shown. [Figure 16B-5] Stress-induced changes as determined by aggregates and fragments measured by capillary gel electrophoresis under denaturing (SDS) conditions (CE-SDS) and after reduction are shown. [Figure 17A] Degranulation is analyzed after 4 h by measuring the percentage of CD107a (lysosomal-associated protein-1, or LAMP-1) positive cells via flow cytometry. [Figure 17B]T cell activation analyzed by measuring the percentage of CD25 positive cells is shown. [Figure 17C] Cytotoxicity is analyzed by determining the percentage of viable target cells after 24 hours via flow cytometry. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] definition The term "human CD123" as used herein refers to the human CD123 protein, also known as the interleukin-3 receptor alpha chain (GenBank Accession No. NM_002183.4, NCBI Reference Sequence: NP_002174.1). The sequence of human CD123 is set forth in SEQ ID NO: 23. The IL3 receptor is a heterodimer of CD123 with a common beta chain, CD131 (NCBI Reference Sequence: NP_000386.1). CD131 is set forth in SEQ ID NO: 24.

[0011] The term "human V52" as used herein refers to the rearranged 52 chain of the Vy9V52-T cell receptor (TCR) (SEQ ID NO: 48). UniProtKB-A0JD36 (A0JD36_HUMAN) shows an example of a variable TRDV2 sequence.

[0012] The term "human Vy9" as used herein refers to the rearranged y9 chain of the Vy9V52-T cell receptor (TCR). UniProtKB-Q99603_HUMAN shows an example of a variable TRGV9 sequence.

[0013] The term "antibody" is intended to refer to an immunoglobulin molecule, a fragment of an immunoglobulin molecule, or any derivative thereof, which has the ability to specifically bind to an antigen under typical physiological conditions for a significant period of time, e.g., a half-life of at least about 30 minutes, at least about 1 hour, at least about 2 hours, at least about 8 hours, at least about 12 hours, about 24 hours or more, about 48 hours or more, about 3, 4, 5, 6, 7 days, or more, or any other relevant functionally defined period of time (e.g., a time sufficient to induce, promote, enhance, and / or modulate a physiological response associated with the antibody binding to the antigen and / or a time sufficient for the antibody to acquire effector activity). The antigen-binding region that interacts with the antigen may include the variable regions of both the heavy and light chains of the immunoglobulin molecule, or may include or consist of a single domain antigen-binding region, e.g., only the heavy chain variable region. The constant region of the antibody, when present, may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (such as effector cells and T cells), as well as components of the complement system, such as C1q, the first component in the classical pathway of complement activation. However, in some embodiments, the Fc region of the antibody is modified to be inactive, where "inactive" means an Fc region that is at least unable to bind any Fcγ receptors or induce Fc-mediated cross-linking of FcRs or induce FcR-mediated cross-linking of target antigens via the two Fc regions of the individual antibodies. In further embodiments, the inactive Fc region is additionally unable to bind C1q. In one embodiment, the antibody contains mutations at positions 234 and 235 (Canfield and Morrison (1991) J Exp Med 173:1483), such as a Leu to Phe mutation at position 234 and a Leu to Glu mutation at position 235 (according to EU numbering, see below). In another embodiment, the antibody contains a Leu to Ala mutation at position 234, a Leu to Ala mutation at position 235, and a Pro to Gly mutation at position 329. In another embodiment, the antibody contains a Leu to Phe mutation at position 234, a Leu to Glu mutation at position 235, and an Asp to Ala mutation at position 265.

[0014] The Fc region of an immunoglobulin is typically defined as the fragment of an antibody generated after digestion of the antibody with papain that contains the two CH2-CH3 regions of the immunoglobulin and the connecting region (e.g., hinge region). The constant domains of the antibody heavy chain define the antibody isotype, e.g., IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgM, IgD, or IgE. The Fc region, together with cell surface receptors called Fc receptors and proteins of the complement system, mediate the effector functions of the antibody.

[0015] The term "hinge region" as used herein is intended to refer to the hinge region of an immunoglobulin heavy chain. Thus, for example, the hinge region of a human IgG1 antibody corresponds to amino acids 216 to 230 according to EU numbering.

[0016] The term "CH2 region" or "CH2 domain" as used herein is intended to refer to the CH2 region of an immunoglobulin heavy chain. Thus, for example, the CH2 region of a human IgG1 antibody corresponds to amino acids 231-340 according to EU numbering. However, the CH2 region may also be of any of the other subtypes described herein.

[0017] The term "CH3 region" or "CH3 domain" as used herein is intended to refer to the CH3 region of an immunoglobulin heavy chain. Thus, for example, the CH3 region of a human IgG1 antibody corresponds to amino acids 341-447 according to EU numbering. However, the CH3 region may also be of any of the other subtypes described herein.

[0018] References to amino acid positions in the Fc region / Fc domain herein are according to EU numbering (Edelman et al., Proc Natl Acad Sci USA. 1969 May; 63(1):78-85; Kabat et al., Sequences of proteins of immunological interest. 5th Edition-1991 NIH Publication No. 91-3242).

[0019] As indicated above, the term antibody as used herein includes fragments of antibodies that retain the ability to specifically bind to an antigen, unless otherwise stated or clearly contradicted by the context. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed by the term "antibody" include (i) Fab' or Fab fragments, i.e., monovalent fragments consisting of the VL, VH, CL and CH1 domains, or monovalent antibodies as described in WO2007059782; (ii) F(ab')2 fragments, i.e., bivalent fragments comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) Fd fragments consisting essentially of the VH and CH1 domains; and (iv) Fv fragments consisting essentially of the VL and VH domains of a single arm of an antibody. Furthermore, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, they can be joined by a synthetic linker that allows them to be produced as a single protein chain using recombinant techniques, with the VL and VH regions pairing to form a monovalent molecule (known as a single-chain antibody or single-chain Fv (scFv)) (see, for example, Bird et al., Science 242, 423-426 (1988) and Huston et al., PNAS USA 85, 5879-5883 (1988)). Such single-chain antibodies are encompassed by the term antibody unless the context clearly indicates otherwise. The term antibody also includes polyclonal antibodies, monoclonal antibodies (mAbs), chimeric antibodies and humanized antibodies, as well as antibody fragments provided by any known technique, such as enzymatic cleavage, peptide synthesis, and recombinant techniques, unless otherwise specified.

[0020] In some embodiments of the antibody of the present invention, the first antigen-binding region or the second antigen-binding region, or both, are single domain antibodies. Single domain antibodies (sdAbs, also called Nanobodies®, or VHHs) are well known to those skilled in the art, see, for example, Hamers-Casterman et al. (1993) Nature 363:446, Roovers et al. (2007) Curr Opin Mol Ther 9:327, and Krah et al. (2016) Immunopharmacol Immunotoxicol 38:21. Single domain antibodies contain a single CDR1, a single CDR2, and a single CDR3. Examples of single domain antibodies are heavy chain only antibodies, antibodies that do not naturally contain light chains, single domain antibodies derived from conventional antibodies, and variable fragments of engineered antibodies. Single domain antibodies can be derived from any species, including mouse, human, camel, llama, shark, goat, rabbit, and cow. For example, naturally occurring VHH molecules can be derived from antibodies produced in Camelidae species, such as camel, dromedary, llama, alpaca, and guanaco. Like whole antibodies, single domain antibodies can selectively bind to a specific antigen. Single domain antibodies can contain only the variable domains of an immunoglobulin chain, i.e., CDR1, CDR2, and CDR3, as well as framework regions.

[0021] The term "immunoglobulin" as used herein is intended to refer to a class of structurally related glycoproteins that typically consist of two pairs of polypeptide chains, one pair of light (L) chains, and one pair of heavy (H) chains, all four potentially interconnected by disulfide bonds, although some mammalian species do not produce light chains and only make heavy chain antibodies. The terms "immunoglobulin heavy chain", "immunoglobulin heavy chain" or "heavy chain" as used herein are intended to refer to one of the chains of an immunoglobulin. A heavy chain typically consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region (abbreviated herein as CH), which defines the immunoglobulin isotype. The heavy chain constant region typically consists of three domains, CH1, CH2, and CH3. The heavy chain constant region further comprises a hinge region. Within the structure of an immunoglobulin (e.g., IgG), the two heavy chains are interconnected via disulfide bonds in the hinge region. Like the heavy chain, each light chain typically consists of several regions, the light chain variable region (VL) and the light chain constant region (CL). The VH and VL regions can be further subdivided into regions of hypervariability (or hypervariable regions that may be hypervariable in sequence and / or form structurally defined loops), also called complementarity determining regions (CDRs), and interspersed with more conserved regions, called framework regions (FRs). Each VH and VL typically consists of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The CDR sequences can be determined using various methods, for example, the methods provided by Choitia and Lesk (1987) J. Mol. Biol. 196:901 or Kabat et al. (1991) Sequence of protein of immunological interest, fifth edition. NIH publication. Various methods for CDR determination and amino acid numbering can be compared on www.abysis.org (UCL).

[0022] The term "isotype" as used herein refers to an immunoglobulin (sub)class (e.g., IgG1, IgG2, IgG3, IgG4, IgD, IgA, IgE, or IgM) or any allotype thereof, such as IgG1m(za) and IgG1m(f), encoded by heavy chain constant region genes. Each heavy chain isotype can be combined with either a kappa (κ) or lambda (λ) light chain. The antibodies of the present invention can have any isotype.

[0023] The term "parent antibody" should be understood as an antibody that is identical to the antibody according to the present invention, but where the parent antibody does not have one or more of the specified mutations. A "variant" or "antibody variant" or "variant of parent antibody" of the present invention is an antibody molecule that contains one or more mutations compared to the "parent antibody". An amino acid substitution may replace a natural amino acid with another naturally occurring amino acid, or a non-naturally occurring amino acid derivative. An amino acid substitution may be conservative or non-conservative. In the context of the present invention, a conservative substitution may be defined by a substitution within a class of amino acids reflected in one or more of the following three tables: [Table 1] [Table 2] [Table 3]

[0024] In the context of the present invention, substitutions in the variants are indicated as follows: Original amino acid - position - replacement amino acid To denote amino acid residues, three letter codes, including the codes Xaa and X, or one letter codes are used. Thus, the designation "T366W" means that the variant contains a substitution of threonine with tryptophan at the variant amino acid position corresponding to amino acid position 366 in the parent antibody.

[0025] Additionally, the term "substitution" includes substitutions with any one of the other 19 naturally occurring amino acids, or with other amino acids, such as unnatural amino acids. For example, substitutions of the amino acid T at position 366 include each of the following substitutions: 366A, 366C, 366D, 366G, 366H, 366F, 366I, 366K, 366L, 366M, 366N, 366P, 366Q, 366R, 366S, 366E, 366V, 366W, and 366Y.

[0026] The term "full length antibody," as used herein, refers to an antibody that contains all the heavy and light chain constant and variable domains corresponding to those normally found in a wild-type antibody of that isotype.

[0027] The term "chimeric antibody" refers to an antibody in which the variable region is derived from a non-human species (e.g., from a rodent) and the constant region is derived from a different species, such as human. Chimeric antibodies can be produced by genetic engineering. Chimeric monoclonal antibodies for therapeutic use are developed to reduce antibody immunogenicity.

[0028] The term "humanized antibody" refers to a genetically engineered non-human antibody containing a human antibody constant domain and a non-human variable domain that has been modified to contain a high level of sequence homology with the human variable domain. This can be achieved by grafting the six non-human antibody complementarity determining regions (CDRs), which together form the antigen binding site, into a homologous human acceptor framework region (FR). Substitution of framework residues from the parent antibody (i.e., non-human antibody) into human framework regions (backmutations) may be required to fully reconstitute the binding affinity and specificity of the parent antibody. Structural homology modeling can help identify amino acid residues in the framework regions that are important for the binding properties of the antibody. Thus, a humanized antibody may comprise non-human CDR sequences, primarily human framework regions optionally containing one or more amino acid backmutations to the non-human amino acid sequences, and optionally a fully human constant region. Optionally, additional amino acid modifications, not necessarily backmutations, can be introduced to obtain a humanized antibody with favorable properties such as affinity and biochemical properties. Humanization of non-human therapeutic antibodies is performed to minimize their immunogenicity in humans, while at the same time maintaining the specificity and binding affinity of the antibody of non-human origin.

[0029] The term "multispecific antibody" refers to an antibody that has specificity for at least two different, e.g., at least three, typically non-overlapping epitopes due to the presence of two or more antigen-binding regions. Such epitopes may be on the same target antigen or on different target antigens. When epitopes are on different targets, such targets may be on the same cell or on different cells or cell types.

[0030] The term "bispecific antibody" refers to an antibody that has specificity for two different, typically non-overlapping epitopes due to the presence of two antigen-binding regions. Such epitopes may be on the same target or on different targets. When epitopes are on different targets, such targets may be on the same cell or on different cells or cell types.

[0031] Examples of different classes of bispecific antibodies include, but are not limited to, (i) IgG-like molecules with complementary CH3 domains to force heterodimerization, (ii) recombinant IgG-like dual targeting molecules, where the two sides of the molecule each contain a Fab fragment or a portion of a Fab fragment of at least two different antibodies, (iii) IgG fusion molecules, where a full-length IgG antibody is fused to an extra Fab fragment or a portion of a Fab fragment, (iv) Fc fusion molecules, where a single chain Fv molecule or a stabilized diabody is fused to a heavy chain constant domain, Fc region or a portion thereof. (v) Fab fusion molecules, where different Fab fragments are fused together and fused to a heavy chain constant domain, Fc region or part thereof, and (vi) scFv and bispecific based antibodies and heavy chain antibodies (e.g. domain antibodies, Nanobodies®) where different single chain Fv molecules or different bispecific antibodies or different heavy chain antibodies (e.g. domain antibodies, Nanobodies®) are fused to each other or to another protein or carrier molecule that is fused to a heavy chain constant domain, Fc region or part thereof.

[0032] Examples of IgG-like molecules with complementary CH3 domain molecules include, but are not limited to, Triomab® (Trion Pharma / Fresenius Biotech), Knobs-into-Hole (Genentech), CrossMAb (Roche) and electrostatically matched (Amgen, Chugai, Oncomed), LUZ-Y (Genentech, Wranik et al. J. Biol. Chem. 2012, 287(52):43331-9, doi:10.1074 / jbc.M112.397869. Epub 2012 Nov 1), DIG-body and PIG-body (Pharmabcine, WO2010134666, WO2014081202), Strand Exchange Engineered Domain body (SEEDbody) (EMD Serono), Biclonics (Merus, WO2013157953), FcΔAdp (Regeneron), bispecific IgG1 and IgG2 (Pfizer / Rinat), Azymetric scaffold (Zymeworks / Merck), mAb-Fv (Xencor), bivalent bispecific antibodies (Roche, WO2009080254) and DuoBody® molecules (Genmab).

[0033] Examples of recombinant IgG-like dual targeting molecules include, but are not limited to, Dual Targeting (DT)-Ig (GSK / Domantis, WO2009058383), Two-in-one Antibody (Genentech, Bostrom, et al 2009. Science 323, 1610-1614), Cross-linked Mabs (Karmanos Cancer Center), mAb2 (F-Star), Zybodies™ (Zyngenia, LaFleur et al. MAbs. 2013 Mar-Apr;5(2):208-18), approaches with common light chain, κλBodies (NovImmune, WO2012023053), and CovX-body® (CovX / Pfizer, Doppalapudi, VR, et al. 2007.Bioorg.Med.Chem.Lett.17,501-506).

[0034] Examples of IgG fusion molecules include, but are not limited to, dual variable domain (DVD)-Ig (Abbott), dual domain dual head antibody (Unilever, Sanofi Aventis), IgG-like Bispecific (ImClone / Eli Lilly, Lewis et al. Nat Biotechnol. 2014 Feb;32(2):191-8), Ts2Ab (MedImmune / AZ, Dimasi et al. J Mol Biol. 2009 Oct 30;393(3):672-92), and BsAb (Zymogenetics, WO2010111625), HERCULES (Biogen Idec), scFv fusion (Novartis), scFv fusion (Changzhou Adam Biotech Inc), and TvAb (Roche).

[0035] Examples of Fc fusion molecules include, but are not limited to, scFv / Fc fusions (Academic Institution, Pearce et al Biochem Mol Biol Int. 1997 Sep;42(6):1179), SCORPION (Emergent BioSolutions / Trubion, Blankenship JW, et al. AACR 100th Annual meeting 2009(Abstract #5465), Zymogenetics / BMS, WO2010111625), Dual Affinity Retargeting Technology (Fc-DART™) (MacroGenics), and Dual(ScFv)2-Fab (National Research Center for Antibody Medicine-China).

[0036] Examples of Fab fusion bispecific antibodies include, but are not limited to, F(ab)2 (Medarex / AMGEN), Dual-Action or Bis-Fab (Genentech), Dock-and-Lock® (DNL) (ImmunoMedics), Bivalent Bispecific (Biotecnol) and Fab-Fv (UCB-Celltech).

[0037] Examples of scFv-based, bispecific-based antibodies, and domain antibodies include, but are not limited to, Bispecific T Cell Engager (BiTE®) (Micromet, Tandem Diabody (Tandab) (Affimed), Dual Affinity Retargeting Technology (DART™) (MacroGenics), Single-chain Diabody (Academic, Lawrence FEBS Lett. 1998 Apr 3; 425(3): 479-84), TCR-like Antibodies (AIT, ReceptorLogics), Human Serum Albumin ScFv Fusion (Merrimack, WO2010059315) and COMBODY molecules (Epigen Biotech, Zhu et al. Immunol Cell Biol. 2010 Aug; 88(6): 667-75), dual targeting nanobodies® (Ablynx, Hmila et al., FASEB J. 2010), and dual targeting heavy chain single domain antibodies. In some embodiments, the multispecific antibodies of the invention are in VHH-Fc format, i.e., the antibodies comprise two or more single domain antigen binding regions linked to each other via a human Fc region dimer. In this format, each single domain antigen binding region is fused to an Fc region polypeptide, and the two fusion polypeptides form a dimeric bispecific antibody via disulfide bridges in the hinge region. Such constructs typically do not contain the complete or any CH1 or light chain sequences. Figure 12B of WO06064136 provides an illustration of one example of this embodiment.

[0038] In the context of an antibody that binds to an antigen, the terms "bind" or "specifically bind" refer to an antibody that binds to an antigen, typically at least about 10 -6 M or less, e.g., 10 -7 M or less, e.g., about 10 -8 M or less, e.g., about 10 -9M or less, about 10 -10 M or less, or about 10 -11 K below M D It refers to the binding of an antibody to a given antigen or target (e.g., human CD123 or Vδ2) with an affinity corresponding to K D The K value can be determined, for example, using surface plasmon resonance (SPR) technology on a BIAcoreT200, or biolayer interferometry (BLI) on an OctetRED96 instrument, using the antigen as the ligand and the binding moiety or binding molecule as the analyte. Specific binding means that the antibody has a K that is at least 10 times lower, e.g., at least 100 times lower, e.g., at least 1,000 times lower, e.g., at least 10,000 times lower, e.g., at least 100,000 times lower, than the affinity for binding to a non-specific antigen other than the given antigen or a closely related antigen (e.g., BSA, casein). D The degree of affinity is determined by the K D depends on the K D If K is very low (i.e., if the binding moiety or binding molecule is very specific), the degree to which the affinity for the antigen is lower than the affinity for a nonspecific antigen may be at least 10,000-fold. D The term "(M)" as used herein refers to the dissociation equilibrium constant of a particular interaction between an antigen and a binding moiety or binding molecule.

[0039] In the context of the present invention, "competition" or "competable" or "competing" refers to a detectably significant decrease in the tendency of a particular binding molecule (e.g., a CD123 antibody) to bind to a particular binding partner (e.g., CD123) in the presence of another molecule (e.g., a different CD123 antibody) that binds to the binding partner. Typically, competition means a decrease of at least about 25%, e.g., at least about 50%, e.g., at least about 75%, e.g., at least 90% of binding caused by the presence of another molecule, such as an antibody, as determined, e.g., by ELISA analysis or flow cytometry using sufficient amounts of two or more competing molecules, e.g., antibodies. Additional methods for determining binding specificity by competitive inhibition can be found, for example, in Harlow et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1988), Colligan et al., eds., Current Protocols in Immunology, Greene Publishing Assoc, and Wiley InterScience NY, (1992, 1993), and Muller, Meth. Enzymol. 92, 589-601 (1983). In one embodiment, the antibody of the invention binds to the same epitope on CD123 as antibody 1D2 or 1A3, and / or the same epitope on V52 as antibody 5C8, 6H4, 6C1, 5D3 (WO2015156673) or 5C8var1 (WO2020060405). The epitope of 5C8 has been determined to include residues S33, S43, and K45 (SEQ ID NO: 48). The epitope of 6H4 has been determined to include residues R139, K152, S189, and S191 (SEQ ID NO: 48). There are several methods available for mapping antibody epitopes on target antigens known in the art, including, but not limited to, cross-linking mass spectrometry, which allows identification of peptides that are part of the epitope, and X-ray crystallography, which identifies individual residues on the antigen that form the epitope.Epitope residues can be determined to be all amino acid residues with at least one atom 5 Å or less from the antibody. 5 Å was chosen as the epitope cutoff distance to account for atoms within the van der Waals radius and possible water-mediated hydrogen bonds. Epitope residues can then be determined to be all amino acid residues with at least one atom 8 Å or less. 8 Å or less is chosen as the epitope cutoff distance to account for the length of the extended arginine amino acid. Crosslinking mass spectrometry begins with conjugating the antibody and antigen with a mass-tagged chemical crosslinker. The presence of the complex is then confirmed using high-mass MALDI detection. After chemical crosslinking, the Ab / Ag complex is very stable, so many different enzymes and digestion conditions can be applied to the complex to provide many different overlapping peptides. Identification of these peptides is performed using high-resolution mass spectrometry and MS / MS techniques. Identification of the crosslinked peptides is determined using mass tags linked to the crosslinking reagent. After MS / MS fragmentation and data analysis, the cross-linked antigen-derived peptide is part of the epitope and the antibody-derived peptide is part of the paratope. All residues between the most N- and C-terminal cross-linked residues from each cross-linked peptide found are considered to be part of the epitope or paratope.

[0040] The terms "first" and "second" antigen-binding region as used herein do not refer to their orientation / location on the antibody, i.e., they have no meaning with respect to the N-terminus or C-terminus. The terms "first" and "second" serve only to accurately and consistently refer to two different antigen-binding regions in the claims and description.

[0041] By "capable of binding to human CD123" is meant that the antibody is capable of binding to human CD123 as a separate molecule and / or as part of a CD123 / CD131 complex. However, the antibody does not bind to CD131 as a separate molecule.

[0042] By "capable of binding to the V52 chain of a Vy9V52-TCR" is meant that the antibody is capable of binding to the V52 chain as a separate molecule and / or as part of a Vy9V52-TCR. However, the antibody does not bind to the Vy9 chain as a separate molecule.

[0043] "Percent sequence identity," as used herein, refers to the number of identical nucleotide or amino acid positions shared by different sequences (i.e., % identity = # of identical positions / total # of positions x 100), taking into account the number of gaps and the length of each gap that need to be introduced for optimal alignment. Percent identity between two nucleotide or amino acid sequences may be determined, for example, using the algorithm of E. Meyers and W. Miller, Comput. Appl. Biosci 4, 11-17 (1988), as incorporated into the ALIGN program (version 2.0), using the PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4.

[0044] Further aspects and embodiments of the invention As mentioned above, in a first main aspect, the present invention relates to a multispecific antibody comprising a first antigen-binding region capable of binding to human CD123 and a second antigen-binding region capable of binding to the V52 chain of the human Vy9V52 T cell receptor.

[0045] In one embodiment, the multispecific antibody is a bispecific antibody. In another embodiment, the multispecific antibody is a trispecific antibody. In another embodiment, the first antigen-binding region is a single domain antibody, e.g., a single domain antibody consisting of a heavy chain variable region. In another embodiment, the second antigen-binding region is a single domain antibody, e.g., a single domain antibody consisting of a heavy chain variable region. In a further embodiment, both the first antigen-antigen binding region and the second antigen-binding region are single domain antibodies, e.g., single domain antibodies each consisting of a heavy chain variable region.

[0046] In a further embodiment, the multispecific antibody is a bispecific antibody, wherein the first antigen-binding region is a single domain antibody and the second antigen-binding region is a single domain antibody, said bispecific antibody optionally comprising further sequences such as a linker and / or an immunoglobulin Fc region.

[0047] In one embodiment, the multispecific antibody competes (i.e. is capable of competing) with an antibody having the sequence set forth in SEQ ID NO: 1 for binding to human CD123, preferably the multispecific antibody binds to the same epitope on human CD123 as the antibody having the sequence set forth in SEQ ID NO: 1. In one embodiment, the multispecific antibody binds to an epitope that includes one or more residues within the region of S203 to R273, such as an epitope contained entirely within the region of S203 to R273, as determined as described in Example 11 herein.

[0048] In another embodiment, the multispecific antibody binds to an epitope on human CD123 comprising one or more residues within the region S203 to T214, one or more residues within the region H221 to K227, one or more residues within the region Y238 to K244, and one or more residues within the region Y268 to R273 (Figure 14).

[0049] In another embodiment, the multispecific antibody binds to an epitope on human CD123 that includes one, more than one or all of residues S203, T209, T214, H221, H225, K227, Y238, K244, Y268, T269 and R273 (Figure 14).

[0050] In one embodiment, the first antigen-binding region comprises a VH CDR1 sequence set forth in SEQ ID NO:2, a VH CDR2 sequence set forth in SEQ ID NO:3, and a VH CDR3 sequence set forth in SEQ ID NO:4.

[0051] In one embodiment, in SEQ ID NO: 2, X1 is G. In another embodiment, X1 is S.

[0052] In one embodiment, in SEQ ID NO: 3, X2 is A. In another embodiment, X2 is T.

[0053] In one embodiment, in SEQ ID NO: 4, X3 is Y. In another embodiment, X3 is F.

[0054] In one embodiment, X1 is G, X2 is A, and X3 is Y.

[0055] In another embodiment, X 1 is G, X 2 is A, and X 3 is F.

[0056] In another embodiment, X1 is G, X2 is T, and X3 is Y.

[0057] In another embodiment, X 1 is G, X 2 is T, and X 3 is F.

[0058] In one embodiment, X1 is S, X2 is A, and X3 is Y.

[0059] In another embodiment, X 1 is S, X 2 is A, and X 3 is F.

[0060] In another embodiment, X 1 is S, X 2 is T, and X 3 is Y.

[0061] In another embodiment, X 1 is S, X 2 is T, and X 3 is F.

[0062] In one embodiment, the first antigen-binding region comprises or consists of a sequence set forth in SEQ ID NO:1 or a sequence having at least 90%, such as at least 92%, for example at least 94%, such as at least 96%, for example at least 98% sequence identity to the sequence set forth in SEQ ID NO:1.

[0063] In another embodiment, the first antigen-binding region comprises or consists of a sequence selected from the group of sequences set forth in SEQ ID NOs: 25 to 34, or a sequence having at least 90%, such as at least 92%, for example at least 94%, for example at least 96%, for example at least 98% sequence identity to a sequence selected from the group of sequences set forth in SEQ ID NOs: 25 to 34.

[0064] In another embodiment, the multispecific antibody competes for binding to human 3CD123 with an antibody having the sequence set forth in SEQ ID NO:9, and preferably the multispecific antibody binds to the same epitope on human CD123 as the antibody having the sequence set forth in SEQ ID NO:9.

[0065] In one embodiment, the multispecific antibody binds to an epitope that includes one or more residues within the region H225-T267, such as an epitope contained entirely within the region H225-T267, as determined as described in Example 11 herein.

[0066] In another embodiment, the multispecific antibody binds to an epitope on human CD123 comprising one or more residues in the regions H225 and R234, and one or more residues in the region T251-T267.

[0067] In another embodiment, the multispecific antibody binds to an epitope on human CD123 that includes one, more than one, or all of residues H225, H231, R234, T251, R255, and T267.

[0068] In one embodiment, the first antigen-binding region comprises a VH CDR1 sequence set forth in SEQ ID NO:10, a VH CDR2 sequence set forth in SEQ ID NO:11, and a VH CDR3 sequence set forth in SEQ ID NO:12.

[0069] In one embodiment, the first antigen-binding region comprises or consists of a sequence set forth in SEQ ID NO:9, or a sequence having at least 90%, such as at least 92%, for example at least 94%, such as at least 96%, for example at least 98% sequence identity to the sequence set forth in SEQ ID NO:9.

[0070] As mentioned above, the multispecific antibody of the invention comprises a second antigen-binding region capable of binding to the V52 chain of the human Vy9V52-T cell receptor. V52 is part of the delta chain of the Vy9V52-TCR. An antibody capable of binding to human V52 may bind to an epitope located entirely within the V52 region or to an epitope that is a combination of residues of the V52 region and the constant region of the delta chain. In one embodiment, the multispecific antibody is capable of activating human Vy9V52 T cells. Activation of Vy9V52 T cells can be measured by gene expression and / or (surface) marker expression (e.g. activation markers such as CD25, CD69, or CD107a) and / or secreted protein (e.g. cytokine or chemokine) profile. In a preferred embodiment, the multispecific antibody is capable of inducing activation (e.g. upregulation of CD69 and / or CD25 expression) by Vy9V52 T cells leading to degranulation characterized by increased CD107a expression (see examples herein) and / or cytokine production (e.g. TNFα, IFNγ). Preferably, the multispecific antibody of the invention is capable of increasing the number of CD107a positive cells by at least 2-fold, such as at least 5-fold, when tested as described in the examples herein. In another preferred embodiment, the multispecific antibody of the invention has an EC50 value for increasing the percentage of CD107a positive cells by 50 pM or less, such as 25 pM or less, such as 20 pM or less, such as 15 pM or less, such as 10 pM or less, when tested using Vy9V52 T cells and C1R-neo target cells as described in the examples herein.

[0071] Several antibodies that bind to V52 are described in WO2015156673, whose antigen-binding regions or at least their CDR sequences can be incorporated into the multispecific antibodies of the invention.

[0072] In one embodiment, the multispecific antibody competes for binding to human V52 with an antibody having the sequence set forth in SEQ ID NO: 17, wherein X4 is Y.

[0073] In a further embodiment, the multispecific antibody binds to the same epitope on human V52 as the antibody having the sequence set forth in SEQ ID NO: 17.

[0074] In one embodiment the multispecific antibody competes for binding to human V52 with an antibody having the sequence set forth in SEQ ID NO: 36, and preferably the multispecific antibody binds to the same epitope on human V52 as the antibody having the sequence set forth in SEQ ID NO: 36.

[0075] In one embodiment the multispecific antibody competes for binding to human V52 with an antibody having the sequence set forth in SEQ ID NO: 37, and preferably the multispecific antibody binds to the same epitope on human V52 as the antibody having the sequence set forth in SEQ ID NO: 37.

[0076] In one embodiment the multispecific antibody competes for binding to human V52 with an antibody having the sequence set forth in SEQ ID NO: 38, and preferably the multispecific antibody binds to the same epitope on human V52 as the antibody having the sequence set forth in SEQ ID NO: 38.

[0077] In one embodiment of the multispecific antibody of the invention, the second antigen-binding region comprises the VH CDR1 sequence set forth in SEQ ID NO: 18, the VH CDR2 sequence set forth in SEQ ID NO: 19, and the VH CDR3 sequence set forth in SEQ ID NO: 20. In one embodiment, X4 of SEQ ID NO: 20 is Y. In another embodiment, X4 of SEQ ID NO: 20 is F. In another embodiment, X4 of SEQ ID NO: 20 is S.

[0078] In one embodiment of the multispecific antibody of the invention, the second antigen-binding region comprises the VH CDR1 sequence as set forth in SEQ ID NO: 40 and the VH CDR3 sequence as set forth in SEQ ID NO: 41, preferably the second antigen-binding region comprises or consists of the sequence as set forth in SEQ ID NO: 36 or a sequence having at least 90%, such as at least 92%, such as at least 94%, such as at least 96%, such as at least 98% sequence identity to the sequence as set forth in SEQ ID NO: 36.

[0079] In one embodiment of the multispecific antibody of the invention, the second antigen-binding region comprises the VH CDR1 sequence set forth in SEQ ID NO: 42, the VH CDR2 sequence set forth in SEQ ID NO: 43 and the VH CDR3 sequence set forth in SEQ ID NO: 44, preferably the second antigen-binding region comprises or consists of a sequence set forth in SEQ ID NO: 37 or a sequence having at least 90%, such as at least 92%, such as at least 94%, such as at least 96%, such as at least 98% sequence identity to the sequence set forth in SEQ ID NO: 37.

[0080] In one embodiment of the multispecific antibody of the invention, the second antigen-binding region comprises the VH CDR1 sequence set forth in SEQ ID NO: 45, the VH CDR2 sequence set forth in SEQ ID NO: 46 and the VH CDR3 sequence set forth in SEQ ID NO: 47, preferably the second antigen-binding region comprises or consists of a sequence set forth in SEQ ID NO: 38 or a sequence having at least 90%, such as at least 92%, such as at least 94%, such as at least 96%, such as at least 98% sequence identity to the sequence set forth in SEQ ID NO: 38.

[0081] In one embodiment of the multispecific antibody of the invention, the second antigen-binding region is humanized.

[0082] In a further embodiment, the second antigen-binding region comprises or consists of a sequence set forth in SEQ ID NO: 17 or a sequence having at least 90%, such as at least 92%, such as at least 94%, such as at least 96%, such as at least 98% sequence identity to the sequence set forth in SEQ ID NO: 17. In one embodiment, X4 of SEQ ID NO: 17 is Y. In another embodiment, X4 of SEQ ID NO: 17 is F. In another embodiment, X4 of SEQ ID NO: 17 is S.

[0083] In a preferred embodiment of the multispecific antibody of the invention, (i) the first antigen-binding region comprises a VH CDR1 sequence set forth in SEQ ID NO:2, a VH CDR2 sequence set forth in SEQ ID NO:3, and a VH CDR3 sequence set forth in SEQ ID NO:4, and the second antigen-binding region comprises a VH CDR1 sequence set forth in SEQ ID NO:18, a VH CDR2 sequence set forth in SEQ ID NO:19, and a VH CDR3 sequence set forth in SEQ ID NO:20, or (ii) the first antigen-binding region comprises a VH CDR1 sequence set forth in SEQ ID NO: 10, a VH CDR2 sequence set forth in SEQ ID NO: 11, and a VH CDR3 sequence set forth in SEQ ID NO: 12, and the second antigen-binding region comprises a VH CDR1 sequence set forth in SEQ ID NO: 18, a VH CDR2 sequence set forth in SEQ ID NO: 19, and a VH CDR3 sequence set forth in SEQ ID NO: 20.

[0084] In a further preferred embodiment of the multispecific antibody of the invention, (i) the first antigen-binding region comprises or consists of the sequence set forth in SEQ ID NO:1, the second antigen-binding region comprises or consists of the sequence set forth in SEQ ID NO:17, and optionally, X4 is Y; or (ii) the first antigen-binding region comprises or consists of the sequence set forth in SEQ ID NO:9 and the second antigen-binding region comprises or consists of the sequence set forth in SEQ ID NO:17, optionally wherein X4 is Y; or (iii) the first antigen-binding region comprises or consists of the sequence set forth in SEQ ID NO: 25 and the second antigen-binding region comprises or consists of the sequence set forth in SEQ ID NO: 17, optionally wherein X4 is Y; or (iv) the first antigen-binding region comprises or consists of the sequence set forth in SEQ ID NO: 26 and the second antigen-binding region comprises or consists of the sequence set forth in SEQ ID NO: 17, optionally wherein X4 is Y; or (v) the first antigen-binding region comprises or consists of the sequence set forth in SEQ ID NO: 27 and the second antigen-binding region comprises or consists of the sequence set forth in SEQ ID NO: 17, optionally wherein X4 is Y; or (vi) the first antigen-binding region comprises or consists of the sequence set forth in SEQ ID NO: 28, and the second antigen-binding region comprises or consists of the sequence set forth in SEQ ID NO: 17, optionally wherein X4 is Y; or (vii) the first antigen-binding region comprises or consists of the sequence set forth in SEQ ID NO: 29, and the second antigen-binding region comprises or consists of the sequence set forth in SEQ ID NO: 17, optionally wherein X4 is Y; or (viii) the first antigen-binding region comprises or consists of the sequence set forth in SEQ ID NO: 30, and the second antigen-binding region comprises or consists of the sequence set forth in SEQ ID NO: 17, optionally wherein X4 is Y; or (ix) the first antigen-binding region comprises or consists of the sequence set forth in SEQ ID NO: 31, and the second antigen-binding region comprises or consists of the sequence set forth in SEQ ID NO: 17, and optionally X4 is Y; or (x) the first antigen-binding region comprises or consists of the sequence set forth in SEQ ID NO: 32, and the second antigen-binding region comprises or consists of the sequence set forth in SEQ ID NO: 17, and optionally X4 is Y; or (xi) the first antigen-binding region comprises or consists of the sequence set forth in SEQ ID NO: 33, and the second antigen-binding region comprises or consists of the sequence set forth in SEQ ID NO: 17, optionally wherein X4 is Y; or (xii) the first antigen-binding region comprises or consists of the sequence set forth in SEQ ID NO: 34, and the second antigen-binding region comprises or consists of the sequence set forth in SEQ ID NO: 17, and optionally X4 is Y.

[0085] In a further preferred embodiment of the multispecific antibody of the invention, (i) the first antigen-binding region comprises or consists of the sequence set forth in SEQ ID NO:1 and the second antigen-binding region comprises or consists of the sequence set forth in SEQ ID NO:36; or (ii) the first antigen-binding region comprises or consists of the sequence set forth in SEQ ID NO:9 and the second antigen-binding region comprises or consists of the sequence set forth in SEQ ID NO:36; or (iii) the first antigen-binding region comprises or consists of the sequence set forth in SEQ ID NO: 25 and the second antigen-binding region comprises or consists of the sequence set forth in SEQ ID NO: 36; or (iv) the first antigen-binding region comprises or consists of the sequence set forth in SEQ ID NO: 26 and the second antigen-binding region comprises or consists of the sequence set forth in SEQ ID NO: 36; or (v) the first antigen-binding region comprises or consists of the sequence set forth in SEQ ID NO: 27 and the second antigen-binding region comprises or consists of the sequence set forth in SEQ ID NO: 36; or (vi) the first antigen-binding region comprises or consists of the sequence set forth in SEQ ID NO: 28 and the second antigen-binding region comprises or consists of the sequence set forth in SEQ ID NO: 36; or (vii) the first antigen-binding region comprises or consists of the sequence set forth in SEQ ID NO: 29 and the second antigen-binding region comprises or consists of the sequence set forth in SEQ ID NO: 36; or (viii) the first antigen-binding region comprises or consists of the sequence set forth in SEQ ID NO: 30 and the second antigen-binding region comprises or consists of the sequence set forth in SEQ ID NO: 36; or (ix) the first antigen-binding region comprises or consists of the sequence set forth in SEQ ID NO: 31 and the second antigen-binding region comprises or consists of the sequence set forth in SEQ ID NO: 36; or (x) the first antigen-binding region comprises or consists of the sequence set forth in SEQ ID NO: 32 and the second antigen-binding region comprises or consists of the sequence set forth in SEQ ID NO: 36; or (xi) the first antigen-binding region comprises or consists of the sequence set forth in SEQ ID NO: 33 and the second antigen-binding region comprises or consists of the sequence set forth in SEQ ID NO: 36; or (xii) the first antigen-binding region comprises or consists of the sequence set forth in SEQ ID NO: 34 and the second antigen-binding region comprises or consists of the sequence set forth in SEQ ID NO: 36.

[0086] In a further preferred embodiment of the multispecific antibody of the invention, (i) the first antigen-binding region comprises or consists of the sequence set forth in SEQ ID NO:1 and the second antigen-binding region comprises or consists of the sequence set forth in SEQ ID NO:37; or (ii) the first antigen-binding region comprises or consists of the sequence set forth in SEQ ID NO:9 and the second antigen-binding region comprises or consists of the sequence set forth in SEQ ID NO:37; or (iii) the first antigen-binding region comprises or consists of the sequence set forth in SEQ ID NO: 25 and the second antigen-binding region comprises or consists of the sequence set forth in SEQ ID NO: 37; or (iv) the first antigen-binding region comprises or consists of the sequence set forth in SEQ ID NO: 26 and the second antigen-binding region comprises or consists of the sequence set forth in SEQ ID NO: 37; or (v) the first antigen-binding region comprises or consists of the sequence set forth in SEQ ID NO: 27 and the second antigen-binding region comprises or consists of the sequence set forth in SEQ ID NO: 37; or (vi) the first antigen-binding region comprises or consists of the sequence set forth in SEQ ID NO: 28 and the second antigen-binding region comprises or consists of the sequence set forth in SEQ ID NO: 37; or (vii) the first antigen-binding region comprises or consists of the sequence set forth in SEQ ID NO: 29 and the second antigen-binding region comprises or consists of the sequence set forth in SEQ ID NO: 37; or (viii) the first antigen-binding region comprises or consists of the sequence set forth in SEQ ID NO: 30 and the second antigen-binding region comprises or consists of the sequence set forth in SEQ ID NO: 37; or (ix) the first antigen-binding region comprises or consists of the sequence set forth in SEQ ID NO: 31 and the second antigen-binding region comprises or consists of the sequence set forth in SEQ ID NO: 37; or (x) the first antigen-binding region comprises or consists of the sequence set forth in SEQ ID NO: 32 and the second antigen-binding region comprises or consists of the sequence set forth in SEQ ID NO: 37; or (xi) the first antigen-binding region comprises or consists of the sequence set forth in SEQ ID NO: 33 and the second antigen-binding region comprises or consists of the sequence set forth in SEQ ID NO: 37; or (xii) the first antigen-binding region comprises or consists of the sequence set forth in SEQ ID NO: 34 and the second antigen-binding region comprises or consists of the sequence set forth in SEQ ID NO: 37.

[0087] In a further preferred embodiment of the multispecific antibody of the invention, (i) the first antigen-binding region comprises or consists of the sequence set forth in SEQ ID NO:1 and the second antigen-binding region comprises or consists of the sequence set forth in SEQ ID NO:38; or (ii) the first antigen-binding region comprises or consists of the sequence set forth in SEQ ID NO: 9 and the second antigen-binding region comprises or consists of the sequence set forth in SEQ ID NO: 38; or (iii) the first antigen-binding region comprises or consists of the sequence set forth in SEQ ID NO: 25 and the second antigen-binding region comprises or consists of the sequence set forth in SEQ ID NO: 38; or (iv) the first antigen-binding region comprises or consists of the sequence set forth in SEQ ID NO: 26 and the second antigen-binding region comprises or consists of the sequence set forth in SEQ ID NO: 38; or (v) the first antigen-binding region comprises or consists of the sequence set forth in SEQ ID NO: 27 and the second antigen-binding region comprises or consists of the sequence set forth in SEQ ID NO: 38; or (vi) the first antigen-binding region comprises or consists of the sequence set forth in SEQ ID NO: 28 and the second antigen-binding region comprises or consists of the sequence set forth in SEQ ID NO: 38; or (vii) the first antigen-binding region comprises or consists of the sequence set forth in SEQ ID NO: 29 and the second antigen-binding region comprises or consists of the sequence set forth in SEQ ID NO: 38; or (viii) the first antigen-binding region comprises or consists of the sequence set forth in SEQ ID NO: 30 and the second antigen-binding region comprises or consists of the sequence set forth in SEQ ID NO: 38; or (ix) the first antigen-binding region comprises or consists of the sequence set forth in SEQ ID NO: 31 and the second antigen-binding region comprises or consists of the sequence set forth in SEQ ID NO: 38; or (x) the first antigen-binding region comprises or consists of the sequence set forth in SEQ ID NO: 32 and the second antigen-binding region comprises or consists of the sequence set forth in SEQ ID NO: 38; or (xi) the first antigen-binding region comprises or consists of the sequence set forth in SEQ ID NO: 33 and the second antigen-binding region comprises or consists of the sequence set forth in SEQ ID NO: 38; or (xii) the first antigen-binding region comprises or consists of the sequence set forth in SEQ ID NO: 34 and the second antigen-binding region comprises or consists of the sequence set forth in SEQ ID NO: 38.

[0088] The first and second antigen-binding regions in a multispecific antibody may be arranged in a variety of ways. In one embodiment, the antigen-binding regions are connected to each other via a linker, such as a covalent linker. In one embodiment, the first antigen-binding region and the second antigen-binding region are covalently linked to each other via a peptide linker, e.g., a linker having a length of 1 to 20 amino acids, e.g., 1 to 10 amino acids, such as 2, 3, 4, 5, 6, 7, 8, or 10 amino acids. In one embodiment, the peptide linker comprises or consists of a sequence of a serine followed by four glycines.

[0089] In some embodiments, the first antigen-binding region capable of binding to human CD123 is located N-terminal to the second antigen-binding region capable of binding to the human V52 chain. In another embodiment, the first antigen-binding region capable of binding to human CD123 is located C-terminal to the second antigen-binding region capable of binding to the human V52 chain.

[0090] Multispecific antibodies of the invention, such as bispecific antibodies, may contain additional molecules, domains or polypeptide sequences beyond the first and second antigen-binding regions. In one embodiment, the multispecific antibody further comprises a half-life prolonging domain, i.e., a domain that extends the half-life of the molecule in the circulation of a human patient. In one embodiment, the multispecific antibody has a terminal half-life of greater than about 168 hours when administered to a human subject. Most preferably, the terminal half-life is 336 hours or greater. As used herein, the "terminal half-life" of an antibody refers to the time it takes for the serum concentration of the polypeptide to decrease by 50% in vivo during the final stages of elimination.

[0091] In one embodiment, the multispecific antibody comprises an Fc region, preferably a human Fc region. In one embodiment, the multispecific antibody is in VHH-Fc format, i.e., the antibody comprises two or more single domain antigen-binding regions linked to each other via a human Fc region dimer, each single domain antigen-binding region is fused to an Fc region polypeptide (not including CH1 or light chain sequences), and the two fusion polypeptides form a dimeric bispecific antibody via disulfide bridges in the hinge regions.

[0092] Various methods for making bispecific antibodies have been described in the art and are discussed, for example, in Brinkmann and Kontermann (2017) MAbs 9:182 and Labrijn et al (2019) Nature Reviews Drug Discovery 18:585. In one embodiment of the invention, the Fc region is a heterodimer comprising two Fc polypeptides, a first antigen-binding region is fused to a first Fc polypeptide and a second antigen-binding region is fused to a second Fc polypeptide, and the first and second Fc polypeptides comprise asymmetric amino acid mutations that favor heterodimer formation over homodimer formation (see, for example, Ridgway et al. (1996) 'Knobs-into-holes' engineering of antibody CH3 domains for heavy chain heterodimerization. Protein Eng 9:617). In further embodiments herein, the CH3 regions of the Fc polypeptides comprise asymmetric amino acid mutations as described above, preferably wherein the first Fc polypeptide comprises a T366W substitution and the second Fc polypeptide comprises a T366S, L368A and Y407V substitution, or vice versa, where the amino acid positions correspond to those of human IgG1 according to the EU numbering system. In further embodiments, the cysteine ​​residue at position 220 of the first and second Fc polypeptides has been deleted or substituted, where the amino acid positions correspond to those of human IgG1 according to the EU numbering system. In further embodiments, the region comprises the sequence set forth in SEQ ID NO:35.

[0093] In some embodiments, the first and / or second Fc polypeptide contains a mutation that renders the Fc region inactive, i.e. unable to mediate effector function. In one embodiment, the first and second Fc polypeptides comprise a mutation at position 234 and / or 235, preferably the first and second Fc polypeptides comprise a L234F and L235E substitution, the amino acid positions corresponding to human IgG1 according to the EU numbering system.

[0094] In a preferred embodiment, the first antigen-binding region comprises a VH CDR1 sequence set forth in SEQ ID NO:2, a VH CDR2 sequence set forth in SEQ ID NO:3, and a VH CDR3 sequence set forth in SEQ ID NO:4; the second antigen-binding region comprises a VH CDR1 sequence set forth in SEQ ID NO:18, a VH CDR2 sequence set forth in SEQ ID NO:19, and a VH CDR3 sequence set forth in SEQ ID NO:20; The first Fc polypeptide comprises the sequence set forth in SEQ ID NO:21 and the second Fc polypeptide comprises the sequence set forth in SEQ ID NO:22, or vice versa.

[0095] In another preferred embodiment, the first antigen-binding region comprises a VH CDR1 sequence set forth in SEQ ID NO: 10, a VH CDR2 sequence set forth in SEQ ID NO: 11, and a VH CDR3 sequence set forth in SEQ ID NO: 12; the second antigen-binding region comprises a VH CDR1 sequence set forth in SEQ ID NO: 18, a VH CDR2 sequence set forth in SEQ ID NO: 19, and a VH CDR3 sequence set forth in SEQ ID NO: 20; The first Fc polypeptide comprises the sequence set forth in SEQ ID NO:21 and the second Fc polypeptide comprises the sequence set forth in SEQ ID NO:22, or vice versa.

[0096] In a further preferred embodiment, the antibody of the invention consists of: (i) a first polypeptide chain comprising a first antigen-binding region having a sequence selected from the group consisting of the sequences set forth in SEQ ID NOs: 1, 9, and 25 to 34, the sequence set forth in SEQ ID NO: 35, and the sequence set forth in SEQ ID NO: 21; and (i) a second polypeptide chain comprising a second antigen-binding region comprising the sequence set forth in SEQ ID NO: 17 (X4 is Y, the sequence set forth in SEQ ID NO: 35, and the sequence set forth in SEQ ID NO: 22).

[0097] In a further preferred embodiment, the antibody of the invention consists of: (i) a first polypeptide chain comprising a first antigen-binding region having a sequence selected from the group consisting of the sequences set forth in SEQ ID NOs: 1, 9, and 25 to 34, the sequence set forth in SEQ ID NO: 35, and the sequence set forth in SEQ ID NO: 22; and (i) a second polypeptide chain comprising a second antigen-binding region comprising the sequence set forth in SEQ ID NO: 17 (X4 is Y, the sequence set forth in SEQ ID NO: 35, and the sequence set forth in SEQ ID NO: 21).

[0098] In one embodiment, a multispecific antibody of the invention is capable of mediating the killing of CD123-expressing cells, such as C1R-neo cells or THP-1 cells, by Vy9V52 T cells.

[0099] Preferably, the antibody is capable of inducing killing of C1R-neo cells upon activation of Vy9V52 T cells with an EC50 value of 50pM or less, such as 25pM or less, for example 20pM or less, such as 15pM or less, for example 10pM or less, or 5pM or less, such as 2pM or less, when tested as described in Example 5 herein.

[0100] In another embodiment, the antibody is capable of inducing the killing of THP-1 cells upon activation of Vy9V52 T cells with an EC50 value of 100pM or less, such as 50pM or less, for example 25pM or less, such as 20pM or less, for example 15pM or less, such as 10pM or less, or even 5pM or less, such as 2pM or less, when tested as described in Example 5 herein.

[0101] In further embodiments, the multispecific antibody is capable of mediating killing of CD123-expressing bone marrow-derived AML tumor cells from a human patient. Such killing may be determined, for example, as described herein in Example 6. In one embodiment, the multispecific antibody of the invention is capable of mediating more than 25%, such as more than 50%, specific cell death at a concentration of 100 fM as determined in the assay described herein in Example 6.

[0102] In a further embodiment, the multispecific antibody is unable to mediate killing of CD123 negative cells, such as CD123 negative human cells.

[0103] In a further embodiment, the multispecific antibodies of the invention may bind to transiently CD123-expressing 293F cells with an EC50 of 50 nM or less, such as 20 nM or less, such as 10 nM or less, for example 5 nM or less, when tested as described in Example 3 herein.

[0104] In a further embodiment, the multispecific antibody of the invention is capable of binding to a recombinant CD123-Fc fusion protein with an EC50 of 50 nM or less, such as 20 nM or less, such as 10 nM or less, for example 5 nM or less, when tested as described in Example 4 herein.

[0105] In a further main aspect, the present invention provides an antibody comprising a first antigen-binding region capable of binding to human CD123, said first antigen-binding region being a single domain antibody, (i) comprising a VH CDR1 sequence as set forth in SEQ ID NO: 2, a VH CDR2 sequence as set forth in SEQ ID NO: 3, and a VH CDR3 sequence as set forth in SEQ ID NO: 4, wherein preferably the first antigen-binding region comprises or consists of a sequence selected from the group of sequences set forth in SEQ ID NOs: 1, 25 to 34, or a sequence having at least 90%, such as at least 92%, for example at least 94%, for example at least 96%, for example at least 98% sequence identity to a sequence selected from the group of sequences set forth in SEQ ID NOs: 1, 25 to 34, or (ii) an antibody comprising the VH CDR1 sequence as set forth in SEQ ID NO: 10, the VH CDR2 sequence as set forth in SEQ ID NO: 11 and the VH CDR3 sequence as set forth in SEQ ID NO: 12, wherein preferably said first antigen-binding region comprises or consists of a sequence as set forth in SEQ ID NO: 9 or a sequence having at least 90%, such as at least 92%, for example at least 94%, such as at least 96%, for example at least 98% sequence identity to said sequence as set forth in SEQ ID NO: 9.

[0106] In one embodiment, in SEQ ID NO: 2, X1 is G. In another embodiment, X1 is S.

[0107] In one embodiment, in SEQ ID NO: 3, X2 is A. In another embodiment, X2 is T.

[0108] In one embodiment, in SEQ ID NO: 4, X3 is Y. In another embodiment, X3 is F.

[0109] In one embodiment, X1 is G, X2 is A, and X3 is Y.

[0110] In another embodiment, X 1 is G, X 2 is A, and X 3 is F.

[0111] In another embodiment, X1 is G, X2 is T, and X3 is Y.

[0112] In another embodiment, X 1 is G, X 2 is T, and X 3 is F.

[0113] In one embodiment, X1 is S, X2 is A, and X3 is Y.

[0114] In another embodiment, X 1 is S, X 2 is A, and X 3 is F.

[0115] In another embodiment, X 1 is S, X 2 is T, and X 3 is Y.

[0116] In another embodiment, X 1 is S, X 2 is T, and X 3 is F.

[0117] In a further main aspect, the present invention relates to a pharmaceutical composition comprising an antibody, such as a multispecific antibody according to the invention as described herein, and a pharma- ceutically acceptable excipient.

[0118] The antibody can be formulated with a pharma- ceutically acceptable excipient according to conventional techniques, such as those disclosed in (Rowe et al., Handbook of Pharmaceutical Excipients, 2012 June, ISBN 9780857110275). The pharma-ceutically acceptable excipient, as well as any other carriers, diluents or adjuvants, should be suitable for the antibody and the selected mode of administration. Compatibility of the excipient and other components of the pharmaceutical composition is determined based on the lack of significant adverse effects on the desired biological properties of the selected antibody or pharmaceutical composition of the present invention (e.g., less than substantial effects on antigen binding (relative inhibition of 10% or less, relative inhibition of 5% or less, etc.)).

[0119] Pharmaceutical compositions may include diluents, fillers, salts, buffers, detergents (e.g., non-ionic detergents such as Tween-20 or Tween-80), stabilizers (e.g., sugars or protein-free amino acids), preservatives, tissue fixatives, solubilizing agents, and / or other materials suitable for inclusion in a pharmaceutical composition. Additional pharma- ceutically acceptable excipients include any and all suitable solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, antioxidants and absorption delaying agents, etc. that are physiologically compatible with the antibodies of the invention.

[0120] In a further main aspect, the present invention relates to a multispecific antibody according to the invention as described herein for use as a medicament.

[0121] The multispecific antibodies according to the invention make it possible to create a microenvironment favourable for the killing of tumour cells, in particular CD123-positive tumour cells, by Vy9V52 T cells.

[0122] Thus, in a further main aspect, the present invention relates to a multispecific antibody according to the invention as described herein for use in the treatment of cancer.In a further main aspect, the present invention relates to a multispecific antibody according to the invention as described herein for use in the treatment of acute myeloid leukemia, B-cell acute lymphoblastic leukemia, hairy cell leukemia, Hodgkin's lymphoma, blastic plasmacytoid dendritic neoplasm, chronic myeloid leukemia, chronic lymphocytic leukemia, B-cell chronic lymphoproliferative disorder or myelodysplastic syndrome.

[0123] Likewise, the present invention relates to a method for the treatment of a disease comprising administering a multispecific antibody according to the invention as described herein to a human subject in need thereof, in one embodiment the disease is cancer, such as acute myeloid leukemia.

[0124] In some embodiments, the antibodies are administered as monotherapy. However, the antibodies of the invention may also be administered in combination therapy, i.e., in combination with other therapeutic agents relevant to the disease or condition being treated.

[0125] "Treat" or "treating" refers to administering an effective amount of an antibody according to the invention for the purpose of alleviating, ameliorating, inhibiting, eradicating (curing) or preventing a symptom or disease state. An "effective amount" refers to an amount effective at a dosage and for a period of time necessary to achieve a desired therapeutic result. An effective amount of a polypeptide, such as an antibody, may vary depending on factors such as the stage of the disease, the age, sex, and weight of the individual, and the ability of the antibody to elicit a desired response in the individual. An effective amount is also one in which any toxic or detrimental effects of the antibody are outweighed by the therapeutically beneficial effects. An exemplary non-limiting range of an effective amount of an antibody of the invention is about 0.1 μg / kg to 100 mg / kg, e.g., about 1 μg / kg to 50 mg / kg, e.g., about 0.01 to 20 mg / kg, e.g., about 0.1 to 10 mg / kg, e.g., about 0.5, about 0.3, about 1, about 3, about 5, or about 8 mg / kg. Administration can be by any suitable route, but is typically parenteral, such as intravenous, intramuscular, or subcutaneous.

[0126] The multispecific antibodies of the invention are typically produced recombinantly, i.e. by expression of a nucleic acid construct encoding the antibody in a suitable host cell, followed by purification of the recombinant antibody produced from the cell culture. The nucleic acid construct may be produced by standard molecular biology techniques well known in the art. The construct is typically introduced into the host cell using an expression vector. Suitable nucleic acid constructs and expression vectors are known in the art. Suitable host cells for recombinant expression of antibodies are well known in the art and include CHO, HEK-293, Expi293F, PER-C6, NS / 0 and Sp2 / 0 cells.

[0127] Thus, in a further aspect, the invention relates to a nucleic acid construct encoding an antibody of the invention, such as a multispecific antibody according to the invention. In one embodiment, the construct is a DNA construct. In another embodiment, the construct is an RNA construct.

[0128] In a further aspect, the present invention relates to an expression vector comprising a nucleic acid construct encoding a multispecific antibody according to the invention.

[0129] In a further aspect, the present invention relates to a host cell comprising one or more nucleic acid constructs encoding a multispecific antibody according to the invention or an expression vector comprising a nucleic acid construct encoding a multispecific antibody according to the invention. [Table 4-1] [Table 4-2] [Table 4-3] [Table 4-4] [Table 4-5] [Table 4-6] [Table 4-7]

[0130] All references, articles, publications, patents, patent publications, and patent applications cited herein are incorporated by reference in their entirety for all purposes. However, the mention of any references, articles, publications, patents, patent publications, and patent applications cited herein is not, and should not be considered as, an admission or any form of suggestion that they constitute valid prior art or form part of the common general knowledge in any country in the world. EXAMPLES

[0131] Example 1: Selection and identification of anti-CD123 VHHs from a phage display library generated from animals immunized with C1R-CD1d cells Llama glama (2 animals) were immunized with CD123 expressing C1R-CD1d cells and then "immune" VHH phage antibody libraries were generated as described (Lameris et al., 2016 Immunology 149:111). These libraries were used for phage selection on captured recombinant human CD123 or directly coated CD123 antigen (extracellular domain, Sino Biological). One or two consecutive selection rounds were performed. After one and two rounds of phage selection, single phage clones were screened for binding to recombinant captured antigen in ELISA. Those clones that scored positive for binding were sequenced and then all clones with different sequences were tested for binding to the cell line used for immunization in flow cytometry. VHH clones showing binding in FACS were then selected for further characterization. Eight different clones were identified and designated: 1E2, 1B4, 1A3, 2D11, 1D2, 1E4, 1H1, and 1F1.

[0132] Example 2: Synthetic gene synthesis, production and purification of bispecific VHHs The sequence of the CD123-specific VHH domain antibody was then reshaped into a bispecific VHH with a V52-specific VHH (5C8var1; SEQ ID NO: 17, where X4 is Y) oriented as follows: N-terminus-anti-CD123 VHH-linker-anti-V52 VHH-C-tag. The V52-specific VHH used is described in SEQ ID NO: 17, where X4 is Y. The linker between the two VHH domains was a glycine (G)-serine (S) stretch with the sequence G4S. cDNAs encoding these proteins were generated by synthetic gene synthesis in Genscript and then cloned by directional cloning into the eukaryotic expression vector pCDNA3.1+ (Thermofisher Scientific). The proteins were expressed by transient transfection in Hek293E cells and then (5 days after expression) purified from conditioned cell culture supernatants using Capture Select C-tag affinity matrix (Thermo Fisher Scientific) according to the supplier's protocol. Purified bispecific VHHs were always >95% pure as determined by SDS-PAGE analysis with Coomassie staining and contained very low levels of endotoxin (<0.5EU / mg).

[0133] Example 3: Specificity of binding of bispecific anti-CD123 x V52 VHHs in ELISA Recombinant purified CD123 antigen (extracellular domain; Sino Biological), or Fc fusion of CD123 antigen (Bio-Techne / R&D Systems) was coated at a concentration of 2 μg / ml onto wells of ELISA plates (Greiner) in PBS. As a negative control, wells were coated with 1% (w / v) BSA. In-house designed, produced, and purified recombinant forms of the extracellular domains of human Vγ9 and Vδ2 TCR chains fused to human Fc were also coated as antigens at 2 μg / ml. After coating and blocking wells with 2% (w / v) BSA, bispecific VHH proteins were tested for binding at a saturating concentration of 50 nM, and bound VHHs were detected using an HRP-labeled anti-VHH antibody (Genscript) and stained with 3,3′,5,5′-tetramethylbenzidine (TMB) / H2O2.

[0134] Figure 1 shows that only 1A3-5C8var1 and 1D2-5C8var1 showed strong and specific binding to both CD123 and γδ-TCR (γδ T cell receptor) in ELISA. The other bispecifics bound weakly or did not bind to CD123.

[0135] Example 4: Specificity of binding of bispecific anti-CD123 x V52 VHHs using flow cytometry Expression constructs for human CD123 and the common beta chain of the receptor (CD131) were purchased from Invivogen. Plasmids were transformed into chemically competent DH5α bacteria and a single colony growing on selective medium was used to inoculate a 50 ml culture to amplify both constructs. Purified DNA was then transfected into Freestyle 293F cells using polyethylenimine (PEI). Either the plasmid alone or a mixture of the two plasmids was used for transfection. As a negative control, non-transfected cells were also used for flow cytometry. One day after transfection, cells were used to test the binding of the bispecific VHHs using staining on a FACS. Briefly, binding of bispecific VHHs at a saturating concentration of 100 nM to transfected cells was detected with an AF647-labeled anti-VHH (Genscript) antibody and staining was visualized using a FACS Celesta (Becton and Dickinson).

[0136] Figure 2 shows that both 1A3-5C8var1 and 1D2-5C8var1 strongly and specifically recognized CD123 when the antigen was expressed alone or in conjunction with CD131. 1D2-5C8var1 gave the strongest signal in flow cytometry.

[0137] To determine the apparent affinity of 1D2-5C8var1 for CD123 using flow cytometry, a range of concentrations of this bispecific VHH were tested for binding to transiently transfected 293F cells expressing CD123, CD131 or both CD123 and -131. The former cells (expressing CD131) were used as a negative control.

[0138] Figure 3 again shows that 1D2-5C8var1 was exquisitely specific for CD123, since only cells (transiently) expressing CD123, but not CD131, were recognized. Furthermore, the data show that the apparent affinity of 1D2-5C8var1 binding to CD123, determined using flow cytometry, was approximately 3 nM. This value was comparable for binding to CD123 alone or to co-expressed CD123 and -131.

[0139] Example 5: Affinity determination of 1D2-5C8var1 for binding CD123 using Biolayer Interferometry (BLI) To determine the kinetics of binding of 1D2-5C8var1 to CD123, recombinant purified CD123-Fc fusion protein (Bio-Techne / R&D Systems) was loaded to a density of 1 nm on an anti-human IgG Fc Capture sensor (using a concentration of 5 μg / ml) for an Octet Red96e (Sartorius) instrument. Different sensors were then soaked in different concentrations of 1D2-5C8var1 and dilutions were performed in 10x kinetic buffer (10×KB) provided by the supplier. From the resulting sensorgrams, the kinetic association and dissociation rate constants were determined by curve fitting.

[0140] Figure 4 shows the actual sensorgram used for curve fitting. The latter is shown as a straight line in the figure. This was used to determine the kinetic association and dissociation rate constants and thus the affinity of anti-CD123 VHH 1D2. Measurements were performed in duplicate and the affinity of 1D2-5C8var1 for CD123 was measured between 3 and 5 nM.

[0141] Example 6: CD123-dependent, 1D2-5C8var1-mediated Vγ9Vδ2 T cell activation and T cell-mediated target cell cytotoxicity Buffy coats were obtained from Sanquin (Amsterdam, the Netherlands). PBMCs were isolated from these buffy coats by Ficoll density gradient centrifugation using a procedure described. Highly pure Vγ9Vδ2 T cells were obtained by MACS using a Vδ2-specific antibody and expanded using published methods (de Bruin et al., 2016 Clin Immunol. 169:128). CD123-positive B lymphoblasts, an EBV-transformed C1R neo cell line (CRL-2369) and a CD123-positive AML-derived THP-1 cell line (TIB-202) were obtained from the American Type Culture Collection and cultured according to the supplier's instructions. Target cells were labeled with cell tracing violet (CTV) for 20 min at 37°C. To measure T cell activation, cells were stained for the activation marker CD107a (or LAMP-1, lysosome-associated membrane protein-1), which is exposed on the cell surface upon cell degranulation. A range of bispecific antibody concentrations were incubated with a 1:1 mixture of target cells and expanded Vy9V52 T cells (50,000 cells each) in the presence of PE-labeled anti-CD107A antibody for 4 hours in a final volume of 100 μl. After incubation, cells were washed and stained with a mixture of fluorescently labeled anti-CD3 and anti-Vy9 antibodies to identify T cells and live / dead staining (7-AAD). Samples were analyzed using a FACS Celesta (Beckton and Dickinson). To assess T cell-mediated target cell cytotoxicity, essentially the same setup was used, only no anti-CD107A antibody was added, and CTV-labeled target cells and Vy9V52 T cells were incubated in the presence of bispecific VHH for 24 hours. At the end of the assay, cells were again stained for CD3 and Vy9 and analyzed using flow cytometry in the presence of live / dead staining (7-AAD).

[0142] Figure 5 shows that 1D2-5C8var1 caused potent T cell activation with an EC50 in the pM range. In the absence of target cells, high concentrations of 1D2-5C8var1 caused only background activation (data not shown). The potency of 1D2-5C8var1 to induce T cell activation was slightly dependent on the T cell donor used; EC50 values ​​ranged from 3 to 13 pM.

[0143] To determine whether the observed T cell activation also resulted in target cell lysis, a cytotoxicity assay was performed using a 1:1 effector to target (E:T) ratio and a 24 h time point. Figure 6 shows that in the presence of expanded Vγ9Vδ2 T cells, 1D2-5C8var1 potently induced lysis of C1R-neo target cells. The EC50 for cytotoxicity was determined by curve fitting between 1 and 2 pM, depending on the T cell donor used (data from two donors are shown).

[0144] The same cytotoxicity assay was repeated with a CD123 positive AML cell line, THP-1. The potency of 1D2-5C8var1 to induce THP-1 target cell lysis was highly comparable. The EC50 was measured to be 1 pM. In contrast, the potency of 1A3-5C8var1 to induce target cell lysis was measured to be approximately 50 pM. Figure 7.

[0145] Example 7: Bispecific VHH-mediated, Vy9V52 T cell activation and T cell-induced lysis of primary AML cells 25,000 bone marrow-derived mononuclear cells from an AML patient were co-cultured overnight at a 1:1 ratio with expanded Vγ9Vδ2 T cells from a healthy donor. Cells were cultured in the presence of PE-labeled CD107a antibody and a range of concentrations of 1D2-5C8var1 (ranging from 10 fM to 100 nM). Cells were harvested, washed, and labeled with an antibody mix containing fluorescently labeled anti-CD45, CD117, CD34, CD33, and CD2 antibodies for 30 s at 4 °C. After washing, cells were resuspended in a mixture of live / dead stain (7AAD) and 123 counting beads and subsequently analyzed using an LSRFortessa flow cytometer.

[0146] Figure 8 shows that both bispecific VHH compounds were able to induce strong T cell activation, depending on CD123 positive primary AML blasts. In addition, the compounds caused high levels of tumor cell lysis, and both showed significant potency in this cytotoxicity. The EC50 values ​​could not be clearly determined from the curves obtained, but were in the fM range (less than 1 pM).

[0147] Example 8: Humanization of anti-CD123 VHH 1D2 using CDR grafting The 1D2 VHH antibody fragment was humanized using CDR grafting technology (see, e.g., U.S. Pat. No. 5,225,539, and Williams, D.G. et al., 2010, Antibody Engineering, volume 1, Chapter 21). First, human germline sequences were identified using IgBLAST (Ye J. et al., 2013, Nucleic Acids Res. 41:W34-40). The closest human germline sequence was the V gene IGVH3-23 * Using this germline sequence, we identified the llama CDR (human germline IGVH3-23 *04) was directly grafted to the 1D2 sequence, resulting in the following cDNA construct: SEQ ID NO: 31. The NCBI NR database (downloaded on September 27, 2020) was then queried using BLASTP (version 2.10.0+) to identify the human template sequence that showed the highest identity with the 1D2 sequence. Two VH sequences were identified that showed a similarity score of 70% or more and similar CDR lengths, preferably identical to those of 1D2 CDR1, CDR2, and CDR3, respectively. The frameworks encoded by GenBank (Benson, DA et al., 2013, Nucleic Acids Res. 41(D1):D36-42) accession numbers CAD60357.1 and AKU38567.1 were selected as templates for grafting of the 1D2 CDRs, resulting in the following cDNA constructs: SEQ ID NO: 28 and 32, respectively. The framework and CDR definitions are those determined by Kabat et al. ("Sequences of Proteins of Immunological Interest", Kabat, E., et al., US Department of Health and Human Services, (1983)). To understand the effect of humanized framework residues on the structure of VHHs, a homology model of 1D2 VHHs was generated using the "Antibody Prediction" tool (default parameters) in BioLuminate 4.2.156 (Schrodinger). The homology model was built based on PDB ID 6GKU. CDRs were grafted in silico to study the effect of human residues on features as loop conformation, surface hydrophobicity, and structural integrity (e.g., increased rigidity) of the CDRs. The obtained constructs were checked for these features, leading to the design of additional constructs. SEQ ID NOs: 25, 26, 27, 29, 30, 33, and 34. These humanized 1D2-VHH sequences were then reshaped into a bispecific VHH with a V52-specific VHH (5C8var1; SEQ ID NO: 17, where X4 is Y) oriented: N-terminus-humanized-anti-1D2 VHH-linker-anti-V52 VHH-C-tag.cDNAs encoding these molecules were then synthesized and cloned into expression vectors for expression in HEK293E cells. Proteins were produced by transient transfection of cells and purified from culture supernatants using C-tag affinity chromatography followed by preparative size-exclusion chromatography.

[0148] Example 9: Affinity determination of humanized 1D2-5C8var1(Y105F) mutant for binding CD123 using Biolayer Interferometry (BLI) To determine the kinetics of binding of humanized 1D2-5C8var1 variants to CD123, recombinant purified CD123-Fc fusion protein (Bio-Techne / R&D Systems) was loaded to a density of 1 nm on an anti-human IgG Fc Capture sensor (using a concentration of 5 μg / ml) for an Octet Red96e (Sartorius) instrument. Different sensors were then soaked with different concentrations of humanized 1D2-5C8var1 variants, starting from 50 nM and its two-fold dilutions. Dilutions were performed in 10x kinetic buffer (10xKB) provided by the supplier. From the resulting sensorgrams, kinetic association and dissociation rate constants were determined by curve fitting. When a fit was possible, the association and dissociation rate constants were used to calculate the affinity of humanized 1D2-5C8var1 variants for binding to CD123. Measurements were performed in duplicate and the affinities of the different humanized 1D2-5C8var1 variants for CD123 ranged from a similar 2.6 nM compared to the parental 1D2 to a non-binding variant, as shown in Table 2. For reference, non-humanized (parental) 1D2 was included in these experiments. [Table 5]

[0149] Example 10: Half-life extended (Fc-containing) bispecific constructs The 1D2-5C8var1 bispecific VHH was reformatted into a therapeutic antibody format containing a human Fc domain. Both VHH domains were coupled to a human IgG1 Fc (i.e., CH2 and CH3) domain with the following characteristics: the VHH was coupled to a modified hinge (AAA followed by SDKTHTCPPCP) and human CH2 and CH3 domains. The CH2 domain was Fc-silenced by the LFLE mutation pair (L234F, L235E) and the CH3 domain was mutated by "knob-into-hole" mutations (knob: T366W and hole: T366S, L368A, and Y407V), which force heterodimerization upon co-expression of the two chains in the same cell. This mutation pair has been described in the scientific literature (Ridgway et al. (1996) Protein Eng 9:617). The C-terminus of the anti-Vy9V52 heavy chain was equipped with a C-terminal tag for purification purposes (AAAEPEA (SEQ ID NO: 53)). The sequence of the construct is set forth in SEQ ID NO: 49 and SEQ ID NO: 50. The resulting antibody construct was designated 1D2-5C8var1(Y105F)-Fc.

[0150] The protein was produced via co-transfection of the two expression vectors encoding the 1D2-5C8var1(Y105F)-Fc proteins in HEK293E cells and purification from the culture supernatant by C-tag affinity chromatography followed by preparative size-exclusion chromatography, resulting in a highly monomeric protein preparation of 1D2-5C8var1(Y105F)-Fc.

[0151] Example 11: 1D2-5C8var1(Y105F)-Fc induces target-dependent T cell activation and causes T cell-mediated target cell cytotoxicity with comparable potency to bispecific VHHs The ability of 1D2-5C8var1(Y105F)-Fc to induce target-dependent Vγ9Vδ2 T cell activation was then tested in co-cultures of Vγ9Vδ2 T cells with THP-1 tumor cells (at a 1:1 ratio). Vγ9Vδ2 T cells were grown from the blood of healthy donors using procedures known in the art. The THP-1 cell line (ATCC Cat. No. Nr.TIB-202) was cultured according to the supplier's recommendations. In a 4-hour co-culture of both cell types, activation of Vγ9Vδ2 T cells was measured by staining for CD107a and measuring the percentage of CD107a positive cells by flow cytometry. Figure 10 shows that 1D2-5C8var1(Y105F)-Fc induced target-dependent T cell activation (no activation was observed in co-cultures of Vγ9Vδ2 T cells with tumor cells in the absence of compound, data omitted). EC50s were typically in the pM range, ranging from 4 to 16 pM (depending on the donor used).

[0152] Of note, the potency of Fc-containing molecules to induce Vγ9Vδ2 T cell activation was not measurably different from bispecific VHHs. This was observed for three different independent T cell donors. To determine whether this T cell activation also resulted in target cell lysis, the viability of THP-1 target cells was measured after 24 h of co-culture with Vγ9Vδ2 T cells (1:1 ratio) in the presence of antibodies. Vγ9Vδ2 T cells were isolated from the blood of healthy donors and expanded using a standardized protocol. The day before the assay, the THP-1 target cell line was labeled with cell tracing violet (CTV) so that it could be distinguished from the effector cell population by flow cytometry. After 24 h of co-culture in the presence of increasing concentrations of compound, the percentage of viable target cells was determined. Figure 11.

[0153] Figure 11 shows that both bispecific VHH and Fc-containing counterparts induce potent T cell-mediated target cell cytotoxicity, and the potency of both molecules in causing target cell lysis is not measurably different. EC50 values ​​ranged from 1 to 3 pM, depending on the donor used. No target cell lysis was observed in co-cultures in the absence of compound (data not shown). After 24 hours, all target cells in the assay were killed.

[0154] Example 12: 1D2-5C8var1(Y105F)-Fc induces preferential killing of tumor cells over target-positive normal cells To determine the extent to which the antibodies induced the killing of CD123-positive normal cells, plasmacytoid dendritic cells (pDCs), known to express CD123 (Collin et al., 2013 Immunology 140,1:22-30), were enriched from peripheral blood mononuclear cell (PBMC) fractions isolated from the blood of two healthy donors using MACS sorting (Miltenyi Biotech, Cat. Nr. 130-097-415). The THP-1 cell line was used as a tumor cell line expressing CD123. Using staining for CD123 and analysis by flow cytometry, it was shown that the expression level of CD123 was approximately 10-fold higher on pDCs than on the THP-1 cell line. Figure 12.

[0155] The cytotoxic effect of CD123-targeted compounds combined with Vγ9Vδ2 T cells against a mixture of target cells was then determined in a co-culture of THP-1 cell line, pDCs, and Vγ9Vδ2 T cells at a ratio of 1:1:2. Vγ9Vδ2 T cells were isolated from the blood of healthy donors and expanded using a standardized protocol. The day before the assay, the THP-1 target cell line was labeled with cell tracing violet (CTV) so that it could be distinguished from effector cell populations and other target cells by flow cytometry. After 24 h of co-culture in the presence of increasing concentrations of compounds, the percentage of live target cells was determined by staining Vγ9 (T cells), CD303 (pDCs), CTV (THP-1) and CD123 (THP-1 and pDCs) and analyzed by flow cytometry.

[0156] Figure 13 shows that the bispecific antibody induced the expected THP-1 target cell lysis (Figure 11) with a potency (EC50) of approximately 1 pM. Surprisingly, however, despite a 10-fold higher expression level of the CD123 target molecule on pDCs (Figure 12), these cells were much less affected. The maximum lysis observed was lower and the EC50 found in the assay was almost 10-fold higher than that found for THP-1 cell lysis. The results for donor #2 were similar (EC50 values ​​of 1 and 11 pM for THP-1 and pDCs, respectively, data omitted). These data indicate preferential induction of lysis of tumor cells over target-positive normal cells by the compound and Vy9V52 T cells.

[0157] Example 13: Epitope mapping reveals 1D2 VHH for binding to membrane-proximal epitopes To determine which epitope on CD123 was recognized by anti-CD123 VHH 1D2, this epitope was mapped using a mass spectrometry-based method (Pimenova et al., 2008 J Mass Spectrom. 43(2):185-95). Several residues within the CD123 molecule were found to be cross-linked by the antibody. FIG. 14.

[0158] Figure 14 identifies the epitope of 1D2 present in the region of amino acids 203-273 of human CD123. These residues are highlighted in the crystal structure of the molecule (PDB ID 5UV8: Broughton et al., 2018 Nat Commun. 9:386), which is the membrane-most proximal region, located 1011 Å away. 2 is mapped onto a second domain covering a surface area of

[0159] Figure 15 shows that the epitope recognized by the lead anti-CD123 antibody is located close to the membrane. The spanned distance is about 40 Å, which is not unusual for an epitope. All CDR regions of 1D2 VHH were found to be cross-linked to the antigen, with a particularly strong signal for CDR3.

[0160] Similar experiments were performed to determine which epitope on CD123 was recognized by anti-CD123 VHH 1A3. Residues H225, H231, R234, T251, R255 and T267 of CD123 were found to cross-link to the 1A3 antibody.

[0161] Example 14: 1D2x5C8var1(Y105F)-Fc shows a good stability profile The thermal stability of 1D2x5C8var1(Y105F)-Fc was analyzed by nano-differential scanning fluorimetry (nano-DSF). The protein showed high thermal stability with an unfolding temperature of >60°C (Table 3). Furthermore, 1D2x5C8var1(Y105F)-Fc was subjected to accelerated stress tests. Samples were incubated at elevated temperature (40°C) and under acidic (50 mM acetate buffer, pH 5.0) and basic (100 mM phosphate buffer, pH 8.5) conditions for 1 week, as well as under oxidative conditions (phosphate buffer, pH 7.4 and 0.05% H2O2) for 6 and 24 hours. Any stress-induced changes were analyzed by measuring aggregates and fragments by size-exclusion chromatography detected by ultraviolet absorption (SEC-UV) under denaturing (SDS) conditions (CE-SDS) and after reduction (A) and (B) capillary gel electrophoresis (Figure 16). No detectable degradation of the stressed protein samples was observed compared to the non-stressed reference samples. The protein was found to be highly stable. [Table 6]

[0162] Example 15: V52-CD123 bispecific antibody 1D2x5C8var1(Y105F) is more potent than 7A5-based Vy9-CD123 bispecific antibody in inducing T cell activation and T cell-mediated tumor cell cytotoxicity The efficacy of the Vδ2-CD123 bispecific antibody 1D2x5C8var1(Y105F)-Fc was compared with the Vγ9-CD123-Fc bispecific antibody based on the Vγ9-binding Fab antibody 7A5. The sequence of 7A5 was kindly provided by Professor Kabelitz. The antibody is characterized in Oberg et al. (2014) Cancer Res 74(5):1349. Mutants of the antibody 7A5 are described in Ganesan et al. (2021) Leukemia 35(8):2274-2284 and WO2020 / 227457. The sequence of Fab7A5 was fused to human CH2 and CH3 sequences containing a knob-into-hole mutation in CH3 and the Fc silencing mutations L234F and L235E. This Vy9-binding "half IgG" (SEQ ID NOs: 51 and 52) was then co-expressed with a CD123-specific VHH-Fc fusion in HEK293E cells to form a bispecific Vy9xCD123 bispecific antibody. The protein was purified via a C-terminal tag present at the C-terminus of CH3 in the Vy9-binding arm and then further purified via preparative size exclusion. This resulted in a pure and essentially endotoxin-free protein. 1D2xFab 7A5-Fc has the same CD123-binding VHH arm as 1D2x5C8var1(Y105F)-Fc. For the assay, 50,000 expanded Vy9V52 T cells were co-cultured with 50,000 Kasumi-3 or THP1 target cells and a dilution series of the two compounds. The results are shown in Figure 17. (A) Degranulation was analyzed after 4 hours by measuring the percentage of CD107a (lysosomal associated protein-1, or LAMP-1) positive cells via flow cytometry. (B) Vy9V52 T cell activation was analyzed by measuring the percentage of CD25 positive cells, and (C) cytotoxicity was analyzed by analyzing the percentage of live target cells via flow cytometry after 24 hours. 1D2x5C8var1(Y105F)-Fc induced more potent activation and degranulation of Vy9V52 T cells, as evidenced by a higher percentage of CD107a positive cells as well as CD25 positive cells, and lower EC50 values ​​than those observed when using Vy9-targeted compounds.Higher potency in cytotoxicity was observed using 1D2×5C8var1(Y105F)-Fc compared to 1D2×Fab7A5-Fc, as evidenced by significantly lower EC50 values.

Claims

1. A single domain antibody capable of binding to human CD123, comprising a complementarity determining region (CDR) 1 sequence set forth in SEQ ID NO: 2, a CDR2 sequence set forth in SEQ ID NO: 3, and a CDR3 sequence set forth in SEQ ID NO:

4.

2. a. X 1 is G; b. X3 is T; and / or c. X 4 is Y; A single domain antibody according to claim 1.

3. a. a first antigen-binding region capable of binding to human CD123, comprising the single domain antibody of claim 1 or 2; b. a second antigen-binding region capable of binding to the V52 chain of a human Vγ9V52 T cell receptor; A multispecific antibody comprising:

4. The multispecific antibody of claim 3 , wherein the multispecific antibody is a bispecific antibody.

5. A multispecific antibody described in claim 3 or 4, wherein the second antigen-binding region is a single domain antibody.

6. A multispecific antibody according to any one of claims 3 to 5, wherein the first antigen-binding region comprises or consists of a sequence selected from the group of sequences set forth in SEQ ID NO: 1 or 33, or a sequence having at least 90%, at least 92%, at least 94%, at least 96%, at least 98% sequence identity to a sequence selected from the group of sequences set forth in SEQ ID NO: 1 or 33.

7. The multispecific antibody of any one of claims 3 to 6, wherein said multispecific antibody is capable of activating human Vγ9Vδ2 T cells.

8. The second antigen-binding region comprises: a. the VH CDR1 sequence set forth in SEQ ID NO: 18, the VH CDR2 sequence set forth in SEQ ID NO: 19, and the VH CDR3 sequence set forth in SEQ ID NO: 20; b. the VH CDR1 sequence set forth in SEQ ID NO:39, the VH CDR2 sequence set forth in SEQ ID NO:40, and the VH CDR3 sequence set forth in SEQ ID NO:41; c. the VH CDR1 sequence set forth in SEQ ID NO: 42, the VH CDR2 sequence set forth in SEQ ID NO: 43, and the VH CDR3 sequence set forth in SEQ ID NO: 44; or d. the VH CDR1 sequence set forth in SEQ ID NO:45, the VH CDR2 sequence set forth in SEQ ID NO:46, and the VH CDR3 sequence set forth in SEQ ID NO:47; The multispecific antibody according to any one of claims 3 to 7, comprising:

9. The second antigen-binding region: a. the sequence set forth in SEQ ID NO: 17, or a sequence having at least 90%, at least 92%, at least 94%, at least 96%, at least 98% sequence identity to the sequence set forth in SEQ ID NO: 17; b. the sequence set forth in SEQ ID NO: 36, or a sequence having at least 90%, at least 92%, at least 94%, at least 96%, at least 98% sequence identity to the sequence set forth in SEQ ID NO: 36; c. the sequence set forth in SEQ ID NO: 37, or a sequence having at least 90%, at least 92%, at least 94%, at least 96%, at least 98% sequence identity to the sequence set forth in SEQ ID NO: 37; or d. the sequence set forth in SEQ ID NO: 38 or a sequence having at least 90%, at least 92%, at least 94%, at least 96%, at least 98% sequence identity to the sequence set forth in SEQ ID NO: 38; The multispecific antibody according to any one of claims 3 to 7, comprising or consisting of:

10. (i) The multispecific antibody of any one of claims 3 to 9, wherein the first antigen-binding region comprises SEQ ID NO: 1, the second antigen-binding region comprises SEQ ID NO: 17, and X 4 is F.

11. The multispecific antibody according to any one of claims 3 to 10, wherein the first antigen-binding region capable of binding to human CD123 is located N-terminal to the second antigen-binding region capable of binding to the human Vδ2 chain.

12. The multispecific antibody of any one of claims 3 to 10, wherein the multispecific antibody further comprises a half-life prolonging domain.

13. The multispecific antibody of claim 12, wherein the half-life extending domain is a human Fc region.

14. 14. The multispecific antibody of claim 13, wherein the Fc region is a heterodimer comprising two Fc polypeptides, the first antigen-binding region being fused to a first Fc polypeptide and the second antigen-binding region being fused to a second Fc polypeptide, and the first and second Fc polypeptides comprising asymmetric amino acid mutations that favor heterodimer formation over homodimer formation.

15. The multispecific antibody of claim 14, wherein the first Fc polypeptide comprises a T366W substitution and the second Fc polypeptide comprises T366S, L368A and Y407V substitutions, or vice versa, and wherein the amino acid positions correspond to human IgG1 according to the EU numbering system.

16. 16. The multispecific antibody of claim 14 or 15, wherein the cysteine ​​residue at position 220 of said first and second Fc polypeptides has been deleted or substituted, said amino acid position corresponding to human IgG1 according to the EU numbering system.

17. The multispecific antibody of any one of claims 14 to 16, wherein the first and second Fc polypeptides further comprise a mutation at position 234 and / or 235.

18. The multispecific antibody of claim 17, wherein the first and second Fc polypeptides comprise L234F and L235E substitutions, and the amino acid positions correspond to human IgG1 according to the EU numbering system.

19. The multispecific antibody of any one of claims 14 to 18, wherein said first Fc polypeptide comprises the sequence set forth in SEQ ID NO:21 and said second Fc polypeptide comprises the sequence set forth in SEQ ID NO:22, or vice versa.

20. The multispecific antibody according to any one of claims 3 to 19, wherein said multispecific antibody is capable of mediating the killing of CD123-expressing cells, such as C1R-neo cells or THP-1 cells, by Vγ9Vδ2 T cells.

21. 3. An antibody comprising a first antigen-binding region capable of binding to human CD123, said first antigen-binding region comprising a single domain antibody according to claim 1 or 2, The antibody, wherein the first antigen-binding region comprises or consists of a sequence selected from the group of sequences set forth in SEQ ID NO: 1 or 33, or a sequence having at least 90%, at least 92%, at least 94%, at least 96%, or at least 98% sequence identity to a sequence selected from the group of sequences set forth in SEQ ID NO: 1 or 33.

22. A pharmaceutical composition comprising a multispecific antibody according to any one of claims 3 to 20, a single domain antibody according to claim 1 or 2, or an antibody according to claim 21, and a pharma- ceutically acceptable excipient.

23. The pharmaceutical composition of claim 22 for use in a method for treating cancer.

24. The pharmaceutical composition of claim 23 for use in the treatment of acute myeloid leukemia, B-cell acute lymphoblastic leukemia, hairy cell leukemia, Hodgkin's lymphoma, blastic plasmacytoid dendritic tumor, chronic myeloid leukemia, chronic lymphocytic leukemia, B-cell chronic lymphoproliferative disease or myelodysplastic syndrome.

25. A nucleic acid construct comprising a nucleotide sequence encoding a multispecific antibody according to any one of claims 3 to 20, a single domain antibody according to claim 1 or 2, or an antibody according to claim 21.

26. A host cell comprising the nucleic acid construct of claim 25.

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