NZ716914A

NZ716914A - Bi-specific monovalent diabodies that are capable of binding cd123 and cd3, and uses therof

Info

Publication number: NZ716914A
Application number: NZ716914A
Authority: NZ
Inventors: Leslie Johnson; Ezio Bonvini; Ling Huang; Gurunadh Chichili; Ralph Alderson; Paul Moore
Original assignee: Macrogenics Inc
Priority date: 2013-08-23
Filing date: 2014-08-20
Publication date: 2021-09-24
Also published as: CL2016000362A1; DK3035965T3; LT3035965T; ES2860973T3; UA119539C2; GEP20197053B; HUE054315T2; HK1225999A1; HRP20210215T1

Abstract

The present invention is directed to sequence-optimized CD 123 x CD3 bi-specific monovalent diabodies that are capable of simultaneous binding to CD 123 and CD3, and to the uses of such diabodies in the treatment of hematologic malignancies.

Description

(12) Accepted (19) NZ (11) 716914 (13) A2 patent speciﬁcaon Title of the Invention: Bi-Speciﬁc Monovalent Diabodies That Are Capable Of Binding CD123 And CD3, And Uses Thereof Cross-Reference to Related ations This Application claims priority to United States Patent Applications No. 61/869,510 (ﬁled on August 23, 2013; pending), 61/907,749 (ﬁled on November 22, 2013; pending), and 61/990,475 (ﬁled on May 8, 2014; pending), and European Patent ation No. 13198784 (ﬁled on December 20, 2013), each of which ations is herein incorporated by reference in its entirety.

Reference to Sequence Listing: This application includes one or more Sequence Listings pursuant to 37 C.F.R. 1.821 et seq., which are disclosed in both paper and computer-readable media, and which paper and computer-readable disclosures are herein incorporated by reference in their entirety.

Background of the Invention: Field of the ion: The present invention is ed to CD123 X CD3 bi-speciﬁc monovalent diabodies that are capable of aneous binding to CD123 and CD3, and to the uses of such molecules in the treatment of logic malignancies.

Description of Related Art: I. CD123 CD123 leukin 3 receptor alpha, IL-3Ra) is a 40 kDa molecule and is part of the interleukin 3 receptor complex (Stomski, F.C. et al. (1996) “Human Interleukin-3 (IL-3) Induces Disulﬁde-Linked IL-3 Receptor Alpha- And Beta-Chain Heterodimerization, Which Is Required For Receptor Activation But Not High-Aﬁnity Binding,” M01. Cell. Biol. 16(6):3035-3046). Interleukin 3 (IL-3) drives early differentiation of multipotent stem cells into cells of the erythroid, myeloid and lymphoid progenitors. CD123 is sed on CD34+ committed progenitors (Taussig, D.C. et al. (2005) “Hematopoietic Stem Cells Express Multiple d s.‘ Implications For The Origin And Targeted Therapy 0f Acute Myeloid Leukemia,” Blood 106:4086-4092), but not by CD34+/CD38- normal poietic stem cells. CD123 is expressed by basophils, mast cells, plasmacytoid dendritic cells, some expression by monocytes, macrophages and eosinophils, and low or no expression by neutrophils and megakaryocytes. Some non-hematopoietic tissues (placenta, Leydig cells of the testis, certain brain cell elements and some endothelial cells) express CD123; however expression is mostly cytoplasmic.

CD123 is reported to be expressed by leukemic blasts and leukemia stem cells (LSC) (Jordan, C.T. et al. (2000) “The Interleukin-3 Receptor Alpha Chain Is A Unique Marker For Human Acute Myelogenous Leukemia Stem Cells,” ia 14:1777-1784; Jin, W. et al. (2009) “Regulation 0f ThI7 Cell Diﬁ’erentiation And EAE Induction By MAP3K NIK,” Blood 113:6603-6610) (Figure 1). In human normal sor populations, CD123 is expressed by a subset of hematopoietic progenitor cells (HPC) but not by normal hematopoietic stem cells (HSC). CD123 is also sed by plasmacytoid dendritic cells (pDC) and basophils, and, to a lesser extent, monocytes and eosinophils (Lopez, A.F. et al. (1989) “Reciprocal Inhibition ing Between Interleukin 3 And Granulocyte-Macrophage Colony-Stimulating Factor To Human Eosinophils,” Proc. Natl. Acad. Sci. (USA) 86:7022-7026; Sun, Q. et al. (1996) lonal Antibody 7G3 Recognizes The N-Terminal Domain Of The Human Interleukin-3 (IL-3) Receptor Alpha Chain And Functions As A Speciﬁc IL-3 or Antagonist,” Blood 87:83-92; Munoz, L. et al. (2001) “Interleukin-3 Receptor Alpha Chain (CD123) Is Widely Expressed In Hematologic ancies,” Haematologica 86(12):1261-1269; Masten, B]. et al. (2006) “Characterization 0f Myeloid And Plasmacytoid tic Cells In Human Lung,” J. Immunol. 177:7784- 7793; Korpelainen, E.I. et al. (1995) “Interferon-Gamma Upregulates Interleukin-3 (IL-3) Receptor Expression In Human Endothelial Cells And Synergizes With IL-3 In Stimulating Major Histocompatibility Complex Class II Expression And Cytokine Production,” Blood 86:176-182).

CD123 has been reported to be overexpressed on malignant cells in a wide range of logic malignancies including acute myeloid leukemia (AML) and ysplastic syndrome (MDS) (Munoz, L. et al. (2001) “Interleukin-3 Receptor Alpha Chain (CD123) Is Widely sed In logic Malignancies,” Haematologica 86(12):1261-1269). Overexpression of CD123 is associated with poorer prognosis in AML (Tettamanti, M.S. et al. (2013) “Targeting 0f Acute Myeloid Leukaemia By Cytokine-Induced Killer Cells Redirected With A Novel CD123-Speciﬁc Chimeric Antigen Receptor,” Br. J. Haematol. 161 :389-401).

AML and MDS are thought to arise in and be perpetuated by a small population of leukemic stem cells (LSCs), which are generally dormant (i.e., not rapidly dividing cells) and therefore resist cell death (apoptosis) and conventional chemotherapeutic agents. LSCs are characterized by high levels of CD123 expression, which is not present in the corresponding normal hematopoietic stem cell population in normal human bone marrow (Jin, W. et al. (2009) “Regulation 0f Th] 7 Cell Diﬁ’erentiation And EAE Induction By MAP3K NIK,” Blood 113:6603-6610; Jordan, C.T. et al. (2000) “The Interleukin-3 or Alpha Chain Is A Unique Marker For Human Acute Myelogenous Leukemia Stem Cells,” Leukemia 7- 1784). CD123 is expressed in 45%-95% of AML, 85% of Hairy cell leukemia (HCL), and 40% of acute B lymphoblastic leukemia (B-ALL). CD123 expression is also associated with multiple other malignancies/pre-malignancies: chronic myeloid leukemia (CML) progenitor cells (including blast crisis CML); Hodgkin’s Reed Stemberg (RS) cells; transformed non-Hodgkin’s lymphoma (NHL); some c lymphocytic leukemia (CLL) (CD11c+); a subset of acute T lymphoblastic leukemia (T-ALL) (16%, most re, mostly adult), plasmacytoid dendritic cell (pDC) (DC2) malignancies and CD34+/CD38- myelodysplastic syndrome (MDS) marrow cell malignancies.

AML is a clonal disease characterized by the proliferation and accumulation of transformed myeloid itor cells in the bone marrow, which ultimately leads to hematopoietic e. The nce of AML increases with age, and older patients typically have worse treatment outcomes than do younger patients (Robak, T. et al. (2009) nt And Emerging Therapies For Acute Myeloid Leukemia,” Clin. Ther. 2:2349-2370). unately, at present, most adults with AML die from their disease.

Treatment for AML lly focuses in the induction of remission (induction therapy). Once remission is achieved, treatment shifts to focus on securing such remission (post-remission or consolidation therapy) and, in some instances, maintenance therapy. The standard remission induction paradigm for AML is chemotherapy with an anthracycline/cytarabine combination, followed by either consolidation chemotherapy (usually with higher doses of the same drugs as were used during the induction period) or human stem cell transplantation, depending on the t's ability to te intensive treatment and the likelihood of cure with chemotherapy alone (see, e.g., Roboz, G.J. (2012) nt Treatment Of Acute Myeloid Leukemia,” Curr. Opin. Oncol. 24:711-719).

Agents frequently used in induction therapy include cytarabine and the anthracyclines. Cytarabine, also known as AraC, kills cancer cells (and other rapidly dividing normal cells) by interfering with DNA synthesis. Side effects ated with AraC treatment include decreased resistance to infection, a result of decreased white blood cell production; ng, as a result of decreased platelet tion; and anemia, due to a potential reduction in red blood cells. Other side effects include nausea and vomiting. Anthracyclines (e.g., daunorubicin, doxorubicin, and idarubicin) have several modes of action including inhibition of DNA and RNA synthesis, disruption of higher order structures of DNA, and production of cell damaging free oxygen radicals. The most consequential e effect of anthracyclines is cardiotoxicity, which considerably limits administered life-time dose and to some extent their usefulness.

Thus, unfortunately, despite substantial ss in the treatment of newly diagnosed AML, 20% to 40% of patients do not achieve remission with the standard induction chemotherapy, and 50% to 70% of patients entering a ﬁrst te remission are expected to relapse within 3 years. The optimum strategy at the time of e, or for patients with the resistant disease, remains uncertain. Stem cell transplantation has been ished as the most effective form of anti-leukemic therapy in patients with AML in first or subsequent remission (Roboz, G]. (2012) “Current Treatment OfAcute Myeloid Leukemia,” Curr. Opin. Oncol. 24:711-719). 11. CD3 CD3 is a T cell co-receptor composed of four distinct chains (Wucherpfennig, KW. et al. (2010) “Structural Biology Of The T-Cell Receptor: Insights Into Receptor Assembly, Ligand Recognition, And Initiation 0f Signaling,” Cold Spring Harb. Perspect. Biol. 2(4):a005140; pages 1-14). In mammals, the complex contains a CD3}! chain, a CD38 chain, and two CD38 chains. These chains associate with a molecule known as the T cell receptor (TCR) in order to te an activation signal in T lymphocytes. In the absence of CD3, TCRs do not assemble properly and are degraded (Thomas, S. et al. (2010) “Molecular Immunology Lessons From Therapeutic T-Cell Receptor Gene Transfer,” Immunology l29(2):170—l77).

CD3 is found bound to the membranes of all mature T cells, and in virtually no other cell type (see, Janeway, CA. et al. (2005) In: IMMUNOBIOLOGY: THE IMMUNE SYSTEM IN HEALTH AND DISEASE,” 6th ed. Garland Science Publishing, NY, pp. 214- 216; Sun, Z. J. et al. (2001) “Mechanisms Contributing To T Cell Receptor Signaling And Assembly Revealed By The Solution Structure OfAn Ectodomain Fragment Of The CD383)» Heterodimer,” Cell :913-923; Kuhns, M.S. et al. (2006) “Deconstructing The Form And Function Of The TCR/CD3 Complex,” Immunity. 2006 Feb;24(2):l33-l39).

III. Bi-Speciﬁc Diabodies The ability of an intact, unmodiﬁed antibody (e.g., an IgG) to bind an epitope of an n s upon the presence of le s on the immunoglobulin light and heavy chains (i.e., the VL and VH domains, respectively). The design of a y is based on the single chain Fv uct (scFv) (see, e.g., Holliger et al. (1993) “’Diabodies Small Bivalent And Bispeciﬁc Antibody Fragments,” Proc. Natl.

Acad. Sci. (USA) 90:6444-6448; US 2004/0058400 (Hollinger et al.); US 220388 (Mertens et al.); Alt et al. (1999) FEBS Lett. 454(1-2):90-94; Lu, D. et al. (2005) “A Fully Human Recombinant IgG-Like Bispeciﬁc dy To Both The Epidermal Growth Factor Receptor And The Insulin-Like Growth Factor or For Enhanced Antitumor Activity,” J. Biol. Chem. 280(20):19665-19672; WO 02/02781 (Mertens et al.); Olafsen, T. et al. (2004) “Covalent Disulﬁde-Linked Anti- CEA Diabody Allows Site-Speciﬁc Conjugation And Radiolabeling For Tumor Targeting Applications,” n Eng. Des. Sel. 17(1):21-27; Wu, A. et al. (2001) “Multimerization Of A Chimeric Anti-CD20 Single Chain Fv-Fv Fusion Protein Is Mediated Through Variable Domain Exchange,” Protein ering 14(2):1025- 1033; Asano et al. (2004) “A Diabody For Cancer Immunotherapy And Its Functional ement By Fusion Of Human Fc Domain,” Abstract 3P-683, J. Biochem. 992; Takemura, S. et al. (2000) “Construction Of A Diabody (Small Recombinant Bispeciﬁc Antibody) Using A Refolding System,” Protein Eng. 13(8):.583-588; Baeuerle, P.A. et al. (2009) “Bispeciﬁc T-Cell Engaging Antibodies For Cancer Therapy,” Cancer Res. :4941-4944).

Interaction of an dy light chain and an antibody heavy chain and, in particular, interaction of its VL and VH domains forms one of the epitope binding sites of the antibody. In contrast, the scFv construct comprises a VL and VH domain of an dy contained in a single polypeptide chain wherein the domains are separated by a ﬂexible linker of sufficient length to allow self-assembly of the two domains into a ﬁanctional epitope binding site. Where self-assembly of the VL and VH domains is rendered impossible due to a linker of insufficient length (less than about 12 amino acid residues), two of the scFv constructs interact with one another other to form a bivalent le in which the VL of one chain associates with the VH of the other (reviewed in Marvin et al. (2005) “Recombinant Approaches To IgG- Like Bispeciﬁc Antibodies, ” Acta Pharmacol. Sin. 26:649-658).

Natural dies are capable of binding to only one epitope species (i.e., mono-specific), although they can bind multiple copies of that s (i.e., exhibiting bi-Valency or multi-valency). The art has noted the capability to produce diabodies that differ from such natural antibodies in being e of binding two or more different epitope species (i. e., exhibiting bi-specif1city or pecif1city in on to bi-Valency or multi-valency) (see, e.g., Holliger et al. (1993) “’Diabodies’. Small Bivalent And Bispeciﬁc Antibody Fragments,” Proc. Natl. Acad. Sci. (USA) 90:6444-6448; US 2004/0058400 (Hollinger et al.); US 2004/0220388 (Mertens et al.); Alt et al. (1999) FEBS Lett. 454(1-2):90-94; Lu, D. et al. (2005) “A Fully Human Recombinant IgG-Like Bispeciﬁc Antibody To Both The Epidermal Growth Factor Receptor And The Insulin-Like Growth Factor Receptor For Enhanced Antitumor Activity,” J. Biol. Chem. 280(20):19665-19672; WO 02/02781 (Mertens et al.); Mertens, N. et al., “New Recombinant Bi- and Trispeciﬁc Antibody Derivatives,” In: NOVEL FRONTIERS IN THE PRODUCTION OF COMPOUNDS FOR ICAL USE, A.

VanBroekhoven et al. (Eds.), Kluwer Academic Publishers, Dordrecht, The Netherlands (2001), pages 195-208; Wu, A. et al. (2001) “Multimerization Of A Chimeric Anti-CD20 Single Chain Fv-Fv Fusion Protein Is Mediated Through Variable Domain Exchange,” Protein Engineering 14(2):1025-1033; Asano et al. (2004) “A Diabody For Cancer therapy And Its onal Enhancement By Fusion OfHuman Fc Domain,” Abstract 3P-683, J. m. 76(8):992; ra, S. et al. (2000) ruction OfA Diabody (Small Recombinant Bispeciﬁc Antibody) Using A Refolding System,” Protein Eng. 13(8):583-588; Baeuerle, P.A. et al. (2009) “Bispeciﬁc T-Cell Engaging Antibodies For Cancer Therapy,” Cancer Res. :4941-4944).

The provision of non-monospeciﬁc diabodies provides a signiﬁcant advantage: the ty to co-ligate and co-localize cells that express different epitopes. Bi-speciﬁc diabodies thus have wide-ranging applications including therapy and immunodiagnosis. ciﬁcity allows for great ﬂexibility in the design and engineering of the diabody in various ations, providing enhanced avidity to multimeric antigens, the cross-linking of differing antigens, and directed targeting to speciﬁc cell types relying on the presence of both target antigens. Due to their increased valency, low iation rates and rapid clearance from the circulation (for diabodies of small size, at or below ~50 kDa), diabody molecules known in the art have also shown particular use in the ﬁeld of tumor imaging (Fitzgerald et al. (1997) “Improved Tumour Targeting By Disulphide Stabilized Diabodies Expressed In Pichia pastoris, ” Protein Eng. 10:1221). Of particular importance is the co-ligating of differing cells, for example, the cross-linking of cytotoxic T cells to tumor cells (Staerz et al. (1985) “Hybrid Antibodies Can Target Sites For Attack By T Cells, ” Nature 314:628-631, and Holliger et al. (1996) “Speciﬁc g OfLymphoma Cells By Cytotoxic T-Cells Mediated By A Bispeciﬁc Diabody, ” Protein Eng. 9:299-305).

Diabody epitope binding domains may also be directed to a e determinant of any immune effector cell such as CD3, CD16, CD32, or CD64, which are expressed on T lymphocytes, natural killer (NK) cells or other mononuclear cells.

In many studies, diabody g to effector cell determinants, e. g., Fcy ors (FcyR), was also found to activate the effector cell (Holliger et al. (1996) “Specific g 0fLymphoma Cells By Cytotoxic T-Cells Mediated By A Bispeciﬁc Diabody, ” Protein Eng. 9:299-305; Holliger et al. (1999) “Carcinoembryonic Antigen (CEA)- c T-cell Activation In Colon Carcinoma Induced By Anti-CD3 x Anti-CEA Bispeciﬁc Diabodies And B7 x Anti-CEA iﬁc Fusion ns, ” Cancer Res. 59:2909-2916; WO 2006/113665; WO 2008/157379; WO 2010/080538; WO 2012/018687; ). Normally, effector cell activation is triggered by the binding of an antigen bound antibody to an effector cell via Fc-FcyR interaction; thus, in this regard, diabody molecules may exhibit e ﬁanctionality independent of whether they comprise an Fc Domain (e. g., as assayed in any effector function assay known in the art or exempliﬁed herein (e.g., ADCC assay)). By cross-linking tumor and effector cells, the diabody not only brings the effector cell within the proximity of the tumor cells but leads to effective tumor killing (see e. g., Cao et al. (2003) “Bispeciﬁc Antibody Conjugates In Therapeutics,” Adv. Drug. DeliV. ReV. 55:171-197).

However, the above advantages come at a salient cost. The formation of such non-monospecif1c diabodies requires the successful assembly of two or more distinct and different polypeptides (i. e., such formation requires that the diabodies be formed h the heterodimerization of different polypeptide chain species). This fact is in contrast to mono-speciﬁc diabodies, which are formed through the homodimerization of identical polypeptide . e at least two dissimilar polypeptides (i.e., two polypeptide species) must be provided in order to form a nonmonospeciﬁc diabody, and e homodimerization of such polypeptides leads to inactive molecules (Takemura, S. et al. (2000) “Construction OfA Diabody (Small Recombinant Bispeciﬁc Antibody) Using A Refolding System,” Protein Eng. l3(8):583-588), the production of such polypeptides must be accomplished in such a way as to t covalent bonding between polypeptides of the same s (i.e., so as to t merization) (Takemura, S. et al. (2000) “Construction Of A Diabody (Small Recombinant Bispeciﬁc Antibody) Using A Refolding System,” Protein Eng. l3(8):583-588). The art has therefore taught the non-covalent ation of such polypeptides (see, e.g., Olafsen et al. (2004) “Covalent de- Linked Anti-CEA y Allows Site-Speciﬁc Conjugation And Radiolabeling For Tumor Targeting Applications,” Prot. Engr. Des. Sel. 17:21-27; Asano et al. (2004) “A Diabody For Cancer Immunotherapy And Its Functional ement By Fusion OfHuman Fc Domain,” Abstract 3P-683, J. Biochem. 76(8):992; Takemura, S. et al. (2000) “Construction OfA Diabody (Small Recombinant Bispeciﬁc Antibody) Using A Refolding System,” Protein Eng. l3(8):583-588; Lu, D. et al. (2005) “A Fully Human inant IgG-Like Bispeciﬁc Antibody To Both The Epidermal Growth Factor Receptor And The Insulin-Like Growth Factor Receptor For Enhanced Antitumor Activity,” J. Biol. Chem. 280(20): 19665-19672).

However, the art has recognized that bi-speciﬁc diabodies composed of non- covalently associated polypeptides are unstable and readily dissociate into non- functional monomers (see, e.g., Lu, D. et al. (2005) “A Fully Human Recombinant IgG-Like Bispeciﬁc Antibody To Both The Epidermal Growth Factor Receptor And The Insulin-Like Growth Factor or For Enhanced Antitumor ty,” J. Biol.

Chem. 280(20):l9665-l9672).

In the face of this challenge, the art has succeeded in developing stable, covalently bonded heterodimeric non-monospeciﬁc diabodies (see, e.g., WO 2006/113665; WO/2008/157379; WO 2010/080538; WO 2012/018687; WO/2012/162068; Johnson, S. et al. (2010) “Eﬂector Cell Recruitment With Novel Fv-Based Dual-Aﬁinity geting Protein Leads To Potent Tumor Cytolysis And In Vivo B—Cell Depletion,” J. Molec. Biol. 399(3):436-449; Veri, MC. et al. (2010) peutic Control OfB Cell Activation Via Recruitment Ochgamma Receptor Hb (CD323) tory on With A Novel Bispeciﬁc Antibody Scaﬂold,” tis Rheum. 62(7):l933-l943; Moore, P.A. et al. (2011) cation Of Dual Aﬁinity Retargeting Molecules To Achieve Optimal Redirected T-Cell Killing 0f B-Cell Lymphoma,” Blood ll7(l7):4542-4551). Such approaches involve engineering one or more cysteine residues into each of the employed polypeptide species. For example, the addition of a cysteine residue to the C-terminus of such constructs has been shown to allow disulﬁde bonding between the polypeptide chains, stabilizing the resulting heterodimer without interfering with the binding characteristics of the bivalent molecule.

Notwithstanding such success, the production of stable, ﬁanctional heterodimeric, non-monospecif1c diabodies can be r optimized by the careful consideration and placement of cysteine residues in one or more of the employed ptide chains. Such zed ies can be produced in higher yield and with greater activity than non-optimized diabodies. The t invention is thus directed to the problem of providing polypeptides that are particularly designed and optimized to form dimeric diabodies. The ion solves this problem through the provision of exemplary, optimized CD123 X CD3 diabodies.

Summary of the Invention: The present invention is directed to CD123 X CD3 bi-specif1c diabodies that are capable of simultaneous g to CD123 and CD3, and to the uses of such molecules in the ent of disease, in particular hematologic malignancies.

The CD123 X CD3 bi-specific diabodies of the invention comprise at least two different polypeptide chains that associate with one another in a heterodimeric manner to form one binding site specific for an epitope of CD 123 and one binding site speciﬁc for an epitope of CD3. A CD123 X CD3 y of the invention is thus monovalent in that it is capable of binding to only one copy of an e of CD123 and to only one copy of an epitope of CD3, but bi-specif1c in that a single diabody is able to bind simultaneously to the epitope of CD123 and to the epitope of CD3. The individual polypeptide chains of the diabodies are ntly bonded to one r, for example by disulﬁde bonding of cysteine residues located within each polypeptide chain. In particular embodiments, the diabodies of the present invention ﬁarther have an immunoglobulin Fc Domain or an Albumin-Binding Domain to extend half-life in vivo .

In detail, the invention also provides a sequence-optimized CD123 X CD3 bi- speciﬁc monovalent diabody capable of specific binding to an epitope of CD123 and to an epitope of CD3, wherein the diabody comprises a ﬁrst polypeptide chain and a second polypeptide chain, covalently bonded to one another, wherein: A. the ﬁrst polypeptide chain comprises, in the inal to C-terminal direction: i. a Domain 1, comprising: (1) a sub-Domain (1A), which comprises a VL Domain of a monoclonal antibody capable of binding to CD3 (VLCD3) (SEQ ID NO:21); and (2) a sub-Domain (1B), which ses a VH Domain of a monoclonal antibody capable of binding to CD123 ) (SEQ ID NO:26); wherein the sub-Domains 1A and 1B are separated from one another by a peptide linker (SEQ ID NO:29); ii. a Domain 2, wherein the Domain 2 is an E-coil Domain (SEQ ID NO:34) or a K-coil Domain (SEQ ID NO:35), wherein the Domain 2 is separated from the Domain 1 by a peptide linker (SEQ ID NO:30); the second polypeptide chain comprises, in the N-terminal to C-terminal direction: i. a Domain 1, comprising: (1) a sub-Domain (1A), which comprises a VL Domain of a monoclonal antibody capable of binding to CD123 ) (SEQ ID NO:25); and (2) a sub-Domain (1B), which comprises a VH Domain of a monoclonal antibody capable of binding to CD3 (VHCDg) (SEQ ID NO:22); wherein the sub-Domains 1A and 1B are separated from one another by a e linker (SEQ ID NO:29); ii. a Domain 2, wherein the Domain 2 is a K-coil Domain (SEQ ID NO:35) or an E-coil Domain (SEQ ID , wherein the Domain 2 is separated from the Domain 1 by a peptide linker (SEQ ID NO:30); and wherein the Domain 2 of the ﬁrst and the second ptide chains are not both E-coil Domains or both K-coil Domains; -1]- and wherein: (a) said VL Domain of said ﬁrst polypeptide chain and said VH Domain of said second polypeptide chain form an Antigen Binding Domain capable of speciﬁcally binding to an e of CD3; and (b) said VL Domain of said second polypeptide chain and said VH Domain of said ﬁrst polypeptide chain form an Antigen Binding Domain capable of speciﬁcally binding to an epitope of CD123.

The invention also provides a non-sequence-optimized CD123 X CD3 bi- speciﬁc monovalent diabody capable of speciﬁc binding to an epitope of CD123 and to an e of CD3, wherein the diabody comprises a ﬁrst polypeptide chain and a second polypeptide chain, covalently bonded to one another, wherein: A. the ﬁrst polypeptide chain comprises, in the N—terminal to inal direction: i. a Domain 1, comprising: (1) a sub-Domain (1A), which comprises a VL Domain of a monoclonal dy e of binding to CD3 (VLCD3) (SEQ ID NO:23); and (2) a sub-Domain (1B), which comprises a VH Domain of a monoclonal antibody capable of binding to CD123 (VHCDm) (SEQ ID NO:28); wherein the sub-Domains 1A and 1B are separated from one r by a peptide linker (SEQ ID NO:29); ii. a Domain 2, wherein the Domain 2 is an E-coil Domain (SEQ ID NO:34) or a K-coil Domain (SEQ ID NO:35), wherein the Domain 2 is separated from the Domain 1 by a peptide linker (SEQ ID NO:30); B. the second polypeptide chain comprises, in the N-terminal to C-terminal direction: i. a Domain 1, comprising: (1) a sub-Domain (1A), which comprises a VL Domain of a onal antibody capable of binding to CD123 (VLCDm) (SEQ ID NO:27); and (2) a sub-Domain (1B), which comprises a VH Domain of a monoclonal antibody capable of binding to CD3 (VHCDg) (SEQ ID NO:24); wherein the mains 1A and 1B are separated from one another by a peptide linker (SEQ ID NO:29); ii. a Domain 2, wherein the Domain 2 is a K-coil Domain (SEQ ID NO:35) or an E-coil Domain (SEQ ID NO:34), wherein the Domain 2 is ted from the Domain 1 by a peptide linker (SEQ ID NO:30); and wherein the Domain 2 of the ﬁrst and the second polypeptide chains are not both E-coil Domains or both K-coil Domains and wherein: (a) said VL Domain of said ﬁrst polypeptide chain and said VH Domain of said second polypeptide chain form an Antigen Binding Domain capable of speciﬁcally binding to an epitope of CD3; and (b) said VL Domain of said second polypeptide chain and said VH Domain of said ﬁrst polypeptide chain form an Antigen Binding Domain capable of speciﬁcally binding to an epitope of CDl23.

The invention additionally provides the ment of the above-described bi-speciﬁc monovalent diabodies, wherein the ﬁrst or second polypeptide chain additionally comprises an Albumin-Binding Domain (SEQ ID NO:36) linked, C- ally to Domain 2 or N-terminally to Domain 1, via a peptide linker (SEQ ID NO:31).

The invention additionally es the embodiment of the above-described bi-speciﬁc monovalent ies wherein the ﬁrst or second polypeptide chain additionally comprises a Domain 3 comprising a CH2 and CH3 Domain of an globulin IgG Fc Domain (SEQ ID NO:37), wherein the Domain 3 is linked, N—terminally, to the Domain lA via a peptide linker (SEQ ID NO:33).

The ion additionally provides the embodiment of the above-described bi-speciﬁc monovalent diabodies wherein the ﬁrst or second polypeptide chain additionally comprises a Domain 3 comprising a CH2 and CH3 Domain of an immunoglobulin IgG Fc Domain (SEQ ID NO:37), wherein the Domain 3 is linked, C-terminally, to the Domain 2 via a peptide linker (SEQ ID NO:32).

The invention additionally provides the embodiment of any of the abovedescribed bi-speciﬁc monovalent ies wherein the Domain 2 of the ﬁrst polypeptide chain is a K-coil Domain (SEQ ID NO:35) and the Domain 2 of the second polypeptide chain is an E-coil Domain (SEQ ID NO:34).

The invention additionally provides the embodiment of any of the above- described bi-speciﬁc monovalent diabodies wherein the Domain 2 of the ﬁrst polypeptide chain is an E-coil Domain (SEQ ID NO:34) and the Domain 2 of the second polypeptide chain is a K-coil Domain (SEQ ID NO:35).

The invention additionally provides the embodiment of a bi-speciﬁc monovalent diabody capable of speciﬁc binding to an epitope of CD123 and to an epitope of CD3, wherein the diabody ses a ﬁrst polypeptide chain and a second polypeptide chain, covalently bonded to one another, n: said bi-speciﬁc diabody ses: A. a ﬁrst polypeptide chain having the amino acid sequence of SEQ ID NO:1; and B. a second polypeptide chain having the amino acid sequence of SEQ ID NO:3; wherein said ﬁrst and said second polypeptide chains are covalently bonded to one another by a disulﬁde bond.

The diabodies of the invention t unexpectedly enhanced onal activities as further described below.

The ies of the invention are preferably capable of cross-reacting with both human and primate CD123 and CD3 proteins, preferably cynomolgus monkey CD123 and CD3 proteins.

The diabodies of the ion are preferably capable of depleting, in an in vitro cell-based assay, cytoid dendritic cells (pDC) from a culture of primary PBMCs with an ICSO of about 1 ng/ml or less, about 0.8 ng/ml or less, about 0.6 ng/ml or less, about 0.4 ng/ml or less, about 0.2 ng/ml or less, about 0.1 ng/ml or less, about 0.05 ng/ml or less, about 0.04 ng/ml or less, about 0.03 ng/ml or less, about 0.02 ng/ml or less or about /ml or less. Preferably, the IC50 is about 0.01ng/ml or less. In the above-described assay, the culture of primary PBMCs may be from cynomolgus monkey in which case said depletion is of cynomolgus monkey cytoid dendritic cells (pDC). Optionally the diabodies of the invention may be capable of depleting cytoid dendritic cells (pDC) from a primary culture of PBMCs as described above wherein the assay is conducted by or in accordance with the protocol of Example 14, as herein described, or by modiﬁcation of such assay as would be understood by those of ordinary skill, or by other means known to those of ry skill.

The diabodies of the invention preferably exhibit cytotoxicity in an in vitro Kasumi-3 assay with an EC50 of about 0.05 ng/mL or less. Preferably, the EC50 is about 0.04 ng/mL or less, about 0.03 ng/mL or less, about 0.02 ng/mL or less, or about 0.01 ng/mL or less. Optionally the diabodies of the ion may exhibit cytotoxicity as described above wherein the assay is conducted by or in accordance with the protocol of Example 3 as herein described, or by modification of such assay as would be understood by those of ordinary skill, or by other means known to those of ordinary skill.

The diabodies of the invention preferably exhibit cytotoxicity in an in vitro Molm-l3 assay with an EC50 of about 5 ng/mL or less. Preferably, the EC50 is about 3 ng/mL or less, about 2 ng/mL or less, about 1 ng/mL or less, about 0.75 ng/mL or less, or about 0.2 ng/mL or less. Optionally the diabodies of the invention may exhibit xicity as described above wherein the assay is ted by or in accordance with the protocol of Example 3 as herein described, or by modification of such assay as would be understood by those of ordinary skill, or by other means known to those of ordinary skill.

The diabodies of the invention are preferably capable of inhibiting the growth of a MOLM-l3 tumor xenograft in a mouse. Preferably the diabodies of the ion may be capable of inhibiting the growth of a MOLM-l3 tumor xenograft in a mouse at a concentration of at least about 20 ug/kg, at least about 4 ug/kg, at least about 0.8 ug/kg, at least about 0.6 ug/kg or at least about 0.4 ug/kg. Preferred antibodies of the invention will inhibit growth of a 3 tumor xenograft in a mouse by at least 25%, but possibly by at least about 40% or more, by at least about 50% or more, by at least about 60% or more, by at least about 70% or more, by at least about 80% or more, by at least about 90% or more, or even by completely inhibiting MOLM-l3 tumor growth after some period of time or by causing tumor regression or disappearance. This inhibition will take place for at least an NSG mouse strain. Optionally, the diabodies of the invention may be capable of inhibiting the growth of a MOLM-l3 tumor xenograft in a mouse in the above-described manner by or in accordance with the protocol of Example 6 as herein described, or by modiﬁcation of such assay as would be understood by those of ordinary skill, or by other means known to those of ordinary skill.

The diabodies of the invention are ably capable of inhibiting the growth of an RS4-ll tumor xenograft in a mouse. Preferably the diabodies of the invention may be capable of inhibiting the growth of a RS4-ll tumor aft in a mouse at a concentration of at least about 0.5 mg/kg, at least about 0.2 mg/kg, at least about 0.1 mg/kg, at least about 0.02 mg/kg or at least about 0.004 mg/kg. Preferred antibodies of the invention will t growth of a RS4-ll tumor aft in a mouse by at least about 25%, but possibly at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or even by completely inhibiting RS4-ll tumor growth after some period of time or by causing tumor regression or disappearance. This inhibition will take place for at least an NSG mouse strain. Optionally, the diabodies of the invention may be capable of inhibiting the growth of a RS4-11 tumor xenograft in a mouse in the above-described manner by or in accordance with the protocol of Example 6 as herein described, or by modiﬁcation of such assay as would be understood by those of ordinary skill, or by other means known to those of ordinary skill.

The ies of the invention are preferably capable of depleting leukemic blast cells in vitro in a primary e of AML bone marrow cells. Preferably the diabodies of the invention may be capable of depleting leukemic blast cells in vitro in a primary culture of AML bone marrow cells at concentrations of at least about 0.01 ng/ml, at least about 0.02 ng/ml, at least about 0.04 ng/ml, at least about 0.06 ng/ml, at least about 0.08 ng/ml or at least about 0.1 ng/ml. Preferably, the diabodies of the invention may be capable of ing leukemic blast cells in vitro in a primary culture ofAML bone marrow cells to less than 20% of the total population of primary leukemic blast cells at diabody concentrations of at least about 0.01 ng/ml, at least about 0.02 ng/ml, at least about 0.04 ng/ml, at least about 0.06 ng/ml, at least about 0.08 ng/ml or at least about 0.1 ng/ml, optionally ing incubation of the primary culture with diabody for about 120 hours. Preferably leukemic blast cells are depleted in vitro in a primary culture of AML bone marrow cells to less than 20% of the total population of primary ic blast cells at diabody concentrations of about 0.01 ng/ml or 0.1 ng/ml following incubation of the primary culture with diabody for about 120 hours.

The diabodies of the invention are preferably capable of inducing an ion of a T cell population in vitro in a primary culture of AML bone marrow cells. ably, such expansion may be to about 70% or more of the maximum T cell population which can be expanded in the assay. Preferably the diabodies of the invention may be capable of inducing an expansion of a T cell population in vitro in a primary culture ofAML bone marrow cells to about 70% or more of the maximum T cell population which can be expanded in the assay at diabody concentrations of at least about 0.01 ng/ml, at least about 0.02 ng/ml, at least about 0.04 ng/ml, at least about 0.06 ng/ml, at least about 0.08 ng/ml or at least about 0.1 ng/ml, optionally following incubation of the y culture with diabody for about 120 hours. ably, a T cell population is expanded in vitro in a primary e ofAML bone marrow cells to about 70% or more of the maximum T cell population which can be expanded in the assay at diabody concentrations of about 0.01 ng/ml or about 0.1 ng/ml following tion of the primary culture with diabody for about 120 hours.

The diabodies of the invention are preferably capable of inducing an activation of a T cell population in vitro in a primary culture of AML bone marrow cells. Such activation may occur at diabody concentrations of at least about 0.01 ng/ml, at least about 0.02 ng/ml, at least about 0.04 ng/ml, at least about 0.06 ng/ml, at least about 0.08 ng/ml or at least about 0.1 ng/ml, ally following incubation of the primary culture with diabody for about 72 hours. Such activation may be measured by the sion of a T cell tion marker such as CD25. Preferably, activation of a T cell population in vitro in a primary culture of AML bone marrow cells as measured by expression of CD25 may occur at diabody concentrations of about 0.01 ng/ml or about 0.1 ng/ml ing incubation of the primary e with diabody for about 72 hours.

The diabodies of the invention are preferably e of depleting leukemic blast cells in vitro in a primary e ofAML bone marrow cells to less than 20% of the total population of primary leukemic blast cells and at the same time inducing an expansion of the T cell population in vitro in the primary culture of AML bone marrow cells to about 70% or more of the maximum T cell population which can be expanded in the assay at diabody concentrations of at least about 0.01 ng/ml, at least about 0.02 ng/ml, at least about 0.04 ng/ml, at least about 0.06 ng/ml, at least about 0.08 ng/ml or at least about 0.1 ng/ml, optionally following tion of the y culture with y for about 120 hours. Preferably, the diabody concentrations are about 0.01 ng/ml or about 0.1 ng/ml and the primary culture is incubated with diabody for about 120 hours.

The diabodies of the invention may be capable of depleting leukemic blast cells in vitro in a primary culture of AML bone marrow cells and/or inducing an expansion of a T cell population in vitro in a primary culture of AML bone marrow cells and/or inducing an activation of a T cell population in vitro in a primary culture of AML bone marrow cells in the above-described manner by or in accordance with the protocol of Example 8 as herein described, or by modiﬁcation of such assay as would be understood by those of ordinary skill, or by other means known to those of ordinary skill.

For the avoidance of any doubt, the diabodies of the invention may exhibit one, two, three, more than three or all of the ﬁanctional attributes described herein.

Thus the diabodies of the invention may exhibit any combination of the ﬁanctional attributes described herein.

The ies of the invention may be for use as a pharmaceutical.

Preferably, the ies are for use in the treatment of a disease or condition associated with or characterized by the sion of CD123. The invention also relates to the use of diabodies of the ion in the manufacture of a pharmaceutical composition, ably for the treatment of a disease or condition associated with or characterized by the expression of CD123 as ﬁarther deﬁned herein.

The disease or condition associated with or characterized by the expression of CD123 may be cancer. For example, the cancer may be selected from the group consisting of: acute myeloid leukemia (AML), chronic myelogenous ia (CML), including blastic crisis of CML and Abelson oncogene associated with CML (Bcr-ABL translocation), myelodysplastic syndrome (MDS), acute B lymphoblastic leukemia (B-ALL), chronic lymphocytic leukemia (CLL), including Richter’s syndrome or Richter’s transformation of CLL, hairy cell leukemia (HCL), c plasmacytoid dendritic cell neoplasm (BPDCN), non-Hodgkin lymphomas (NHL), including mantel cell ia (MCL), and small lymphocytic lymphoma (SLL), Hodgkin’s lymphoma, systemic mastocytosis, and Burkitt’s lymphoma.

The disease or condition ated with or characterized by the expression of CD123 may be an inﬂammatory condition. For example, the inﬂammatory ion may be selected from the group consisting of: Autoimmune Lupus (SLE), allergy, asthma and rheumatoid arthritis.

The invention additionally provides a pharmaceutical composition comprising any of the above-described diabodies and a physiologically acceptable carrier.

The invention additionally provides a use of the above-described pharmaceutical composition in the treatment of a disease or condition associated with or characterized by the expression of CD123.

The invention is particularly ed to the embodiment of such use, wherein the disease or condition associated with or characterized by the sion of CD123 is cancer (especially a cancer selected from the group ting of: acute myeloid leukemia (AML), chronic myelogenous leukemia (CML), including blastic crisis of CML and Abelson oncogene associated with CML BL translocation), myelodysplastic syndrome (MDS), acute B blastic leukemia (B-ALL), chronic lymphocytic leukemia (CLL), including Richter’s syndrome or Richter’s transformation of CLL, hairy cell leukemia (HCL), blastic plasmacytoid dendritic cell neoplasm (BPDCN), non-Hodgkin lymphomas (NHL), including mantel cell leukemia (MCL), and small lymphocytic lymphoma (SLL), Hodgkin’s lymphoma, systemic mastocytosis, and Burkitt’s lymphoma).

The invention is also particularly ed to the embodiment of such use, wherein the disease or condition associated with or terized by the expression of CD123 is an atory condition (especially an inﬂammatory condition selected from the group consisting of: Autoimmune Lupus (SLE), allergy, asthma, and rheumatoid arthritis).

Terms such as “about” should be taken to mean within 10%, more preferably within 5%, of the speciﬁed value, unless the context requires otherwise.

Brief Description of the Drawings: Figure 1 shows that CD123 was known to be expressed on leukemic stem cells.

Figure 2 illustrates the structures of the ﬁrst and second polypeptide chains of a two chain CD123 X CD3 ciﬁc lent diabody of the t invention.

Figures 3A and 3B illustrate the structures of two versions of the ﬁrst, second and third ptide chains of a three chain CD123 X CD3 bi-speciﬁc monovalent Fc diabody of the present invention on 1, Figure 3A; Version 2, Figure 3B).

Figure 4 (Panels A-E) shows the y of different CD123 X CD3 bi- speciﬁc diabodies to mediate T cell redirected killing of target cells displaying varying amount of CD123. The Figure provides dose-response curves indicating that the sequence-optimized CD123 X CD3 bi-speciﬁc diabody (“DART-A”) having an Albumin-Binding Domain (DART-A with ABD “w/ABD”) exhibited greater cytotoxicity than a control bi-specif1c diabody (Control DART) or a non-sequenceoptimized CD123 X CD3 bi-specific diabody -B”) in multiple target cell types: RS4-ll (Panel A); TF-l (Panel B); Molm-l3 (Panel C); Kasumi-3 (Panel D); and THP-l (Panel E) at an E:T (effector : target) ratio of 10: 1.

Figure 5 (Panels A-D) shows the ability of the sequence-optimized CD123 X CD3 bi-specif1c diabody A), sequence-optimized CD123 X CD3 bi-specif1c diabody having an Albumin-Binding Domain (DART-A with ABD “w/ABD”) and ce-optimized CD123 X CD3 bi-specif1c diabody having an immunoglobulin IgG Fc Domain (DART-A with Fe “w/Fc”) to mediate T cell activation during redirected killing of target cells. The Figure presents dose-response curves showing the cytotoxicity mediated by DART-A, DART-A w/ABD and DART-A w/Fc in Kasumi-3 (Panel A) and THP-l (Panel B) cells and d CD8 T cells at an E:T (effector cell : target cell) ratio of 10:1 (18 hour incubation). Panels C and D show dose-response curves of T cell activation using the marker CD25 on CD8 T cells in the ce (Panel D) and absence (Panel C) of target cells.

Figure 6 (Panels A-B) shows Granzyme B and Perforin levels in CD4 and CD8 T cells after treatment with the sequence-optimized CD123 X CD3 bi-specific diabody (DART-A) (Panel A) or a control bi-specif1c diabody (Control DART) (Panel B) in the presence of Kasumi-3 target cells and resting T cells at an E:T ratio of10:l.

Figure 7 s A-B) shows the in viva antitumor activity of the sequence- optimized CD123 X CD3 bi-specif1c diabody (DART-A) at nanogram per kilogram dosing levels. MOLM-l3 cells mediate CD123 expression) were co-mixed with T cells and implanted subcutaneously (T:E 1:1) in NSG mice. Intravenous treatment was once daily for 8 days (QDX8) starting at tation. Various concentrations of DART-A were compared to a control bi-specific diabody (Control DART). Panel A shows the Molm-l3 cells alone or with T cells, and the effect of various doses of DART-A on tumor volume even to times beyond 30 days. Panel B shows the effect of increasing doses of DART-A on tumor volume seen in NSG mice receiving MOLM-l3 cells and T cells (T:E 1:1) for a time course of 0-18 days. -2]- Figure 8 shows the in viva mor activity of the sequence-optimized CD123 X CD3 ciﬁc diabody (DART-A) on RS4-11 cells (ALL with monocytic features). Cells were co-mixed with T cells and implanted subcutaneously (T:E 1:1) in NSG mice. Intravenous treatment was once daily for 4 days (QDx4) starting at implantation. Various trations of DART-A were compared to a control bi- speciﬁc diabody (Control DART).

Figure 9 (Panels A-B) shows CD123+ blasts in bone marrow mononucleocytes (BM MNC) and peripheral blood mononucleocytes ) from AML patient 1 (Panel A) compared to Kasumi-3 AML cell line (Panel B).

Figure 10 (Panels A-C) shows the ability of the sequence-optimized CD123 X CD3 bi-speciﬁc diabody (DART-A) to mediate blast ion in primary AML at 120h (Panel A), drive T cell expansion in primary AML at 120h (Panel B) and induce T cell activation in AML at 48h and 72 h (Panel C).

Figure 11 (Panels A-H) shows the identiﬁcation of the CD123+ blast population in a primary sample of ALL PBMCs. Panels A and E show the forward and side scatter of the input population of normal PBMC (Panel A) and ALL PBMCs (Panel E). Panels B and F show the identiﬁcation of the lymphocyte population as primarily B cells (Panel B) and leukemic blast cells (Panel F). Panels C and G show identiﬁcation of the population of lymphocytes that are CD123+. Panels D and H show the identiﬁcation of CD19+ cells and CD123+ cells.

Figure 12 (Panels A-B) shows the identiﬁcation of the CD4 and CD8 populations of T cells in a primary sample of ALL PBMCs. Panel A shows the forward and side scatter of the input ALL PBMCs. Panel B shows the CD4 or CD8 populations of T cells present in the samples. The numbers indicate that CD4 T cells ent approximately 0.5% of the total cells and CD8 T cells represent approximately 0.4% of the total cells present in the ALL PBMC .

Figure 13 (Panels A-H) shows the ability of the sequence-optimized CD123 X CD3 ciﬁc diabody (DART-A) to mediate ALL blast depletion with autologous CTL. Panels A and E show the forward and side scatter of the input tion of normal PBMC (Panel A) and ALL PBMCs (Panel E). The PBMCs were untreated (Panels B and F), treated with a control bi-speciﬁc diabody (Control DART) (Panels C and G) or treated with DART-A (Panels D and H) and incubated for 7 days followed by staining for CD34 and CD19.

Figure 14 (Panels A-L) shows the ability of the sequence-optimized CD123 X CD3 bi-speciﬁc diabody A) to mediate T cell ion (Panels A, B, C, G, H and I) and activation (Panels D, E, F, J, K and L) in normal PBMC (Panels A- F) and ALL PBMC (Panels G-L). The cells were untreated (Panels A, D, G and J), or treated with a control bi-speciﬁc y ol DART) (Panels B, E, H and K) or DART-A s C, F, I and L) for 7 days.

Figure 15 (Panels A-C) shows the identiﬁcation of the AML blast tion and T cells in a primary AML sample. Panel A shows the forward and side scatter of the input AML PBMCs. Panel B shows the identiﬁcation of the AML blast population in the AML sample. Panel C shows the identiﬁcation of the T cell population in the AML sample.

Figure 16 s A-C) shows the ability of the sequence-optimized CD123 X CD3 bi-speciﬁc diabody (DART-A) to mediate AML blast depletion with autologous CTL and T cell expansion. Primary AML PBMCs from patient 2 were incubated with PBS, a l bi-speciﬁc diabody (Control DART) or DART-A for 144h. Blast cells (Panel A), CD4 T cells (Panel B) and CD8 T cells (Panel C) were counted.

Figure 17 (Panels A-D) shows the ability of the sequence-optimized CD123 X CD3 bi-speciﬁc diabody (DART-A) to mediate T cell activation in AML. CD25 (Panel A) and Ki-67 (Panel B) expression was determined for the CD4 and CD8 T cells from AML patient 2 following incubation with a control bi-speciﬁc diabody (Control DART) or DART-A with autologous PBMCs. The level of perforin (Panel C) and granzyme B (Panel D) was determined for the CD4 and CD8 T cells from AML patient 2 following incubation with Control DART or DART-A with autologous PBMCs.

Figure 18 s A-D) shows that the ce-optimized CD123 X CD3 bi-speciﬁc diabody (DART-A) is capable of cross-reacting with both human and primate CD123 and CD3 proteins. The panels show BIACORETM sensogram traces of the s of analyses conducted to assess the ability of DART-A to bind to human (Panels A and C) and non-human primate (Panels B and D) CD3 (Panels A and B) and CD123 (Panels C and D) ns. The KD values are provided.

Figure 19 (Panels A-B) shows the ability of the sequence-optimized CD123 X CD3 bi-speciﬁc diabody (DART-A) to mediate autologous monocyte depletion in vitro with human and cynomolgus monkey PBMCs. The Panels present the results of dose-response curves of DART-A-mediated cytotoxicity with primary human PBMCs (Panel A) or cynomolgus monkey PBMCs (Panel B).

Figure 20 (Panels A-N) shows the ability of the sequence-optimized CD123 X CD3 ciﬁc diabody (DART-A) to mediate the depletion ofpDC in cynomolgus monkeys without systemic cytokine induction. Panels A-D show l results obtained at 4h and 4d with vehicle and carrier. Panels E-H show control results obtained at 4h and 4d with a control bi-speciﬁc diabody (Control DART). Panels I-N show results obtained at 4h and 4d at 10 ng/kg/d and at 4d with 30 ng/kg/d of DART- Figure 21 (Panels A-D) shows the ability of the sequence-optimized CD123 X CD3 bi-speciﬁc diabody (DART-A) to mediate dose-dependent depletion ofpDC in lgus s. Cynomolgus monkeys were dosed with DART-A at 0.1, 1, 10, 100, 300, or 1000 ng/kg. PBMCs were evaluated at the ted time and total B cells (Panel A), monocytes (Panel B), NK cells (Panel C) and pDC (Panel D) were counted.

Figure 22 (Panels A-D) shows the ability of the ce-optimized CD123 X CD3 bi-speciﬁc diabody (DART-A) to intermittently modulate T cells in cynomolgus monkeys. Cynomolgus monkeys were dosed with DART-A at at 0.1, 1, , 30 100, 300, or 1000 ng/kg. PBMCs were evaluated at the indicated time and total T cells (Panel A), CD4 T cells (Panel B), CD69 cells (Panel C) and CD8 T cells (Panel D) were counted.

Figure 23 shows the SDS-PAGE analysis of puriﬁed DART-A protein under reducing (left) and non-reducing (right) conditions.

Figures 24A-24B show the physicochemical characterization of puriﬁed DART-A. Figure 24A: SEC proﬁle of DART-A protein on a ated TSK WXL column. Figure 24B: Mass spectrum of DART-A protein.

Figures 25A-25D show SPR analysis of DART-A binding to immobilized human or cynomolgus monkey CD123 and CD3. Dashed lines represent the global ﬁt to a 1:1 Langmuir model of the experimental binding curves obtained at DART-A concentrations of 0, 6.25, 12.5, 25, 50 or 100nM (continuous lines). The data are representative of three independent experiments.

Figures 26A-26E show that DART-A was capable of simultaneously binding both CD3 and CD123. Figure 26A-26B provides the results of a bifunctional ELISA and demonstrates simultaneous engagement of both target antigens of DART- A. ELISA plates were coated with human CD123 (Figure 26A) or cynomolgus monkey CD123 (Figure 26B). ing DART-A and l DART concentrations were followed by ion with human CD3-biotin. Figures 26C-26E demonstrate cell-surface binding of DART-A on CD123+ Molm—13 target cell (Figure 26C), human T cells (Figure 26D) and cynomolgus T cells (Figure 26E). Binding was detected by FACS is using a monoclonal antibody speciﬁc to E-coil and K-coil region of the DART-A or Control DART molecule.

Figures 27A-27H show the ability of DART-A to mediate redirected target cell killing by human or monkey effector cells against CD123+ Kasumi-3 leukemic cell lines, demonstrate the ability of the molecules to bind to subsets of normal circulating leukocytes, ing pDCs and monocytes and demonstrate the ability of the molecules to deplete CD14'CD123high cells (pDC and basophils) without affecting monocytes (CD14+ . Figure 27A shows the relative anti-CD123-PE binding sites on U937 and -3 leukemic cell lines as ined by QFACS analysis.

Figure 27B shows the relatively low percent cytotoxicity mediated by DART-A or Control DART on an AML cell line (U937 cells) which, as shown in Figure 27A have relatively few CD123 binding . Figure 27C shows the percent cytotoxicity mediated by DART-A or Control DART in the presence of puriﬁed human T cells (as effector cells) on an AML cell line (Kasumi-3 cells) which, as shown in Figure 27A have a substantial number of CD123 binding sites. In Figures 27B-27C, the ET ratio is 10: 1. Figure 27D shows the percent cytotoxicity mediated by DART-A or Control DART in the presence of puriﬁed cynomolgus monkey PBMCs (as effector cells) on Kasumi-3 cells (the ET ratio is 15:1), and demonstrates that DART-A can bind cynomolgus monkey T cells. Figure 27E shows the relative anti-CD123-PE g sites on -3 cells, human monocytes, human plasmacytoid dendritic cells ), cynomolgus monkey monocytes and cynomolgus monkey plasmacytoid dendritic cells as determined by QFACS is. Figure 27F shows the ability of DART-A to deplete CD14’ CD12310 cells. Figure 27G shows the ability of DART-A to deplete human CD14’ CD123Hi cells. Figure 27H shows the ability of DART-A to deplete cynomolgus monkey CD14’ CD123Hi cells. Cytotoxicity was determined by LDH release, with ECSO values determined using GraphPad PRISM® software.

Figure 28 shows the use of a two-compartment model to estimate pharmacokinetic parameters of DART-A. The data show the end of inﬁlsion (EOI) serum concentrations of DART-A in cynomolgus monkeys after receiving a 96-hour inﬁJsion at 100 ng/kg/day 300 ng/kg/day, 600 ng/kg/day, and 1000 ng/kg/day Dose.

Each point represents an individual animal; horizontal lines represent the mean value for the dose group.

Figures C show the effect of DART-A infusions on the production of the cytokine, IL-6. Serum IL-6 levels (mean :: SEM) in monkeys inﬁlsed with DART-A are shown by treatment group. Cynomolgus monkeys were treated with vehicle control on Day 1, followed by 4 weekly infusions of either e (Group 1) (Figure 29A) or DART-A administered as 4-day weekly ons starting on Days 8, , 22, and 29 (Groups 2-5) e 29B) or as a 7-day/week on for 4 weeks starting on Days 8 (Group 6) (Figure 29C). Treatment intervals are indicated by the filled gray bars.

Figures 30A-30F show the effect of DART-A inﬁlsions on the depletion of CD14-/CD123+ cells (Figures 30A-30C) and CD303+ cells (Figures 30D-30F). The mean :: SEM of the circulating levels of CD123+ (Figures 30A-30C) or CD303+ (Figures 30D-30F) by Study Day and by group is shown. Cynomolgus monkeys were treated with vehicle control on Day 1, followed by 4 weekly inﬁJsions of either vehicle (Group 1) (Figures 30A and 30D) or DART-A administered as 4- day weekly inﬁlsions starting on Days 8, 15, 22, and 29 (Groups 2-5) (Figures 30A and 30E) or as a 7-day/week on for 4 weeks starting on Days 8 (Group 6) es 30C and 30F). Treatment intervals are indicated by the ﬁlled gray bars.

Figures 31A-311 show the observed changes in T cell tions (Figures 31A-31C), CD4+ cell populations (Figures 31D-31F) and CD8+ cell populations (Figures 31G-311) receiving DART-A administered as 4-day infusions starting on Days 8, 15, 22, and 29. Legend: CD25+ (gray squares); CD69+ (gray triangles), PD- 1+ (white triangles); Tim-3+ (white squares). T cells were enumerated via the CD4 and CD8 markers, rather than the canonical CD3, to eliminate possible interference the DART-A. Cynomolgus monkeys were treated with e l on Day 1, followed by 4 weekly inﬁJsions of either e (Group 1) or DART-A administered as 4-day weekly inﬁlsions starting on Days 8, 15, 22, and 29 (Group 5) or as a 7- day/week inﬁlsion for 4 weeks starting on Days 8 (Group 6). Treatment intervals are indicated by the filled gray bars. The mean :: SEM of the absolute number of total circulating T cells by Study Day and group is shown (Figures 31A-31C). Relative values (mean t :: SEM) of CD25+, CD69+, PD-1+ and Tim-3+ of CD4 (Figures 31D-31E) or CD8 T cells es 31F-31H) by Study Day and by group is shown.

Figures 32A-32F show the observed changes in T CD4+ cell populations (Figures 32A-32C) and CD8+ cell populations (Figures 32D-32F) during and after a continuous 7-day inﬁasion of DART-A. The mean :: SEM percent of CD25+, CD69+, PD-1+ and Tim-3+ on CD4 (Figures 32A-32C) or CD8 (Figures 32D-32F) T cells by Study Day for Groups 2, 3 and 4 are shown. Treatment als are indicated by the filled gray bars. Legend: CD25+ (gray squares); CD69+ (gray triangles), PD-1+ (white triangles); Tim-3+ (white squares).

Figures 33A-33F show the observed changes in T CD4+ cell populations es 33A-33C) and CD8+ cell tions (Figures 33D-33F) during and after a continuous 7-day inﬁlsion of DART-A. The mean :: SEM percent of CD4+ Naive (CD95-/CD28+), CMT (CD95+/CD28+), and EMT (CD95+/CD28-) T cells in CD4+ population (Figures 33A-33C) or CD8 population (Figures 33D-33F) by Study Day for Groups 2, 3 and 4 are shown. Cynomolgus monkeys were treated with vehicle control on Day 1, followed by 4 weekly inﬁlsions of or DART-A administered as 4- day weekly infusions ng on Days 8, 15, 22, and 29 s 2-4). Treatment als are indicated by the ﬁlled gray bars. Legend: Naive (white triangles); CMT (black triangles), EMT (gray squares).

Figure 34 shows -mediated cytotoxicity against Kasumi-3 cells with PBMCs from either naive monkeys or monkeys treated with multiple inﬁlsions of DART-A.

Figures 35A-35F show that DART-A exposure increased the relative frequency of central memory CD4 cells and effector memory CD8+ cells at the expense of the corresponding naive T cell population. The mean :: SEM percent of CD4+ Naive (CD95-/CD28+), CMT (CD95+/CD28+), and EMT (CD95+/CD28-) T cells in CD4+ population (Figures 35A-35C) or in CD8+ population (Figures 35D- 35F) by Study Day and by Group is shown. Cynomolgus monkeys were treated with vehicle l on Day 1, followed by 4 weekly infusions of either vehicle (Group 1) or DART-A administered as 4-day weekly inﬁlsions starting on Days 8, 15, 22, and 29 (Group 5) or as a 7-day/week SlOIl for 4 weeks starting on Days 8 (Group 6).

Treatment intervals are indicated by the ﬁlled gray bars. Legend: Naive (white triangles); CMT (black triangles), EMT (gray s).

Figures 36A-36F show the effect of DART-A on red cell parameters in monkeys that had ed infusions of the molecules. Circulating RBCs (Figures 36A-36C) or reticulocytes (Figures 36D-36F) levels (mean :: SEM) in samples collected at the indicated time points from monkeys treated with DART-A are shown.

Figures 37A-37B show that the frequency (mean t :: SEM) of CDl23+ cells (Figure 37A) or HSC (CD34+/CD38-/CD45-/CD90+ cells) (Figure 373) within the Lin- cell population in bone marrow samples collected at the indicated time points from monkeys treated with DART-A. lgus monkeys were treated with vehicle control on Day 1, ed by 4 weekly ons of either vehicle (Group 1) or DART-A administered as 4-day weekly infusions ng on Days 8, 15, 22, and 29 (Groups 2-5) or as a 7-day/week infusion for 4 weeks starting on Days 8 (Group 6).

Detailed Description of the Invention: The present invention is directed to sequence-optimized CD123 X CD3 bi- specific monovalent diabodies that are capable of simultaneous binding to CD123 and CD3, and to the uses of such les in the ent of hematologic malignancies.

Although non-optimized CD123 X CD3 bi-specific diabodies are fully functional, analogous to the improvements obtained in gene expression through codon optimization (see, e.g., Grosjean, H. et al. (1982) “Preferential C0d0n Usage In Prokaryotic Genes: The Optimal C0d0n-Anticod0n Interaction Energy And The Selective C0d0n Usage In Eﬁiciently Expressed Genes” Gene 18(3):l99-209), it is possible to further enhance the stability and/or function of CD123 X CD3 bi-specif1c diabodies by modifying or reﬁning their sequences.

The preferred CD123 X CD3 bi-specif1c diabodies of the t invention are composed of at least two polypeptide chains that associate with one another to form one binding site ic for an epitope of CD123 and one binding site specific for an epitope of CD3 (Figure 2). The individual polypeptide chains of the diabody are covalently bonded to one another, for example by disulf1de bonding of cysteine residues located within each polypeptide chain. Each polypeptide chain contains an Antigen Binding Domain of a Light Chain Variable Domain, an Antigen Binding Domain of a Heavy Chain Variable Domain and a heterodimerization domain. An ening linker peptide (Linker 1) separates the Antigen Binding Domain of the Light Chain Variable Domain from the Antigen g Domain of the Heavy Chain le Domain. The Antigen Binding Domain of the Light Chain Variable Domain of the ﬁrst polypeptide chain interacts with the Antigen Binding Domain of the Heavy Chain Variable Domain of the second polypeptide chain in order to form a first functional antigen binding site that is ic for the first antigen (i. e., either CD123 or CD3). Likewise, the Antigen Binding Domain of the Light Chain le Domain of the second polypeptide chain interacts with the n Binding Domain of the Heavy Chain Variable Domain of the ﬁrst polypeptide chain in order to form a second ﬁlnctional antigen binding site that is speciﬁc for the second antigen (i.e., either CD123 or CD3, depending upon the identity of the ﬁrst antigen). Thus, the ion of the Antigen Binding Domain of the Light Chain Variable Domain and the Antigen Binding Domain of the Heavy Chain Variable Domain of the ﬁrst and second polypeptide chains are coordinated, such that the two polypeptide chains tively comprise Antigen g Domains of light and Heavy Chain Variable Domains capable of binding to CD123 and CD3.

The formation of heterodimers of the ﬁrst and second polypeptide chains can be driven by the heterodimerization s. Such s include GVEPKSC (SEQ ID NO:50) (or VEPKSC; SEQ ID NO:51) on one polypeptide chain and GFNRGEC (SEQ ID NO:52) (or FNRGEC; SEQ ID NO:53) on the other polypeptide chain 7/0004909). Alternatively, such domains can be engineered to contain coils of opposing charges. The heterodimerization domain of one of the polypeptide chains comprises a sequence of at least six, at least seven or at least eight positively charged amino acids, and the heterodimerization domain of the other of the polypeptide chains comprises a sequence of at least six, at least seven or at least eight negatively charged amino acids. For example, the ﬁrst or the second heterodimerization domain will preferably comprise a sequence of eight positively charged amino acids and the other of the heterodimerization domains will preferably comprise a sequence of eight negatively charged amino acids. The positively charged amino acid may be lysine, ne, histidine, etc. and/or the negatively d amino acid may be glutamic acid, aspartic acid, etc. The positively charged amino acid is preferably lysine and/or the negatively charged amino acid is preferably glutamic acid.

The CD123 X CD3 bi-speciﬁc diabodies of the present invention are engineered so that such ﬁrst and second polypeptide chains covalently bond to one another via cysteine residues along their length. Such ne residues may be introduced into the intervening linker that separates the VL and VH domains of the polypeptides. Alternatively, and more ably, a second peptide (Linker 2) is introduced into each polypeptide chain, for example, at the amino-terminus of the polypeptide chains or a on that places Linker 2 between the heterodimerization domain and the Antigen Binding Domain of the Light Chain Variable Domain or Heavy Chain Variable Domain.

In particular embodiments, the sequence-optimized CD123 X CD3 bi-speciﬁc monovalent diabodies of the present invention ﬁlrther have an immunoglobulin Fc Domain or an Albumin-Binding Domain to extend half-life in viva.

The CD123 X CD3 bi-speciﬁc monovalent diabodies of the present invention that comprise an immunoglobulin Fc Domain (2'.e., CD123 X CD3 bi-speciﬁc monovalent Fc diabodies) are ed of a ﬁrst polypeptide chain, a second polypeptide chain and a third polypeptide chain. The ﬁrst and second polypeptide chains associate with one another to form one binding site speciﬁc for an epitope of CD123 and one binding site speciﬁc for an epitope of CD3. The ﬁrst polypeptide chain and the third polypeptide chain associate with one another to form an immunoglobulin Fc Domain (Figure 3A and Figure 3B). The ﬁrst and second polypeptide chains of the bi-speciﬁc monovalent Fc diabody are covalently bonded to one another, for example by disulﬁde bonding of cysteine residues located within each polypeptide chain.

The ﬁrst and third polypeptide chains are covalently bonded to one another, for example by de bonding of cysteine residues located within each polypeptide chain. The ﬁrst and second polypeptide chains each contain an Antigen Binding Domain of a Light Chain le Domain, an Antigen Binding Domain of a Heavy Chain Variable Domain and a heterodimerization domain. An intervening linker e r 1) separates the Antigen Binding Domain of the Light Chain Variable Domain from the n Binding Domain of the Heavy Chain Variable Domain.

The Antigen Binding Domain of the Light Chain Variable Domain of the ﬁrst ptide chain interacts with the Antigen Binding Domain of the Heavy Chain Variable Domain of the second polypeptide chain in order to form a ﬁrst functional antigen g site that is speciﬁc for the ﬁrst antigen (2'.e., either CD123 or CD3).

Likewise, the n Binding Domain of the Light Chain Variable Domain of the second polypeptide chain interacts with the Antigen Binding Domain of the Heavy Chain Variable Domain of the ﬁrst polypeptide chain in order to form a second functional antigen binding site that is speciﬁc for the second n (i.e., either CD3 or CD123, ing upon the identity of the ﬁrst n). Thus, the selection of the Antigen Binding Domain of the Light Chain Variable Domain and the Antigen Binding Domain of the Heavy Chain Variable Domain of the ﬁrst and second polypeptide chains are coordinated, such that the two polypeptide chains collectively comprise Antigen g s of light and Heavy Chain Variable Domains capable of binding to CD123 and CD3. The ﬁrst and third polypeptide chains each contain some or all of the CH2 Domain and/or some or all of the CH3 Domain of a complete immunoglobulin Fc Domain and a cysteine-containing peptide. The some or all of the CH2 Domain and/or the some or all of the CH3 Domain associate to form the immunoglobulin Fc Domain of the bi-speciﬁc monovalent Fc diabodies of the t ion. The ﬁrst and third polypeptide chains of the bi-speciﬁc monovalent Fc ies of the present invention are covalently bonded to one another, for example by disulﬁde bonding of cysteine residues located within the cysteine-containing peptide of the polypeptide chains. 1. The Sequence-Optimized CD123 x CD3 Bi-Speciﬁc Diabody, “DART-A” The invention es a sequence-optimized ciﬁc diabody capable of simultaneously and speciﬁcally binding to an epitope of CD123 and to an epitope of CD3 (a “CD123 x CD3” bi-speciﬁc diabody or DART-A). As discussed below, DART-A was found to exhibit enhanced ﬁlnctional activity relative to other non- sequence-optimized CD123 X CD3 bi-speciﬁc diabodies of similar composition, and is thus termed a “sequence-optimized” CD123 X CD3 bi-speciﬁc diabody.

The sequence-optimized CD123 X CD3 bi-speciﬁc diabody (DART-A) comprises a ﬁrst ptide chain and a second polypeptide chain. The ﬁrst polypeptide chain of the bi-speciﬁc diabody will comprise, in the N-terminal to C- terminal direction, an N—terminus, a Light Chain le Domain (VL Domain) of a monoclonal antibody capable of binding to CD3 (VLCDg), an intervening linker peptide (Linker l), a Heavy Chain Variable Domain (VH Domain) of a monoclonal antibody capable of binding to CD123 (VHCDm), and a C-terminus. A preferred sequence for such a VLCD3 Domain is SEQ ID NO:21: ?A PSLTVS PGGTVTLTCRS S TGAVTT SWYANWVQQKPGQAPRGL 22GG TNKRAPWT PARFSGSLLGGKAALT: TGAQA*12D *iADYYCALWYSNLWVFGGGT KLTVLG The Antigen Binding Domain of VLCD3 comprises CDRl SEQ ID NO:38: RSSTGAVTTSNYAN, CDRZ SEQ ID NO:39: GTNKRAP, and CDR3 SEQ ID NO:40: ALWYSNLWV.

A preferred ce for such Linker l is SEQ ID NO:29: GGGSGGGG. A preferred sequence for such a VHCDm Domain is SEQ ID NO:26: EVQLVQSGAELKKPGASVKVSCKASGYTFTDYYMKWVRQAPGQGT.*1W GD 9SNGATFYNQKFKGRVT KSTSTAYM'.T. SST.RS *iDTAVYYCA'RSl-ILLRA SWFAYWGQGTLVTVSS The Antigen Binding Domain m ses CDRl SEQ ID NO:47: DYYMK, CDR2 SEQ ID NO:48: 2D PSNGATFYNQKFKG, and CDR3 SEQ ID NO:49: SHLLRAS.

The second polypeptide chain will comprise, in the N—terminal to C-terminal direction, an N—terminus, a VL domain of a monoclonal antibody e of binding to CD123 (VLCDm), an intervening linker peptide (e.g., Linker l), a VH domain of a monoclonal antibody capable of binding to CD3 (VHCDg), and a C-terminus. A red sequence for such a VLCDm Domain is SEQ ID NO:25: 2DFVMTQS 92DSLAVSLGERVTMSC{SSQSLLNSGNQ <NYLTWYQQ<PGQ29PKL L2. YWAST {.L'SGVPDRFSGSGSGT 2DFTT.T SSTQA*.2DVAVYYCQNDYSY?YTF GQGTKT *1 < The Antigen Binding Domain ofVLCDm comprises CDRl SEQ ID NO:44: KSSQSLLNSGNQKNYLT, CDR2 SEQ ID NO:45: WASTRES, and CDR3 SEQ ID NO:46: QNDYSYPYT.

A preferred sequence for such a VHCD3 Domain is SEQ ID NO:22: V'...SGGGTVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGTWIVGR R SKYNNYATYYADSVKDRFT SRDDSKNSLYLQMNSLKTEDTAVYYCVRHGNF GNSYVSWFAYWGQGTLVTVS S The n Binding Domain of VHCD3 comprises CDRl SEQ ID NO:41: TYAMN, CDR2 SEQ ID NO:42: RIIRSKYNNYATYYADSVKD, and CDR3 SEQ ID HGNFGNSYVSWFAY.

The sequence-optimized CD123 X CD3 bi-specif1c diabodies of the present invention are engineered so that such first and second polypeptides ntly bond to one another via cysteine residues along their length. Such cysteine residues may be introduced into the intervening linker (e.g., Linker 1) that separates the VL and VH domains of the polypeptides. Alternatively, and more preferably, a second peptide (Linker 2) is introduced into each polypeptide chain, for example, at a position N- terminal to the VL domain or C-terminal to the VH domain of such ptide chain.

A preferred sequence for such Linker 2 is SEQ ID NO:30: GGCGGG.

The formation of heterodimers can be driven by further engineering such polypeptide chains to contain polypeptide coils of opposing . Thus, in a preferred ment, one of the polypeptide chains will be engineered to contain an “E-coil” domain (SEQ ID NO:34: EVAALEKEVAALEKEVAALEKEVAALEK) whose residues will form a negative charge at pH 7, while the other of the two polypeptide chains will be engineered to contain an “K-coil” domain (SEQ ID NO:35: EVAN.E'JEVAAT.E'JEVAATETEVAATE?) whose residues will form a positive charge at pH 7. The presence of such charged domains promotes association between the ﬁrst and second polypeptides, and thus fosters heterodimerization.

It is immaterial which coil is provided to the ﬁrst or second polypeptide chains. However, a red sequence-optimized CD123 X CD3 bi-specif1c diabody of the present invention (“DART-A”) has a first polypeptide chain having the sequence (SEQ ID NO:1): QAVVTQIEPSLTVSPGGTVTLTCRSSTGAVTTSWYANWVQQKPGQAPRGLIIGG TNKRAPWTPARFSGSLLGGKAALTI TGAQA*DﬁADYYCALWYSWLWVFGGGT KLTVLGGGGSGGGGIEVQLVQSGAIELKKPGASV{VSC{ASGYTFTDYYM<WVR QAPGQGTﬁ'W GD ?SNGATFYNQKFKGRVTIITVDKSTSTAYM*TSST{8&3 TAVYYCAIRSHLLRASWFAYWGQGTLVTVSSGGCGGGﬁ'VAALﬁKmVAALﬁKﬁV AALﬁKﬁ'VAAT.m< DART-A Chain 1 is composed of: SEQ ID NO:21 — SEQ ID NO:29 — SEQ ID NO:26 — SEQ ID NO:30 — SEQ ID NO:34. A DART-A Chain 1 encoding polynucleotide is SEQ ID NO:2: caggctgtggtgac:caggagccttcactgaccg,g,ccccaggcggaactg tgaccctgacatgcagatccagcacaggcgcagtgaccaca:ctaactacgc caattgggtgcagcagaagccaggacaggcaccaaggggcc,gaccgggggt aaaagggc:ccctggacccctgcacgg,,,,c,ggaag:ctgctgg gcggaaaggccgcchgaccacLaccggggcacaggccgaggacgaagccga LLngchcgcgg,a,agcaa,chngg,gc“cgggggtggcaca aaaccgacchgccgggagggggcggacccggcggcggaggcgaggtgcagc cgngcagcccggggccgagccgaagaaacccggagcccccgcgaaggtgtc ttgcaaagccagtggc:acacct:cacagacLacLa,a,gaag,ggchagg caggctccaggacagggac,ggaa,ggachgcgaLa,ca,Lch,ccaacg gggccacccchacaaccagaagccLaaaggcagggcgaccaccaccgcgga caaatcaacaagcac,ch,aca,ggachgagc,cchgcgccccgaagat acagccgcgcacLaL,g,chcgg,cacacctgc:gagagccagc,gg,L,g CL,a,ngggacagggcaccc,gg,gacagchc,Lccggaggatgtggcgg tggagaag:ggccgcac:ggagaaagaggt:gctgctttggagaaggagg:c gc,gcacL,gaaaaggaggtcgcagccctggagaaa The second polypeptide chain of DART-A has the sequence (SEQ ID th3) DFVMTQS?DSLAVSLG.§{VTMSCKSSQSLLNSGNQ<NYLTWYQQKPGQ?P{L L: YWAST{LSGVRDRFSGSGSGTDFTTT SSTQA*.DVAVYYCQNDYSY?YTF GQGT<Pm <GGGSGGGGﬁVQPVﬁSGGGTVQPGGSLRLSCAASGFTFSTYA N WVRQA?G<GP*'WVG{ YATYYADSVKDRFT SRDDSKNSLYLQMWS LKTEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSSGGCGGGKVAALK.EK VAAT.<7KVAAT.K7KVAAT.Km DART-A Chain 2 is ed of: SEQ ID NO:25 — SEQ ID NO:29 — SEQ ID NO:22 — SEQ ID NO:30 — SEQ ID NO:35. A DART-A Chain 2 encoding polynucleotide is SEQ ID NO:4: gac,ch,gaLgacacachLcc,gaLag,chgccgtgagtctgggggagc ggg,gac,a,g,cL,gcaagagc:cccag:cactgctgaacagcggaaatca gaaaaaccacccgaccEggcaccagcagaagccaggccagccccccaaaccg chaLcLaLcgggc,Lccaccagggaa,c,ggcg,gcccgacaga:tcagcg gcagcggcagcggcacaga,LSnaccc,gacaaL,Lc,achLgcaggccga ggachggc,g,gLac,aL,gccagaa,ga,Lacagc,aLcchacacLL,c ggccaggggaccaagc:ggaaa:taaaggaggcggatccggcggcggaggcg aggtgcagccggcggachcgggggagchcggcccagcc:ggagggccccc gagac,chch,gcagcc,chgaL,cachLcagcaca,accha,gaa, tgggtccgccaggc2ccagggaaggggccggagcgggccggaaggaccaggc ccaagtacaacaattatgcaaccLac,a,gccgactc:gtgaagga,agaL, ctcaagagatgattcaaagaac,caccg,aLc,gcaaa:gaacagc ctgaaaaccgaggacacggccgLgLa,Lachcg,gagacacgg:aacttcg gcaa,,c,,achch,ngLLuchLaLngggacaggggacactggtgac ,g,g,cL,ccggagga:gtggcggtggaaaagtggccgcactgaaggagaaa gL,gc,gc,LLgaaagagaaggtcgccgcact:aaggaaaaggtcgcagccc :gaaagag As discussed below, the sequence-optimized CD123 X CD3 bi-speciﬁc diabody (DART-A) was found to have the ability to simultaneously bind CD123 and CD3 as arrayed by human and monkey cells. Provision of DART-A was found to cause T cell activation, to mediate blast reduction, to drive T cell expansion, to induce T cell tion and to cause the redirected killing of target cancer cells. 11. Comparative Non-Sequence-Optimized CD123 x CD3 Bi-Speciﬁc Diabody, “DART-B” ] DART-B is a non-sequence-optimized CD123 X CD3 bi-speciﬁc diabody having a gross structure that is similar to that of DART-A. The ﬁrst polypeptide chain of DART-B will se, in the N—terminal to C-terminal direction, an N- terminus, a VL domain of a monoclonal antibody capable of binding to CD3 (VLCDg), an intervening linker e (Linker l), a VH domain of a monoclonal antibody capable of binding to CD123 (VHCDm), an ening Linker 2, an E-coil Domain, and a C-terminus. The VLCD3 Domain of the ﬁrst polypeptide chain of DART-B has the sequence (SEQ ID NO:23): DIQLTQS?AIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIIYDTS< YQFSGSGSGTSYSTT SSMmAﬁDAATYYCQQWSSNPLTFGAGTKLE The VHCDm Domain of the ﬁrst polypeptide chain of DART-B has the sequence (SEQ ID NO:28): QVQLVQSGAIELKKPGASVKVSCKASGYTFTDYYMKWVQQAPGQGT*W GD PSNGATFYNQKFKGRVTIITVDKSTSTAYM*TSSTRSmDTAVYYCAQSHLLRA SWFAYWGQGTLVTVSS Thus, DART-B Chain 1 is composed of: SEQ ID NO:23 — SEQ ID NO:29 — SEQ ID NO:28 — SEQ ID NO:30 — SEQ ID NO:34. The sequence of the ﬁrst polypeptide chain of DART-B is (SEQ ID NO:5): DIIQLTQS?AIMSASPGEKVTMTCQASSSVSYMNWYQQKSGTSPKQW_ YDTS A VASGVPYQFSGSGSGTSYSTT SSM*A*DAATYYCQQWSSWPLTFGAGTKLLL-El GGGGQVQLVQSGAIELKK ?GASV{VSCKASGYTFTDYYM<WVQQAPG QGLﬁ'W GD PSNGATFYNQKFKGRVTITVDKSTSTAYM*TSST{SﬁDTAVY YCARSHLLRASWFAYWGQGTLVTVSSGGCGGGﬁVAALﬁKﬁVAALﬁKﬁVAAPﬂ KﬁVAALﬁK A DART-B Chain 1 encoding polynucleotide is SEQ ID NO:6: gacattcagc:gacccachLccagcaaLcaLchLgcaLchcaggggaga aggtcaccatgacctgcagagccagLLcaagLgLaagLLacaLgaachgLa ccagcagaagtcaggcacctcccccaaaagangaLLLaLgacacatccaaa ngchLCngachch,achcL,canggcangggLC,gggacctcat actctc:cacaa:cagcagcatggaggCCgaagaLgCLgccaCLLa,Lach ccaacanggagLagLaacccchcacg ggaccaagc2ggag ctgaaaggaggcgga:ccggcggcggaggccagg:gcagcngLgcachcg agctgaagaaacccggagcLLcchgaagngLcLLgcaaagccag tggCCacaccttcacagacLacLaLa,gaagngchaggcaggctccagga cagggac,ggaa,ggachgcgaLa,caLLch,ccaacggggccaCLLLCL agaagLLLaaaggcaggngacLaLLacchggacaaatcaacaag caCLchLaLa,ggachgagc,cchgcchc,gaagatacagcchgLac LaLLgLchcgg,cacacctgc:gagagccagc,ggLL,chLaLngggac cccngLgacangLcLLccggaggatgtggcggtggagaagtggc cgcaCngagaaagaggt:gctgctttggagaaggagg,cgc,gcacL,gaa aaggaggtcgcagccctggagaaa The second polypeptide chain of DART-B will comprise, in the N—terminal to C-terminal direction, an N—terminus, a VL domain of a monoclonal antibody capable of binding to CD123 (VLCDm), an intervening linker peptide (Linker l) and a VH domain of a monoclonal antibody e of binding to CD3 ), an intervening Linker 2, a K-coil Domain, and a C-terminus.

The VLCDm Domain of the second polypeptide chain of DART-B has the sequence (SEQ ID NO:27): DFVMTQS9DSLAVSLGERVTMSC{SSQSLLNSGNQ<NYLTWYQQ<PGQ?PKL L: YWAST<£SGVPDRFSGSGSGTDFTTT SSTQAmDVAVYYCQNDYSY?YTF GQGTKT.m < The VHCD3 Domain of the second polypeptide chain of DART-B has the sequence (SEQ ID NO:24): D: {LQQSGAELARLDGASVKMSCKT SGYTFTRYTMHWV <QRPGQGT. *1W GY N ?S?GYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCAQYYDDHY CLDYWGQGTTLTVSS ] Thus, DART-B Chain 2 is composed of: SEQ ID NO:27 — SEQ ID NO:29 — SEQ ID NO:24 — SEQ ID NO:30 — SEQ ID NO:35. The sequence of the second polypeptide chain of DART-B is (SEQ ID NO:7): DFVMTQS9DSLAVSLG.§{VTMSC{SSQSLLNSGNQ<NYLTWYQQ<PGQ9PKL L: YWAST{LSGVPDRFSGSGSGTDFTTT .DVAVYYCQNDYSY9YTF GQGTKTﬁ' {GGGSGGGGI <LQQSGA.ELAR9GASV{MSCKTSGYTFTRYTMI WV<QRPGQGT*'W GY N9S{GYTWYNQKFKDKATLTTDKSSSTAYMQLSSLT SEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSGGCGGGKVAAPKEKVAAPK? KVAAT.K9KVAAPK? ] A DART-B Chain 2 encoding polynucleotide is SEQ ID NO:8: gac,ch,gaLgacacachLcc,gaLag,chgccgtgagtctgggggagc ggg,gac,a,g,cL,gcaagagc:cccag:cactgctgaacagcggaaatca gaaaaaccacccgacccggcaccagcagaagccaggccagccccccaaaccg chaLcLaLcgggc,Lccaccagggaa,c,ggcg,gcccgacaga:tcagcg gcagcggcagcggcacaga,Lguaccc,gacaaL,Lc,achLgcaggccga ggachggc,g,gLac,aL,gccagaa,ga,Lacagc,aLcchacacLL,c ggccaggggaccaagc:ggaaa:taaaggaggcgga:ccggcggcggaggcg aaCCgcagcagtcaggggccgaaccggcaagacc:ggggcc:cagt gaagaLg,cc,gcaagacL,chgc,acach,,ac,agg,acacga:gcac aaacagaggcc:ggacagggLC,ggaa,ggaL,ggaLacaL,aa,c ctagccgcggccacaccaaccacaaccagaagc“caaggacaaggccacacc gac:acagacaaaccccccagcacagcccacacgcaac:gagcagcc:gaca :ctgaggach,gcag,cLacLachLgcaaga,a,,a,ga,gacca,Lac, gccccgacLaccggggccaaggcaccac:ctcacagccccccccggaggatg tggcggtggaaaagtggccgcactgaaggagaaag,,chchL,gaaagag aaggtcgccgcacttaaggaaaaggtcgcagccctgaaagag 111. Modiﬁed Variants of Sequence-Optimized CD123 x CD3 ciﬁc y (DART-A) A. Sequence-Optimized CD123 x CD3 Bi-Speciﬁc Diabody Having An Albumin-Binding Domain (DART-A with ABD “w/ABD”) In a second embodiment of the invention, the sequence-optimized CD123 X CD3 bi-speciﬁc y (DART-A) will comprise one or more Albumin-Binding Domain (“ABD”) (DART-A with ABD “w/ABD”) on one or both of the polypeptide chains of the diabody.

As disclosed in WO 2012/018687, in order to improve the in viva pharmacokinetic properties of diabodies, the diabodies may be modiﬁed to contain a polypeptide portion of a serum-binding protein at one or more of the termini of the diabody. Most preferably, such polypeptide portion of a serum-binding protein will be installed at the C-terminus of the diabody. A particularly preferred polypeptide portion of a binding protein for this purpose is the Albumin-Binding Domain (ABD) from streptococcal protein G. The Albumin-Binding Domain 3 (ABD3) of protein G of Streptococcus strain G148 is particularly preferred.

The Albumin-Binding Domain 3 (ABD3) of protein G of Streptococcus strain G148 consists of 46 amino acid residues forming a stable three-helix bundle and has broad albumin-binding speciﬁcity (Johansson, M.U. et al. (2002) “Structure, Speciﬁcity, And Mode 0f Interaction For Bacterial Albumin-Binding Modules,” J.

Biol. Chem. ):8114-8120). Albumin is the most abundant protein in plasma and has a half-life of 19 days in humans. Albumin possesses l small molecule binding sites that permit it to non-covalently bind to other proteins and y extend their serum half-lives.

Thus, the first polypeptide chain of such a sequence-optimized CD123 X CD3 bi-specific diabody having an Albumin-Binding Domain contains a third linker (Linker 3), which separates the E-coil (or ) of such polypeptide chain from the Albumin-Binding Domain. A preferred sequence for such Linker 3 is SEQ ID NO:31: GGGS. A red Albumin-Binding Domain (ABD) has the sequence (SEQ H)N(h36kPAEAKVPANREPDKYGVSDYYKNL:DNAKSAEGVKAP Dd PAAPR Thus, a preferred first chain of a sequence-optimized CD123 X CD3 bi- 1c diabody having an Albumin-Binding Domain has the sequence (SEQ ID NO:9): QAVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSWYANWVQQKPGQAPRGL: GG TNKRAPWTPARFSGSLLGGKAALTI TGAQAﬁ'DmADYYCALWYSWLWVFGGGT KLTVLGGGGSGGGG_EVQLVQSGA_ELK<PGASV<VSC{ASGYTFTDYYM<WVR Tﬁ'W GD 9SNGATbYNQKb<G<VT TVDKSTSTAYMTSST<SﬁD TAVYYCARS{LLRASWFAYWGQGTLVTVSSGGCGGGWVAATﬁKVAATﬁKﬁV AAPﬁKﬁVAAPm<GGGSTAGAKVTANRGTDKYGVSDYYKNLIDNAKSAAGV<A T. )0 PAAP? A sequence-optimized CD123 X CD3 diabody having an Albumin-Binding Domain is composed of: SEQ ID NO:21 — SEQ ID NO:29 — SEQ ID NO:26 — SEQ ID NO:30 — SEQ ID NO:34 — SEQ ID NO:31 — SEQ ID NO:36. A pdymmbmﬂmemmmngmmhasammweommnmﬂCIH23XCD3dﬁhﬂyhmﬂgan Albumin-Binding Domain derivative is SEQ ID NO:10: caggctgtggtgactcaggagccttcactgaccgtgtccccaggcggaactg tgaccctgacatgcagatccagcacaggcgcagtgaccaca:ctaactacgc caattgggtgcagcagaagccaggacaggcaccaaggggcc,gaecgggggt acaaacaaaagggc:ccctggacccctgcacgg,L,Lceggaagtctgctgg gcggaaaggccgcechaceaeLaccggggcacaggccgaggacgaagccga e,ac,aLLngc,cegeggea,agcaaech,ggg,geecgggggtggcaca acengcegggagggggeggaeccggcggcggaggcgaggtgcagc egngcag,ccggggcegagcegaagaaacccggagceecchgaaggtgtc eegcaaagccageggceacaccttcacagaceacLaeaegaageggchagg caggctccaggacagggaceggaa,ggachgcgaLaecaeLch,ccaacg gggccac,L,cLacaa,cagaag,,Laaaggcaggg,gac,aLeaccgegga caaatcaacaagcac,ch,aea,ggachgagc,cchgcgcecegaagat acagccgegeacLaLegechcgg,cacacctgc:gagagccagc,gg,,,g gggacagggcacccegg,gacagech,Lccggaggatgtggcgg tggagaag:ggccgcac:ggagaaagaggLechchLeggagaaggaggtc gcegcaceegaaaaggaggtcgcagccctggagaaaggcggcgggechegg caaaagtgctggccaaccgcgaaceggaLaaa,angcg,gagcga L,aLeaLaagaacc:gattgacaacgcaaaatccgcggaaggcgtgaaagca c,ga,LgaLgaaaL,chgccgccctgcct The second polypeptide chain of such a sequence-optimized CD123 X CD3 diabody having an Albumin-Binding Domain has the sequence described above (SEQ ID NO:3) and is encoded by a polynucleotide having the sequence of SEQ ID NO:4.

B. Sequence-Optimized CD123 x CD3 Bi-Speciﬁc Diabodies Having An IgG Fc Domain (DART-A with Fe “w/Fc”) In a third ment, the invention provides a ce-optimized CD123 X CD3 bi-speciﬁc diabody composed of three polypeptide chains and possessing an IgG Fc Domain (DART-A with Fe “W/Fc” Version 1 and Version 2) (Figure 3A-3B).

In order to form such IgG Fc Domain, the ﬁrst and third polypeptide chain of the diabodies contain, in the N-terminal to inal direction, a cysteine-containing peptide, (most preferably, Peptide 1 having the amino acid sequence (SEQ ID : DKTHTCPPCP), some or all of the CH2 Domain and/or some or all of the CH3 Domain of a complete immunoglobulin Fc Domain, and a C-terminus. The some or all of the CH2 Domain and/or the some or all of the CH3 Domain associate to form the immunoglobulin Fc Domain of the bi-speciﬁc monovalent Fc Domain- ning diabodies of the present invention. The ﬁrst and second polypeptide chains of the cific monovalent Fc ies of the present invention are covalently bonded to one another, for example by disulﬁde bonding of cysteine residues located within the cysteine-containing peptide of the polypeptide chains.

The CH2 and/or CH3 Domains of the first and third polypeptides need not be identical, and advantageously are modiﬁed to foster complexing between the two polypeptides. For e, an amino acid substitution (preferably a substitution with an amino acid comprising a bulky side group forming a ‘knob’, e.g., tryptophan) can be introduced into the CH2 or CH3 Domain such that steric interference will prevent interaction with a similarly mutated domain and will te the mutated domain to pair with a domain into which a complementary, or accommodating mutation has been engineered, i.e., “the hole’ (e.g., a substitution with glycine). Such sets of mutations can be engineered into any pair of polypeptides comprising the bi-specific monovalent Fc diabody molecule, and ﬁarther, engineered into any portion of the polypeptides chains of said pair. Methods of protein engineering to favor heterodimerization over homodimerization are well known in the art, in particular with respect to the engineering of immunoglobulin-like molecules, and are encompassed herein (see e. g., Ridgway et al. (1996) “‘Knobs-Into-Holes’ Engineering 0f Antibody CH3 Domains For Heavy Chain Heterodimerization,” Protein Engr. 9:6l7-621, Atwell et al. (1997) e Heterodimers From Remodeling The Domain Interface Of A Homodimer Using A Phage Display Library,” J. Mol. Biol. 270: 26-35, and Xie et al. (2005) “A New Format 0f Bispeciﬁc Antibody: Highly nt Heterodimerization, Expression And Tamor Cell Lysis, ” J. Immunol. Methods 296:95-101; each of which is hereby incorporated herein by reference in its ty). Preferably the ‘knob’ is engineered into the CH2-CH3 Domains of the first polypeptide chain and the ‘hole’ is engineered into the CH2-CH3 Domains of the third ptide chain. Thus, the ‘knob’ will help in preventing the first polypeptide chain from homodimerizing Via its CH2 and/or CH3 Domains. As the third polypeptide chain preferably contains the ‘hole’ substitution it will heterodimerize with the first polypeptide chain as well as homodimerize with itself A preferred knob is created by ing a native IgG Fc Domain to contain the modification T366W. A preferred hole is created by modifying a native IgG Fc Domain to contain the modification T366S, L368A and Y407V. To aid in purifying the third polypeptide chain homodimer from the ﬁnal bi-speciﬁc monovalent Fc diabody comprising the ﬁrst, second and third polypeptide chains, the protein A binding site of the CH2 and CH3 Domains of the third polypeptide chain is preferably mutated by amino acid substitution at position 435 ). Thus, the third polypeptide chain homodimer will not bind to n A, Whereas the bi-speciﬁc monovalent Fc diabody will retain its ability to bind protein A via the protein A binding site on the ﬁrst polypeptide chain.

A red sequence for the CH2 and CH3 Domains of an antibody Fc Domain present in the ﬁrst polypeptide chain is (SEQ ID NO:56): APLAAGGPSVFLFPPKPKDTL SRTP*‘VTCVVVDVSH4DP*‘V<EWWYV3GV 'UI—IILUVTNAKTK9<4'QYWSTYRVVSVLTVL{QDWLNG<LYKC<VSN<AL9A9 4K :SKAKGQ9{L9QVYTTWP9SRL*MTKVQVSLWCLV<GEY9SD AV4W4SWGQ -3WNYKTT 99VLDSDGSFFLYS<LTVD<S9WQQGWVFSCSV HEALTVTYTQ <SLSLSPG< A preferred sequence for the CH2 and CH3 Domains of an antibody Fc Domain present in the third polypeptide chain is (SEQ ID NO: 11): APLAAGGPSVFLFPPKPKDTT SRTPLVTCVVVDVSH4DP4V<EWWYVJGV 'UI—IILUVTNAKTK9<4'QYWSTYRVVSVLTVL{QDWLNG<LYKC<VSN<AL9A9 4K :SKAKGQW9{L9QVYTTP9SRL*MTKVQVSLSCAV<GEY9SD AV4W4SWGQ .3WNYKTT 99VLDSDGSFFLVS<LTVD<S9WQQGWVFSCSV HLALTV<YTQ C. DART-A w/Fc Version 1 uct In order to illustrate such Fc diabodies, the invention provides a DART-A W/Fc version 1 construct. The ﬁrst polypeptide of the DART-A w/Fc n 1 uct comprises, in the N—terminal to C-terminal direction, an N—terminus, a VL domain of a monoclonal antibody e of binding to CD123 (VLCDm), an intervening linker peptide (Linker l), a VH domain of a monoclonal antibody capable of binding to CD3 (VHCDg), a Linker 2, an E—coil Domain, a Linker 5, Peptide l, a polypeptide that contains the CH2 and CH3 Domains of an Fc Domain and a C- terminus. A red Linker 5 has the sequence (SEQ ID NO:32): GGG. A preferred polypeptide that contains the CH2 and CH3 Domains of an Fc Domain has the ce (SEQ ID NO:37): APEAAGGPSVFLFPPKPKDTP SRTPL1VTCVVVDVSH4DPL1V<EWWYVDGV 'Ul—ZIL-ljViNAKTK9&4L1QYNSTY9VVSVLTVL{QDWLNG<EYKC<VSN<AL9A9 4K :SKAKGQW9{_L9QVYTTP9SRLLMTKNQVSLWCLV<GEY9SD WGQ .3WNYKTT99VLDSDGSFFLYS<LTVD<S9WQQGWVFSCSV HEALdWiYTQ <SLSLSPG< Thus, the ﬁrst polypeptide of such a DART-A W/Fc version 1 construct is ed of: SEQ ID NO:25 — SEQ ID NO:29 — SEQ ID NO:22 — SEQ ID NO:30 — SEQ ID NO:34 — SEQ ID NO:32 — SEQ ID NO:55 — SEQ ID NO:37.

A preferred ce of the ﬁrst polypeptide of such a DART-A w/Fc version 1 construct has the sequence (SEQ ID NO:13): DFVMTQS9DSLAVSLG.£{VTMSC{SSQSLLNSGNQ<NYLTWYQQ<PGQ9PKL L: YWAST<LSGVP39FSGSGSGTDFTTT MSTQAL.DVAVYYCQNDYSY9YTF GQGT<P4 <GGGSGGGG4VQPVLSGGGTVQPGGSLRLSCAASGFTFSTYAMW WVRQA9G<GPL1WVG< RS<YNNYATYYADSVKDRFT NSLYLQ NS LKTEDTAVYYCVR{GWFGWSYVSWFAYWGQGTLVTVSSGGCGGGLVAATLK4 VAAP4<4VAAPLKLVAAT4<GGGD<TdTC99C9APLAAGG9SVFLFPPK9KD TR S<TP4VTCVVVDVS{4DP4V<EWWYVDGVEV{NAKT<9<4L1QYWSTY9 VVSVLTVL{QDWLWG<EY<C<VSW<AP9A9 4<T SKAKGQ9<L9QVYTLP9 SR4L1MTKWQVSLWCLV<GEY9SD AV4W4SWGQ 9EWNYKTT99VLDSDGSFF LYS<LTVD<S9WQQGWVFSCSV {EALdeYTQ<SLSLSPG< A preferred polynucleotide ng such a polypeptide is (SEQ ID NO:14): gac,chLgaLgacacachLcc,gaLag,chgccgtgagtctgggggagc ggngaCLaLgLCL,gcaagagc:cccag:cactgctgaacagcggaaatca gaaaaacvaLCLgachggLaccagcagaagccaggccagccccc:aaactg chaLcLaLnggc,LccaccagggaaLC,ggchgcccgacagattcagcg gcagcggcagcggcacagaLLLLaCCCLgacaaLLLCLagLCLgcaggccga ggachggCLngLaCLaL,gvcagaa,ga,Lacagc:atccctacactttc ggccaggggaccaagc:ggaaa:taaaggaggcggatccggcggcggaggcg aggtgcagcngvggechnggggagchngvccagcc:ggagggvcccv gagacLCLCCLngcegCCLCngaL,cach,cagcacaLacchaLgaa, tgggtccgccaggc2ccagggaaggggcngagvgggvngaaggavcaggv ccaagtacaacaattetgcaachacLa,gccgactc:gtgaagga,agaLL caccaLCLcaagagatgattcaaagaacLcachLaLchcaaa:gaacagc ctgaaaaccgaggacecggcchgLaLLachLngagacacggLaacLch gcaa,LC,LachgLCLngLLLchLa,ngggacaggggacactggtgac LngLcLLccggagge:gtggcggtggagaagtggccgcac:ggagaaagag gLLchgCLLngageaggaggvcchgcacLLgaaaaggaggtcgcagccc :ggagaaaggcggcggggacaaaac:cacacatgcccaccgtgcccagcacc cgcggggggecchcachLLCCLCLLccccccaaaacccaaggac accctcatgatctcccggacccc,gagchacaLgchgngg,ggacgtga gccacgaagacchgegchaag,Lcaac,ggLachggacggcgtggaggt gcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccg'(I agcgtcc:caccgtcctgcaccaggactggc:gaatggcaaggag'(I acaagtgcaaggtctccaacaaagccctcccagccccca:cgagaaaacca(I c:ccaaagccaaagggcagccccgagaaccacaggtg:acaccctgccccca tcccgggaggagatgaccaagaaccaggtcagccchggchcvggvcaaag ch,cLa,cccagcgacatcgccgtggag:gggagagcaatgggcagccgga gaacaac2acaagaccacgccLccchgccggac ,ccgacggc,cc,Lc,Lc ctc:acagcaagctcaccg:ggacaagagcaggtggcagcaggggaacg:ct LC,CaLgc,ccg,gaLgca,gaggctctgcacaaccactacacgcagaagag cc,chcc,ch,ccgggtaaa The second chain of such a DART-A W/Fc version 1 construct will comprise, in the N—terminal to C-terminal direction, an N—terminus, a VL domain of a monoclonal antibody capable of binding to CD3 (VLCDg), an intervening linker peptide (Linker l), a VH domain of a monoclonal antibody capable of binding to CD123 (VHCDm), a Linker 2, a K—coil Domain, and a C-terminus. Thus, the second polypeptide of such a DART-A W/Fc version 1 construct is composed of: SEQ ID NO:21 — SEQ ID NO:29 — SEQ ID NO:26 — SEQ ID NO:30 — SEQ ID NO:35.

Such a polypeptide has the sequence (SEQ ID NO:15): QAVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSWYANWVQQKPGQAPRGL_IGG TNKRAPWTPARFSGSLLGGKAALTI TGAQAﬁDﬁADYYCALWYSWLWVFGGGT KLTVLGGGGSGGGG_EVQLVQSGA_ELKKPGASV{VSCKASGYTFTDYYMKWVR QAPGQGTﬁ'W GD FYNQKFKGRVT--TVDKSTSTAY SmD TAVYYCARSHLLRASWFAYWGQGTLVTVSSGGCGGGKVAATK:‘J <VAAT.Km<V AAPKTKVAATKm A preferred polynucleotide ng such a polypeptide has the sequence (SEQ ID : caggctgtggtgactcaggagccttcactgaccgtgtccccaggcggaactg tgaccctgacatgcagatccagcacaggcgcag:gaccaca:ctaactacgc caattgggtgcagcagaagccaggacaggcaccaaggggcc “gaucgggggt acaaacaaaagggc:ccctggacccctgcacgg ggaag:ctgctgg gcggaaaggccgcchgaccavLaccggggcacaggccgaggacgaagccga LLngc,cvgvgg,a,agcaa,ch,ggg,gv“cgggggtggcaca aaaccgacchgccgggagggggvggavccggcggcggaggcgaggtgcagc ,gngcag,ccggggc“gagc,gaagaaacccggagcv“cchgaaggtgtc vcgcaaagccagvggcvacaccttcacagacvacLavaGgaagGggchagg caggctccaggacagggac,ggaa,ggachgcgaLa,ca, LCCL,ccaacg gggccac,L,cLacaa,cagaag,,Laaaggcaggg,gac,aLvaccg,gga caaatcaacaagcacvchcavavggachgagc ,cchgcgc,c,gaagat acagccg,g,acLaL,g,chcgg,cacacctgc:gagagccagcvgngvg ctta:tggggacagggcaccccgg,gacag,ch vLccggaggatgtggcgg tggaaaag1ggccgcac:gaaggagaaagt:gctgctttgaaagagaagg:c gccgcacvvaaggaaaaggtcgcagccctgaaagag The third polypeptide chain of such a DART-A w/Fc version 1 will comprise the CH2 and CH3 s of an IgG Fc Domain. A preferred polypeptide that is composed of Peptide l (SEQ ID NO:55) and the CH2 and CH3 Domains of an Fc Domain (SEQ ID NO:11) and has the sequence of SEQ ID NO:54: D<T{TC99CPA9EAAGGPSVFLFPPKPK3TL VTCVVVDVSH4DPﬁ V<FWWYVDGVEVINAKTK9<m'QYWSTYRVVSVLTVLIQDWLNG<EYKC<VS W<AL9A9 4KT SKAKGQ9<j9QVYTTP9SRﬁHﬁMTKVQVSLSCAV<GFY9S3 AVﬁWﬁSWGQ 9EWNYKTT99VLDSDGSFFLVS<LTVD<S9WQQGWVFSCSV {jAL{W{YTQ<SLSLSPG< A preferred polynucleotide that encodes such a polypeptide has the sequence (SEQ ID NO:12): gacaaaac:cacacatgcccaccgtgcccagcacctgaagccgcggggggac c3Lcc,cLoccccccaaaacccaaggacaccctcatgatctcccg gacccc,gagchacaLgchggngngacgtgagccacgeagaccc:gag chaagoLcaac,gg,acgngacggcgtggaggtgcataatgccaagacaa gggaggagcagtacaacagcacgLaccg,gogchegcgtcc:cac cgtcctgcaccaggactggc:gaatggcaaggag:acaagtgcaaggtctcc aacaaagccctcccagcccccatcgagaaaaccacccccaaegccaaagggc gagaaccacaggtg:acaccctgcccccatcccgggaggagatgac caagaaccaggtcagccogagL,gcgcagtcaaaggcttcte:cccagcgac atcgccgtggag:gggagagcaatgggcagccggagaacaac:acaagacca cgchccchgc,ggac,ccgacgchcc,Lchchcgocegcaagctcac cg:ggacaagagcaggtggcagcaggggaacg“CLLC,caLgcoccg,gaLg catgaggctctgcacaaccgctacacgcagaagagcc,cocccoch,ccgg gtaaa D. DART-A w/Fc Version 2 Construct ] As a second example of such a DART-A w/Fc diabody, the invention provides a three chain diabody, “DART-A w/Fc Diabody Version 2” (Figure 3B).

The ﬁrst polypeptide of such a DART-A W/Fc version 2 construct comprises, in the N—terminal to C-terminal direction, an N—terminus, a peptide linker (Peptide l), a polypeptide that contains the CH2 and CH3 Domains of an Fc Domain linked (via a Linker 4) to the VL domain of a monoclonal antibody capable of binding to CD123 ), an intervening linker peptide (Linker l), a VH domain of a monoclonal antibody capable of binding to CD3 (VHCDg), a Linker 2, a K—coil Domain, and a C- terminus .

A preferred polypeptide that contains the CH2 and CH3 Domains of an Fc Domain has the sequence (SEQ ID NO:37): APEAAGGPSVFLFPPKPKDTP SRTP*‘VTCVVVDVSH4DP4'V<EWWYV3GV 'Ul—ZIL-ljV4NAKTK994'QYWSTY9VVSVLTVL{QDWLNG<EYKC<VSN<AP9A9 4K :SKAKGQ9949QVYTTWP9SR4*MTKWQVSLWCLV<GEY9SD AV4W4SWGQ -3WNYKTT 99VLDSDGSFFLYS<LTVD<S9WQQGWVFSCSV HEAL4W4YTQ <SLSLSPG< “Linker 4” will ably se the amino acid sequence (SEQ ID NO:57): APSSS. A preferred “Linker 4” has the sequence (SEQ ID NO:33): ME. Thus, the first polypeptide of such a DART-A W/Fc version 2 construct is composed of: SEQ ID NO:55 — SEQ ID NO:37 — SEQ ID NO:33 — SEQ ID NO:25 — SEQ ID NO:29 — SEQ ID NO:22 — SEQ ID NO:30 — SEQ ID NO:35.

A polypeptide having such a sequence is (SEQ ID NO:17): D<T{TC99CPAPEAAGGPSVFLFPPKP{DTP SRTP4VTCVVVDVSH4DP4 V<FWWYVJGVEV4NAKTK994'QYWSTY9VVSVLTVL{QDWLNG<EYKC{VS 9 4KT SKAKGQ99.49QVYTT 99SR*H*MT<WQVSLWCLV<GFY9SD AV4W4SWGQ9EWNYKTT 99VLDSDGSFFLYS<LTVD<S9WQQGWVFSCSV {EAL4W4YTQ<SLSLSPG<A9SSS9 Lti DFVMTQS9DSLAVSLG49VTMSC<S SQSLLWSGNQ<WYLTWYQQ<9GQ99<LLI_YWAST94SGV9D9FSGSGSGT3F TTT SSTQA4.3VAVYYCQWDYSY9YTFGQGT<14 <GGGSGGGG4VQPV4SG GGLVQPGGSL9LSCAASGFTFSTYA NWVRQA9G<GP4WVG9 {SKYNNYAT YYADSVKDRFT SRDDSKWSLYLQMWSLKT4DTAVYYCV9HGNFGNSYVSWF AYWGQGTLVTVSSGGCGGGKVAAT.K4KVAAT.<4KVAAT.K4KVAATK4 ] A preferred polynucleotide encoding such a polypeptide has the sequence (SEQ ID NO:18): gacaaaac:cacacatgcccaccgtgcccagcacctgaagccgcggggggac chcag,c4Lcc,cL,ccccccaaaacccaaggacaccctcatgatctcccg gacccc,gagchacaLgchggngngacgtgagccacgeagaccc:gag chaag,Lcaac,gg,acgngacggcgtggaggtgcataatgccaagacaa agccgcgggaggagcagtacaacagcacgLaccg,g,gchegcgtcc:cac cgtcctgcaccaggactggc:gaatggcaaggag:acaagtgcaaggtctcc aacaaagccctcccagcccccatcgagaaaaccacccccaaegccaaagggc gagaaccacaggtg:acaccctgcccccatcccgggaggagatgac caagaaccaggtcagcc,ngg,gcc,gg,caaagch,cLe,cccagcgac atcgccgtggag:gggagagcaatgggcagccggagaacaac:acaagacca cgchccchgc,ggac,ccgacggc,cc,Lchchc,acegcaagctcac cg:ggacaagagcaggtggcagcaggggaacg“CLLC,caLgc,ccg,gaLg gc4ccgcacaaccactacacgcagaagagcc4c“cccchccccgg gtaaagcccc:tccagctcccc:atggaagac,,cg,ga,gacacag:ctcc ,gaLag,c,ggccgtgagtctgggggagcggg,gac,acg,cL,gcaagagc :cccag,cac,gcLgaacagcggaaatcagaaaaac,a,c,gachggLacc agcagaagccaggccagccccc,aaachcha,cLaL,gggc,Lccaccag ggaa,chgcg,gcccgacagattcagcggcagcggcagcggcacaga,Lo, aCCCCgacaa,,Lc,agochcaggccgaggacgoggc,ngLac,aL,g,c agaa,gaoLacagc:atccctacactttcggccaggggaccaagc:ggaaa: taaaggaggcggatccggcggcggaggcgaggtgcagcngvggachvggg ggagchngvccagcc2ggagggvcccLgagacchchngcagccvcvg gattcaccttcagcaca,accha,gaa,ngchcgccagg02ccagggaa ggggcvggagvgggvngaaggavcagg“ccaagtacaacaattatgcaacc Lac,augccgactc:gtgaagga,agaL“caccaococaagagatgattcaa agaacvcacLgvaLcLgcaaa:gaacagcc:gaaaaccgaggacacggccg: goa,Lachogogagacacgguaac,chgcaa,,c,,achchungLL, ch,a,ngggacaggggacachg,gac,gugocL,ccggaggatgtggcg gtggaaaagtggccgcactgaaggagaaag3LgcvgchLgaaagagaagg: cgccgcact:aaggaaaaggtcgcagccc:gaaagag The second polypeptide chain of such a DART-A w/Fc version 2 construct comprises, in the N—terminal to C-terminal direction, the VL domain of a monoclonal antibody capable of binding to CD3 (VLCDg), an intervening linker peptide (Linker l) and a VH domain of a monoclonal dy capable of binding to CDl23 ).

This portion of the molecule is linked (via Linker 2) to an E-coil Domain. Thus, the third polypeptide of such a DART-A w/Fc n 2 construct is composed of: SEQ ID NO:21 — SEQ ID NO:29 — SEQ ID NO:26 — SEQ ID NO:30 — SEQ ID NO:34. A polypeptide having such a sequence is (SEQ ID NO:1), and is preferably encoded by a polynucleotide having the sequence of SEQ ID NO:2.

The third polypeptide chain will comprise the CH2 and CH3 Domains of an IgG Fc Domain. A red polypeptide is composed of Peptide l (SEQ ID NO:55) and the CH2 and CH3 Domains of an Fc Domain (SEQ ID NO:11) and has the sequence of SEQ ID NO:54.

In order to assess the ty of the above-mentioned CD123 X CD3 bi- specific diabodies (DART-A, DART-A w/ABD, DART-A w/Fc, DART-B), a control bi-specific y (Control DART) was ed. The l DART is capable of simultaneously binding to FITC and CD3. Its two polypeptide chains have the following respective sequences: Control DART Chain 1 (SEQ ID NO: 19).

DVVMTQTPFSL9VSLGDQAS: SCRSSQSLVHSNGWTYLRWYLQKPGQSPKVL :YKVSWRFSGV9DRFSGSGSGTDETTK S<VﬁAﬁDTGVYFCSQSTHVPWTFG GGTKT.*' KGGGSGGGGﬁ'VQLVmSGGGLVQ9GGSLRLSCAASGFTFNTYAMNW VRQAPGKGTﬁ'WVA{ RSKYNNYATYYADSVKDRFT SRDDSKNSLYLQMNSL KTEDTAVYYCVR{GNFGWSYVSWFAYWGQGTLVTVSSGGCGGGﬁVAALﬁKﬁV AALﬁKﬁVAALﬁKﬁVAALﬁK Control DART Chain 2 (SEQ ID : QAVVTQ_EPSLTVSPGGTVTLTCRSSTGAVTTSWYANWVQQKPGQAPRGL_IGG TN<RAPWTPARFSGSLLGG<AALTI TGAQAmD*ADYYCALWYSNLWVFGGGT {LTVLGGGGSGGGGﬁ'VKTDﬁTGGGTVQPGR? SGFTFSDYW NWVR QS?*KGT*WVAQ RW<PYWYETYYSDSVKG<ET SRDDSKSSVYLQMWNLRV ﬂ) G YYCTGSYYG DYWGQGTSVTVSSGGCGGGKVAALKTKVAALKTKVAA L<T<VAAT.Km IV. Pharmaceutical Compositions The compositions of the invention include bulk drug compositions useful in the manufacture of pharmaceutical compositions (e.g., impure or non-sterile compositions) and pharmaceutical compositions (z'.e., compositions that are suitable for administration to a subject or patient) which can be used in the preparation of unit dosage forms. Such compositions se a prophylactically or therapeutically ive amount of the sequence-optimized CD123 X CD3 bi-speciﬁc diabodies of the present invention, or a combination of such agents and a pharmaceutically acceptable carrier. Preferably, compositions of the invention comprise a prophylactically or eutically effective amount of the sequence-optimized CD123 X CD3 ciﬁc diabody of the ion and a pharmaceutically acceptable carrier.

The invention also encompasses pharmaceutical compositions comprising sequence-optimized CD123 X CD3 bi-speciﬁc ies of the invention, and a second therapeutic antibody (e.g., tumor speciﬁc monoclonal antibody) that is c for a particular cancer antigen, and a pharmaceutically acceptable carrier.

In a speciﬁc embodiment, the term “pharmaceutically able” means approved by a regulatory agency of the Federal or a state government or listed in the US. Pharmacopeia or other generally recognized copeia for use in animals, and more particularly in humans. The term “carrier” refers to a diluent, adjuvant (e. g., Freund’s adjuvant (complete and incomplete), excipient, or vehicle with which the therapeutic is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic , such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously.

Saline solutions and aqueous dextrose and ol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, ﬂour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering . These compositions can take the form of solutions, sions, emulsion, s, pills, es, powders, sustained-release formulations and the like.

Generally, the ingredients of compositions of the invention are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent. Where the composition is to be administered by infusion, it can be dispensed with an inﬁJsion bottle containing e pharmaceutical grade water or saline. Where the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.

The compositions of the invention can be formulated as neutral or salt forms. ceutically acceptable salts include, but are not limited to those formed with anions such as those derived from hydrochloric, phosphoric, acetic, , tartaric acids, etc, and those formed with cations such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2- ethylamino ethanol, histidine, procaine, etc.

The invention also es a pharmaceutical pack or kit sing one or more containers filled with sequence-optimized CD123 X CD3 bi-specif1c diabodies of the invention alone or with such pharmaceutically acceptable carrier. onally, one or more other prophylactic or therapeutic agents useful for the treatment of a disease can also be ed in the pharmaceutical pack or kit. The invention also provides a pharmaceutical pack or kit comprising one or more containers ﬁlled with one or more of the ingredients of the pharmaceutical compositions of the ion.

Optionally associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of ceuticals or biological products, which notice reﬂects approval by the agency of manufacture, use or sale for human administration.

The present invention provides kits that can be used in the above methods. A kit can comprise sequence-optimized CD123 X CD3 bi-specif1c diabodies of the invention. The kit can ﬁarther comprise one or more other prophylactic and/or therapeutic agents useful for the treatment of cancer, in one or more containers; and/or the kit can ﬁarther comprise one or more xic antibodies that bind one or more cancer antigens associated with cancer. In certain ments, the other prophylactic or therapeutic agent is a chemotherapeutic. In other embodiments, the prophylactic or therapeutic agent is a biological or hormonal therapeutic.

V. Methods of Administration The compositions of the present invention may be provided for the ent, prophylaxis, and amelioration of one or more symptoms ated with a disease, disorder or infection by administering to a subject an effective amount of a fusion protein or a conjugated molecule of the invention, or a pharmaceutical composition comprising a ﬁJsion n or a conjugated le of the invention. In a preferred aspect, such compositions are substantially ed (z'.e., substantially free from substances that limit its effect or produce undesired side effects). In a speciﬁc embodiment, the t is an animal, preferably a mammal such as non-primate (e.g., bovine, equine, feline, canine, rodent, etc.) or a primate (e. g., monkey such as, a cynomolgus monkey, human, etc.). In a preferred embodiment, the subject is a human.

Various delivery systems are known and can be used to administer the compositions of the invention, e.g, encapsulation in mes, microparticles, microcapsules, recombinant cells capable of expressing the antibody or fusion protein, receptor-mediated endocytosis (See, e.g., Wu et al. (1987) tor-Mediated In Vitro Gene Transformation By A e DNA Carrier System,” J. Biol. Chem. 262:4429-4432), construction of a nucleic acid as part of a retroviral or other vector, etc.

Methods of administering a le of the invention include, but are not limited to, parenteral administration (e.g., intradermal, uscular, intraperitoneal, intravenous and subcutaneous), epidural, and mucosal (e.g, intranasal and oral ). In a speciﬁc embodiment, the ce-optimized CD123 X CD3 bi-speciflc diabodies of the invention are administered intramuscularly, intravenously, or subcutaneously. The compositions may be administered by any ient route, for example, by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and may be administered together with other biologically active agents. Administration can be systemic or local. In addition, pulmonary administration can also be employed, e.g., by use of an r or nebulizer, and formulation with an aerosolizing agent. See, e.g., US. Patent Nos. 968; 5,985, 320; 5,985,309; 5,934,272; 5,874,064; ,855,913; 5,290,540; and 4,880,078; and PCT Publication Nos. WO 92/19244; WO 97/32572; WO 97/44013; WO 98/31346; and WO 03, each of which is incorporated herein by nce in its entirety.

The invention also provides that the sequence-optimized CD123 X CD3 bi- specific diabodies of the invention are packaged in a ically sealed ner such as an ampoule or sachette indicating the quantity of the molecule. In one embodiment, the sequence-optimized CD123 X CD3 bi-specif1c diabodies of the invention are supplied as a dry sterilized lyophilized powder or water free concentrate in a hermetically sealed container and can be reconstituted, e.g., with water or saline to the appropriate concentration for administration to a subject. Preferably, the sequence-optimized CD123 X CD3 bi-speciflc diabodies of the invention are supplied as a dry sterile lized powder in a hermetically sealed container at a unit dosage of at least 5 ug, more preferably at least 10 ug, at least 15 ug, at least 25 ug, at least 50 ug, at least 100 ug, or at least 200 ug.

The lyophilized sequence-optimized CD123 X CD3 bi-specif1c diabodies of the invention should be stored at between 2 and 8°C in their original container and the molecules should be administered within 12 hours, preferably within 6 hours, within 5 hours, within 3 hours, or within 1 hour after being tituted. In an alternative embodiment, sequence-optimized CD123 X CD3 bi-specific diabodies of the invention are supplied in liquid form in a hermetically sealed container indicating the quantity and concentration of the molecule, fusion protein, or conjugated molecule.

Preferably, the liquid form of the sequence-optimized CD123 X CD3 bi-specific diabodies of the ion are supplied in a hermetically sealed ner in which the molecules are present at a concentration of least 1 ug/ml, more preferably at least 2.5 ug/ml, at least 5 ug/ml, at least 10 ug/ml, at least 50 ug/ml, or at least 100 ug/ml.

The amount of the composition of the invention which will be effective in the treatment, prevention or amelioration of one or more symptoms associated with a disorder can be determined by standard clinical ques. The precise dose to be employed in the ation will also depend on the route of administration, and the seriousness of the condition, and should be decided according to the judgment of the practitioner and each patient’s circumstances. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems.

For sequence-optimized CDl23 X CD3 bi-specific diabodies encompassed by the invention, the dosage administered to a patient is preferably ined based upon the body weight (kg) of the recipient subject. The dosage stered is typically from at least about 0.3 ng/kg per day to about 0.9 ng/kg per day, from at least about 1 ng/kg per day to about 3 ng/kg per day, from at least about 3 ng/kg per day to about 9 ng/kg per day, from at least about 10 ng/kg per day to about 30 ng/kg per day, from at least about 30 ng/kg per day to about 90 ng/kg per day, from at least about 100 ng/kg per day to about 300 ng/kg per day, from at least about 200 ng/kg per day to about 600 ng/kg per day, from at least about 300 ng/kg per day to about 900 ng/kg per day, from at least about 400 ng/kg per day to about 800 ng/kg per day, from at least about 500 ng/kg per day to about 1000 ng/kg per day, from at least about 600 ng/kg per day to about 1000 ng/kg per day, from at least about 700 ng/kg per day to about 1000 ng/kg per day, from at least about 800 ng/kg per day to about 1000 ng/kg per day, from at least about 900 ng/kg per day to about 1000 ng/kg per day, or at least about 1,000 ng/kg per day.

In r embodiment, the patient is administered a treatment regimen comprising one or more doses of such prophylactically or therapeutically effective amount of the sequence-optimized CD123 X CD3 bi-specif1c diabodies encompassed by the invention, wherein the treatment regimen is administered over 2 days, 3 days, 4 days, 5 days, 6 days or 7 days. In certain embodiments, the treatment regimen ses intermittently administering doses of the prophylactically or therapeutically effective amount of the sequence-optimized CD123 X CD3 bi-specif1c diabodies encompassed by the invention (for example, administering a dose on day 1, day 2, day 3 and day 4 of a given week and not administering doses of the lactically or therapeutically effective amount of the sequence-optimized CD123 X CD3 bi-speciﬁc diabodies encompassed by the ion on day 5, day 6 and day 7 of the same week).

Typically, there are 1, 2, 3, 4, 5 or more s of treatment. Each course may be the same regimen or a different regimen.

In another embodiment, the administered dose escalates over the ﬁrst quarter, first half or first two-thirds or three-quarters of the regimen(s) (e.g., over the ﬁrst, second, or third regimens of a 4 course treatment) until the daily prophylactically or therapeutically effective amount of the sequence-optimized CD123 X CD3 bi- speciﬁc ies encompassed by the invention is achieved.

Table 1 provides 5 examples of different dosing regimens described above for a typical course of treatment.

Table 1 Regimen ND “93“02% y Dosage (ng y per k sub} ect we1 ht per day)'. p—A \14; 100 100 100 100 100 3 mimNm -—, 300 500 none none none none none 1, “N 300 500 700 900 u 1,000 U‘I @0300.) \l-hN-hN-h none none none none none The dosage and frequency of administration of sequence-optimized CD123 X CD3 bi-specif1c diabodies of the invention may be d or altered by enhancing uptake and tissue penetration of the sequence-optimized CD123 X CD3 bi-specif1c ies by modiﬁcations such as, for example, lipidation.

The dosage of the sequence-optimized CD123 X CD3 bi-specif1c diabodies of the ion administered to a patient may be calculated for use as a single agent y. Alternatively, the sequence-optimized CD123 X CD3 bi-specif1c diabodies of the invention are used in combination with other therapeutic compositions and the dosage administered to a patient are lower than when said molecules are used as a single agent therapy.

The ceutical compositions of the invention may be administered locally to the area in need of treatment; this may be achieved by, for example, and not by way of limitation, local 111ﬁ1S1011, by injection, or by means of an implant, said implant being of a porous, non-porous, or gelatinous material, including membranes, such as sialastic membranes, or ﬁbers. Preferably, when administering a molecule of the invention, care must be taken to use als to which the molecule does not The itions of the invention can be delivered in a vesicle, in particular a me (See Langer (1990) “New Methods OfDrag Delivery,” Science 249: 1527- 1533); Treat et al., in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 353- 365 (1989); Lopez- Berestein, ibid., pp. 3 17-327; see generally .

The compositions of the invention can be red in a controlled-release or sustained-release system. Any technique known to one of skill in the art can be used to produce sustained-release formulations comprising one or more sequence- optimized CD123 X CD3 bi-specif1c ies of the invention. See, e.g., US. Patent No. 4,526,938; PCT publication WO 91/05548; PCT publication WO 96/20698; Ning et al. (1996) “Intratamoral Radioimmanotherapby Of A Human Colon Cancer aft Using A Sustained-Release Gel,” Radiotherapy & Oncology 39:179-189, Song et al. (1995) “Antibody Mediated Lang Targeting 0f Long-Circulating Emulsions, ” PDA Journal of Pharmaceutical Science & Technology 50:372-397; Cleek et al. (1997) “Biodegradable Polymeric Carriers For A bFGF Antibody For Cardiovascular Application, ” Pro. Int’l. Symp. Control. Rel. Bioact. Mater. 24:853-854; and Lam et al. (1997) “Microencapsulation 0fRecombinant Humanized Monoclonal dy For Local Delivery, ” Proc. Int’l. Symp. Control Rel. Bioact.

Mater. 24:759-760, each of which is incorporated herein by reference in its entirety.

In one embodiment, a pump may be used in a controlled-release system (See Langer, supra; Sefton, (1987) “Implantable ” CRC Crit. Rev. Biomed. Eng. 14:201- 240; Buchwald et al. (1980) “Long-Term, Continuous Intravenous Heparin stration By An Implantable Infusion Pump In Ambulatory Patients With Recurrent Venous Thrombosis,” Surgery 88:507-516; and Saudek et al. (1989) “A Preliminary Trial Of The Programmable Implantable Medication System For n Delivery,” N. Engl. J. Med. 321:574-579). In another embodiment, ric materials can be used to achieve controlled-release of the molecules (see e. g., MEDICAL APPLICATIONS OF CONTROLLED RELEASE, Langer and Wise (eds.), CRC Pres., Boca Raton, Florida (1974); CONTROLLED DRUG BIOAVAILABILITY, DRUG PRODUCT DESIGN AND PERFORMANCE, Smolen and Ball (eds.), Wiley, New York (1984); Levy et al. (1985) “Inhibition 0f Calciﬁcation OfBioprosthetic Heart Valves By Local Controlled-Release Diphosphonate, ” Science 228:190-192; During et al. (1989) “Controlled Release 0fDopamine From A ric Brain Implant.‘ In Vivo Characterization,” Ann. Neurol. 25:351-356; Howard et al. (1989) “Intracerebral Drug Delivery In Rats With Lesion-Induced Memory Deﬁcits, ” J. Neurosurg. 7(1):105-112); US. Patent No. 5,679,377; US. Patent No. 5,916,597; US. Patent No. ,912,015; US. Patent No. 5,989,463; US. Patent No. 5,128,326; PCT Publication No. W0 99/15154; and PCT Publication No. WO 99/20253). Examples of rs used in sustained-release formulations include, but are not limited to, poly(2-hydroxy ethyl methacrylate), poly(methyl methacrylate), poly(acrylic acid), thylene-co- vinyl acetate), poly(methacrylic acid), polyglycolides (PLG), polyanhydrides, poly(N— vinyl pyrrolidone), poly(vinyl l), rylamide, poly(ethylene glycol), ctides (PLA), poly(lactide-co-glycolides) , and polyorthoesters. A controlled-release system can be placed in proximity of the therapeutic target (e.g., the lungs), thus requiring only a fraction of the systemic dose (see, e.g., Goodson, in MEDICAL ATIONS OF CONTROLLED RELEASE, supra, vol. 2, pp. 115-138 (1984)). Polymeric compositions useful as lled-release implants can be used according to Dunn et al. (See US. 5,945,155). This ular method is based upon the therapeutic effect of the in situ controlled-release of the bioactive material from the polymer system. The implantation can generally occur re within the body of the patient in need of eutic treatment. A non-polymeric sustained ry system can be used, whereby a non-polymeric implant in the body of the subject is used as a drug delivery system. Upon implantation in the body, the c solvent of the implant will dissipate, disperse, or leach from the composition into surrounding tissue ﬂuid, and the non-polymeric material will gradually coagulate or precipitate to form a solid, microporous matrix (See US. 5,888,533).

Controlled-release systems are discussed in the review by Langer (1990, “New Methods OfDrug Delivery, ” Science 249:1527-1533). Any technique known to one of skill in the art can be used to produce sustained-release ations comprising one or more therapeutic agents of the invention. See, e.g., US. Patent No. 4,526,938; International Publication Nos. WO 48 and WO 96/20698; Ning et al. (1996) “Intratumoral Radioimmunotberapby Of A Human Colon Cancer Xenograft Using A Sustained-Release Gel,” Radiotherapy & Oncology -189, Song et al. (1995) ody Mediated Lung ing 0f Long-Circulating Emulsions,” PDA Journal of Pharmaceutical Science & Technology 50:372-397; Cleek et al. (1997) “Biodegradable Polymeric Carriers For A bFGF Antibody For vascular Application, ” Pro. Int’l. Symp. Control. Rel. Bioact. Mater. -854; and Lam et al. (1997) “Microencapsulation 0fRecombinant Humanized Monoclonal Antibody For Local Delivery, ” Proc. Int’l. Symp. Control Rel. Bioact.

Where the composition of the invention is a nucleic acid encoding a sequence-optimized CD123 X CD3 bi-specif1c diabody of the invention, the nucleic acid can be administered in vivo to promote expression of its encoded sequence- optimized CD123 X CD3 bi-specif1c diabody, by constructing it as part of an appropriate nucleic acid expression vector and administering it so that it becomes intracellular, e. g., by use of a retroviral vector (See US. Patent No. 286), or by direct injection, or by use of microparticle bombardment (e.g., a gene gun; Biolistic, Dupont), or coating with lipids or cell-surface receptors or transfecting agents, or by administering it in e to a homeobox-like peptide which is known to enter the nucleus (See e.g, Joliot et al. (1991) “Antennapedz'a H0me0b0x e Regulates Neural Morphogenesis,” Proc. Natl. Acad. Sci. (U.S.A.) 88:1864-1868), etc.

Alternatively, a nucleic acid can be introduced intracellularly and incorporated within host cell DNA for expression by gous recombination.

Treatment of a subject with a therapeutically or prophylactically ive amount of sequence-optimized CD123 X CD3 bi-specif1c diabodies of the invention can include a single treatment or, ably, can include a series of treatments. In a preferred example, a subject is treated with sequence-optimized CD123 X CD3 bi- speciﬁc diabodies of the invention one time per week for between about 1 to 10 weeks, preferably between 2 to 8 weeks, more preferably between about 3 to 7 weeks, and even more preferably for about 4, 5, or 6 weeks. The pharmaceutical compositions of the invention can be administered once a day, twice a day, or three times a day. Alternatively, the pharmaceutical compositions can be administered once a week, twice a week, once every two weeks, once a month, once every six weeks, once every two months, twice a year or once per year. It will also be appreciated that the effective dosage of the molecules used for treatment may increase or decrease over the course of a particular treatment.

VI. Uses of the itions of the Invention The sequence-optimized CD123 X CD3 bi-specif1c diabodies of the present ion have the ability to treat any disease or condition associated with or characterized by the expression of CD123. Thus, without limitation, such molecules may be employed in the sis or treatment of acute d leukemia (AML), chronic enous leukemia (CML), including blastic crisis of CML and Abelson oncogene associated with CML (Bcr-ABL translocation), myelodysplastic syndrome (MDS), acute B lymphoblastic leukemia (B-ALL), chronic cytic leukemia (CLL), including Richter’s syndrome or Richter’s transformation of CLL, hairy cell ia (HCL), blastic plasmacytoid dendritic cell neoplasm (BPDCN), non- n lymphomas (NHL), including mantel cell leukemia (MCL), and small lymphocytic lymphoma (SLL), Hodgkin’s lymphoma, systemic mastocytosis, and Burkitt’s lymphoma (see Example 2); Autoimmune Lupus (SLE), allergy, asthma and rheumatoid arthritis. The bi-specific diabodies of the present invention may additionally be used in the manufacture of medicaments for the treatment of the above-described conditions.

] Having now generally described the invention, the same will be more readily understood through reference to the following examples, which are provided by way of illustration and are not intended to be limiting of the present invention unless speciﬁed.

Example 1 Construction Of CD123 x CD3 Bi-Speciﬁc Diabodies And Control n Table 2 contains a list of bi-specif1c diabodies that were expressed and puriﬁed. Sequence-optimized CD123 X CD3 bi-specif1c diabody (DART-A) and non- sequence-optimized CD123 X CD3 bi-specif1c diabody (DART-B) are capable of simultaneously g to CD123 and CD3. The control bi-specif1c diabody (Control DART) is capable of simultaneously binding to FITC and CD3. The bi-specific diabodies are heterodimers or heterotrimers of the d amino acid sequences.

Methods for forming bi-specif1c diabodies are provided in WC 13665, WO 2008/157379, , , and WO 2012/162067.

Polypeptide Chain EncoNtiiﬂelgfggnces. .

Bi—Speciﬁc ies Amino Acid g q Se u uences ce-Optimized CD123 x CD3 .

Bi—Speciﬁc Diabody (DART-A) Egg £3 £8;° Egg £3 £8:.

(Binds to CD3 at epitope 1) ° Non-Sequence-Optimized CD123 x . .

CD3 ciﬁc Diabody (DART-B) Egg :3 $3; $3 :3 £33 (Binds to CD3 at epitope 2) ° ° Sequence-Optimized CD123 x CD3 Bi—Speciﬁc Diabody Having an n-Binding Domain (DART-A w/ABD) SEQ ID N0:9 SEQ ID N0:10 (Binds to CD3 at epitope 1) SEQ ID N0:3 SEQ ID N0:4 Comprises an Albumin-Binding Domain (ABD) for extension of half- life in vivo Table 2 Polypeptide Chain c Acid Bi-Speciﬁc Diabodies Amino Acid Encoding Sequences Se u uences Sequence-Optimized CD123 x CD3 Bi-Speciﬁc Diabody Having an IgG Fc Domain Version 1 (DART-A w/Fc SEQ ID N0:54 SEQ ID N0:12 version 1) SEQ ID N0:13 SEQ ID N0:14 (Binds to CD3 at epitope 1) SEQ ID N0:15 SEQ ID N0:16 Comprises an Fc Domain for extension of half-life in vivo Sequence-Optimized CD123 x CD3 Bi-Speciﬁc Diabody Having an IgG Fc Domain Version 2 (DART-A w/Fc SEQ ID N0:54 SEQ ID N0:12 version 2) SEQ ID N0:17 SEQ ID N0:18 (Binds to CD3 at epitope 1) SEQ ID N0:1 SEQ ID N0:2 Comprises an Fc Domain for ion of half-life in vivo Control Bi-Speciﬁc Diabody (Control DART (or Control DART) SEQ ID N0:19 (Binds to CD3 at epitope 1) SEQ ID N0:20 (Binds to an irrelevant target - FITC) Example 2 Antibody Labeling Of Target Cells For Quantitative FACS (QFACS) A total of 106 target cells were ted from the culture, resuspended in % human AB serum in FACS buffer (PBS + 1% BSA+ 0.1% NaAzide) and incubated for 5 min for blocking Fc receptors. Antibody labeling of microspheres with different antibody binding capacities (QuantumTM Simply Cellular® (QSC), Bangs tories, Inc., Fishers, IN) and target cells were d with anti-CD123 PE antibody (BD Biosciences) according to the manufacturer’s instructions. Brieﬂy, one drop of each QSC phere was added to a 5 mL polypropylene tube and PE labeled-anti-CD123 antibody was added at 1 ug/rnL concentration to both target cells and microspheres. Tubes were incubated in the dark for 30 minutes at 4 °C. Cells and microspheres were washed by adding 2mL FACS buffer and centriﬁaging at 2500 x G for 5 minutes. One drop of the blank microsphere population was added after washing. Microspheres were analyzed ﬁrst on the ﬂow cytometer to set the test- c instrument settings (PMT voltages and sation). Using the same instrument settings, geometric mean ﬂuorescence values of microspheres and target cells were recorded. A standard curve of antibody binding sites on microsphere populations was generated from geometric mean ﬂuorescence of microsphere populations. Antibody binding sites on target cells were calculated based on geometric mean ﬂuorescence of target cells using the standard curve generated for microspheres in al spreadsheet (Bangs Laboratories).

To determine suitable target cell lines for evaluating CD123 X CD3 bi- speciﬁc diabodies, CD123 surface expression levels on target lines Kasumi-3 (AML), Molml3 (AML), THP-l (AML), TF-l (Erythroleukemia), and RS4-ll (ALL) were evaluated by quantitative FACS (QFACS). Absolute numbers of CD123 antibody binding sites on the cell surface were calculated using a QFACS kit. As shown in Table 3, the absolute number of CD123 dy binding sites on cell lines were in the order of Kasumi-3 (high) > Molml3 (medium) > THP-l(medium) > TF-l (medium low) > RS4-ll (low). The three t expressing cell lines were the AML cell lines: Kasumi-3, MOLM13, and THP-l. The non-AML cell lines: TF-l and RS4- 11 had medium—low/ low expression of CD123, respectively.

Table 3 Target Cell CD123 Surface Expression Line Antibod Bindin_ Sites Kasumi-3 1 18620 Molml 3 273 1 1 THP-l 58316 TF-l 14163 RS4-1 1 957 A498 Neative HT29 Neative CTL Cytotoxicity Assay (LDH Release Assay) ] Adherent target tumor cells were detached with 0.25% Trypsin-EDTA solution and ted by centriﬁlgation at 1000 rpm for 5 min. Suspension target cell lines were harvested from the culture, washed with assay medium. The cell concentration and viability were measured by Trypan Blue ion using a Beckman Coulter l counter. The target cells were diluted to 4 x105 cells/mL in the assay medium. 50 uL of the d cell suspension was added to a 96-well U- bottom cell culture treated plate (BD Falcon Cat#353077).

Three sets of controls to measure target maximal release (MR), antibody independent cellular cytotoxicity (AICC) and target cell spontaneous release (SR) were set up as follows: 1) MR: 200 uL assay medium without CD123 X CD3 bi-speciﬁc diabodies and 50 uL target cells; ent added at the end of the experiment to ine the maximal LDH release. 2) AICC: 50 uL assay medium t CD123 X CD3 bi-speciﬁc diabodies, 50 [LL target cells and 100 uL T cells. 3) SR: 150 uL medium without CD123 X CD3 bi-speciﬁc diabodies and 50 [LL target cells.

CD123 X CD3 bi-speciﬁc diabodies (DART-A, DART-A w/ABD and DART-B) and controls were initially diluted to a concentration of 4 ug/mL, and serial dilutions were then prepared down to a ﬁnal concentration of 0.00004 ng/mL (z'.e., 40 fg/mL). 50 [LL of dilutions were added to the plate ning 50 uL target cells/well.

Puriﬁed T cells were washed once with assay medium and resuspended in assay medium at cell density of 2 x 106 cells/mL. 2 x 105 T cells in 100 uL were added to each well, for a ﬁnal effector-to-target cell (E:T) ratio of 10:1. Plates were ted for approximately 18hr at 37°C in 5% C02.

Following incubation, 25 uL of 10x lysis solution (Promega # G182A) or 1 mg/mL digitonin was added to the m release control wells, mixed by pipetting 3 times and plates were incubated for 10 min to tely lyse the target cells. The plates were centrifuged at 1200 rpm for 5 minutes and 50 uL of supernatant were transferred from each assay plate well to a ﬂat bottom ELISA plate and 50 ul of LDH substrate on (Promega #G1780) was added to each well. Plates were incubated for 10-20 min at room temperature (RT) in the dark, then 50 [LL of Stop solution was added. The optical density (O.D.) was measured at 490 nm within 1 hour on a Victor2 Multilabel plate reader (Perkin Elmer #1420-014). The percent cytotoxicity was calculated as described below and dose-response curves were generated using GraphPad PRISM5® software.

Speciﬁc cell lysis was calculated from OD. data using the following formula: CytotOXiCity (%) = 100 X (OD of Sample - OD of AICC)/ (OD of MR - OD of SR) Redirected Killing of Target Cell Lines with Different Levels of CD123 e Levels: The CD123 X CD3 cif1c diabodies ted a potent redirected killing ability with concentrations required to achieve 50% of maximal activity (EC50s) in sub-ng/mL range, regardless of CD3 epitope binding speciﬁcity A versus DART-B) in target cell lines with high CD123 expression, Kasumi-3 (EC50=0.0l ng/mL) (Figure 4 Panel D), medium CD123-expression, Molml3 (EC50=0.l8 ng/mL) and THP-l (EC50=0.24 ng/mL) (Figure 4, Panel C and E, respectively) and medium low or low CD123 expression, TF-l (EC50=0.46 ng/mL) and RS4-ll (EC50=0.5 ng/mL) (Figure 4, Panel B and A, respectively). Similarly, CD123 X CD3 bi-specif1c molecules mediated redirected g was also observed with multiple target cell lines with T cells from ent donors and no redirected killing activity was ed in cell lines that do not express CD123. Results are summarized in Table 4.

Target cell line CD123 surface EC50 of Sequence- Max % killing expression optimized CD123 x (antibody binding CD3 bi-speciﬁc sites) diabodies (ng/mL) Kasumi-3 118620 94 M01m13 43 THP-l 40 TF-l 46 Rs4-11 A498 No activit HT29 No activit Should it be necessary to replicate this example it will be appreciated that one of skill in the art may, within reasonable and acceptable limits, vary the above- described protocol in a manner riate for replicating the described results. Thus, the exempliﬁed protocol is not intended to be adhered to in a precisely rigid manner.

Example 4 T Cell Activation During Redirected Killing By Sequence-Optimized CD123 x CD3 Bi-Speciﬁc Diabodies (DART-A, DART-A w/ABD and DART-A w/Fc) The sequence-optimized CD123 X CD3 bi-speciﬁc diabodies exhibited a potent redirected killing ability regardless of the ce or absence of half-life extension technology (DART-A versus DART-A w/ABD versus DART-A w/Fc) in target cell lines with high CD123 expression, Kasumi-3, and medium, THP-l, CD123-expression, (Figure 5, Panels A and B, respectively) To characterize T cell activation during ce-optimized CD123 X CD3 bi-speciﬁc diabody mediated redirected killing process, T cells from redirected killing assays were stained for T cell activation marker CD25 and analyzed by FACS. As shown in Figure 5, Panel D, CD25 was up-regulated in CD8 T cells in a dose-dependent manner indicating that ce-optimized CD123 X CD3 bi-speciﬁc diabodies induce T cell tion in the process of redirected killing. Conversely, in the absence of target cells there is no activation of CD8 T cells (Figure 5, Panel C) indicating the sequence-optimized CD123 X CD3 bi-speciﬁc diabodies do not activate T cells in the absence of target cells. Likewise, CD8 T cells are not activated when incubated with target cells and a control bi-speciﬁc diabody (Control DART) (Figure 5, Panel D) indicating the requirement of cross-linking the T cell and target cell with the sequence-optimized CD123 X CD3 bi-speciﬁc diabodies.

Example 5 ellular ng for Granzyme B and Perforin To determine the intracellular levels of granzyme B and in in T cells, a CTL assay was setup as described above. After approximately 18 h, cells from the assay plate were stained with anti-CD4 and anti-CD8 dies by incubating for 30 minutes at 4°C. Following surface staining, cells were incubated in 100 uL n and permeabilization buffer (BD ences) for 20 min at 4°C. Cells were washed with permeabilization/wash buffer (BD BioSciences) and incubated in 50 uL of me B and a perforin antibody mixture (prepared in 1X permeabilization/wash buffer) at 4°C for 30 s. Then cells were washed with 250 uL permeabilization/wash buffer and resuspended in permeabilization/wash buffer for FACS acquisition.

Upregulation Of Granzyme B And Perforin By Sequence-Optimized CD123 x CD3 ciﬁc Diabody (DART-A) In T Cells During Redirected Killing To investigate the possible mechanism for sequence-optimized CD123 X CD3 bi-speciﬁc diabody (DART-A) ed cytotoxicity by T cells, intracellular granzyme B and perforin levels were measured in T cells after the redirected killing.

Dose-dependent upregulation of granzyme B and perforin levels in both CD8 and CD4 T cells was observed following tion of T cells and Kasumi-3 cells with DART-A (Figure 6, Panel A). Interestingly, the upregulation was almost two-fold higher in CD8 T cells compared to CD4 T cells e 6, Panel A). When the assay was med in the presence of granzyme B and perforin inhibitors no cell killing was observed. There was no upregulation of me B or perforin in CD8 or CD4 T cells when T cells were incubated with Kasumi-3 target cells and a control bi- speciﬁc diabody (Control DART) e 5, Panel B). These data indicate that DART-A mediated target cell killing may be mediated through granzyme B and perforin mechanisms.

Example 6 in vivo mor Activity Of Sequence-Optimized CD123 x CD3 Bi-Speciﬁc Diabody (DART-A) Isolation of PBMCs and T Cells from Human Whole Blood PBMCs from healthy human donors were isolated from whole blood by using Ficoll gradient centriﬁlgation. In brief, whole blood was diluted 1:1 with sterile PBS. Thirty-ﬁve mL of the diluted blood was layered onto 15 mL Ficoll-PaqueTM Plus in 50-mL tubes and the tubes were centrifuged at 1400 rpm for 20 min with the brake off. The buffy coat layer between the two phases was collected into a 50 mL tube and washed with 45 mL PBS by centrifuging the tubes at 600 x g (1620 rpm) for min. The supernatant was ded and the cell pellet was washed once with PBS and viable cell count was determined by Trypan Blue dye exclusion. The PBMCs were resuspended to a ﬁnal concentration of 2.5x106 cells/mL in complete medium (RPMI 1640, 10%FBS, 2 mM Glutamine, 10mM HEPES, 00u/mL penicillin/Streptomycin (P/S).

T cell isolation: Untouched T cells were isolated by negative selection from PBMCs from human whole blood using Dynabeads Untouched Human T Cell isolation kit (Life Technologies) according to cturer’s ctions. After the isolation, T cells were cultured overnight in RPMI medium with 10% PBS, 1% penicillin/Streptomycin.

Tumor Model Human T cells and tumor cells (Molm13 or RS4-11) were combined at a ratio of 1:5 (1 x 106 and 5 x 106, respectively) and suspended in 200 uL of sterile saline and injected subcutaneously (SC) on Study Day 0 (SDO). Sequence-optimized CD123 X CD3 bi-speciﬁc diabody (DART-A) or a control bi-speciﬁc diabody (Control DART) were administered intravenously (IV) via tail vein injections in 100 uL as outlined in Table 5 (MOLM13) and Table 6 (RS4-11).

Stud Desi_n for MOLM13 Model Number of Schedule Animals Vehicle l (MOLM-13 cells alone implanted SDO, 1, 2, 3 or + T cells) DART A 0 U1 SDO, 1, 2, 3 oo - HM SDO, 1, 2, 3 - SDO, 1, 2, 3 0-02 SDO, 1, 2, 3 SDO, 1, 2, 3 SDO, 1, 2, 3 DART-A 0.00016 SDO, 1, 2, 3 Table 6 Stud Desi_n for RS4-11 Model Number of Treatment Group Dose (mg/kg) Schedule Animals Vehicle Control SDO, 1, 2, 3 (RS4-11 cells alone implanted) —_-—Vehicle Control SDO, 1, 2, 3 1 + T cells implanted) Control DART SDO, 1, 2, 3 DART-A SDO, 1, 2, 3 ———-_DART-A SDO, 1, 2, 3 ———-_DART-A SDO, 1, 2, 3 ———-_DART-A SDO, 1, 2, 3 ———-_DART-A SDO, 1, 2, 3 Data Collection and Statistical Analysis: Animal s - Individual animal weights were ed twice weekly until study completion beginning at the time of tumor cell injection.

Moribundity/Mortality - Animals were observed twice weekly for general moribundity and daily for mortality. Animal deaths were assessed as drug-related or technical based on factors ing gross observation and weight loss; animal deaths were ed daily.

Tumor volume - Individual tumor volumes were recorded twice weekly beginning within one week of tumor implantation and continuing until study tion.

Length (mm)><width2 Tumor Volume (mm3) = Animals experiencing technical or drug-related deaths were censored from the data calculations.

Tumor growth inhibition - Tumor growth inhibition (TGI) values were ated for each group containing treated animals using the formula: _Mean Final Tumor Volume (Treated) - Mean Initial Tumor Volume (Treated)x 1 100 Mean Final Tumor Volume (Control) - Mean Initial Tumor Volume (Control) Animals experiencing a partial or complete response, or animals experiencing technical or drug-related deaths were censored from the TGI calculations. The National Cancer ute criteria for compound activity is TGI>58% tt et al. (2004) Anticancer Drug Development Guide; Totowa, NJ: Humana ).

Partial/Complete Tumor Response - Individual mice possessing tumors measuring less than 1mm3 on Day 1 were classiﬁed as having partial response (PR) and a t tumor regression (%TR) value was determined using the formula: Final Tumor Volume (mm3) 1- X’IOO% Initial Tumor Volume (mm3) ] Individual mice lacking palpable tumors were classiﬁed as undergoing a complete response (CR).

Tumor Volume Statistics — Statistical es were carried out between d and control groups comparing tumor s. For these analyses, a two-way analyses of variance followed by a Bonferroni post-test were employed. All analyses were performed using GraphPad PRISM® software (version 5.02). Weight and tumor data from individual animals experiencing technical or drug-related deaths were censored from analysis. However, tumor data from animals reporting partial or complete responses were included in these calculations.

MOLM13 s The AML cell line MOLM13 was pre-mixed with activated T cells and implanted SC in NOD/SCID gamma (NSG) knockout mice (N = 8/group) on SDO as ed above. The MOLM13 tumors in the vehicle-treated group (MOLM13 cells alone or plus T cells) demonstrated a relatively aggressive growth proﬁle in vivo (Figure 7, Panels A and B). At SD8, the average volume of the tumors in the vehicle-treated group was 129.8 :: 29.5 mm3 and by SD15 the tumors had reached an average volume of 786.4 :: 156.7 mm3. By the end of the experiment on SD18, the tumors had reached an average volume of 1398.8 :: 236.9 mm3.

Treatment with DART-A was initiated on the same day the tumor cell/T cell mixture was implanted [(SDO)] and proceeded subsequently with daily injections for an additional 7 days for a total of 8 daily injections. The animals were treated with DART-A at 9 dose levels (0.5, 0.2, 0.1, 0.02, and 0.004 mg/kg and 20, 4, 0.8 and 0.16 [Lg/kg). Results are shown in Figure 7, Panel A (0.5, 0.2, 0.1, 0.02, and 0.004 mg/kg) and Figure 7, Panel B (20, 4, 0.8 and 0.16 ug/kg). By Study Day 11, the growth of the MOLM13 tumors was signiﬁcantly inhibited at the 0.16, 0.5, 0.2, 0.1, 0.02, and 0.004 mg/kg dose levels (p < 0.001). Moreover, the treatment of the MOLM13 tumor-bearing mice at the 20 and 4 [Lg/kg dose levels resulted in 8/8 and 7/8 CRs, respectively. By the end of the experiment on SD18, the average volume of the tumors treated with DART-A at the 0.8 — 20 ug/kg) ranged from 713.60 :: 267.4 to 0 mm3, all of which were signiﬁcantly smaller than the tumors in the e-treated control group. The TGI values were 100, 94, and 49% for the 20, 4, and 0.8 [Lg/kg dose groups, tively. In comparison to the e-treated MOLM13 tumor cell group, the groups that received DART-A at the 20 and 4 [Lg/kg dose level reached statistical signiﬁcance by SD15 while the group treated with 0.8 [Lg/kg reached signiﬁcance on SD18.

RS4-11 Results The ALL cell line RS4-11 was pre-mixed with activated T cells and implanted SC in NOD/SCID gamma knockout mice (N = 8/group) on SDO as detailed above. The RS4-11 tumors in the vehicle-treated group (RS4-11 cells alone or plus T cells) demonstrated a relatively sive growth proﬁle in vivo (Figure 8).

Treatment with DART-A was initiated on the same day the tumor cell/T cell mixture was implanted [(SDO)] and proceeded subsequently with daily injections for an additional 3 days for a total of 4 daily injections. The s were treated with DART-A at 5 dose levels (0.5, 0.2, 0.1, 0.02, and 0.004 . Results are shown in Figure 8.

Sequence-optimized CD123 X CD3 bi-speciﬁc diabody (DART-A) effectively inhibited the growth of both MOLM13 AML and RS4-11ALL tumors implanted SC in ID mice in the context of the Winn model when dosing was initiated on the day of implantation and continued for 3 or more consecutive days.

Based on the criteria established by the National Cancer Institute, DART-A at the 0.1 mg/kg dose level and higher (TGI >58) is considered active in the RS4-ll model and an DART-A dose of 0.004 mg/kg and higher was active in the MOLMl3 model. The lower DART-A doses associated with the inhibition of tumor growth in the MOLMl3 model compared with the RS4-ll model are consistent with the in vitro data demonstrating that MOLMl3 cells have a higher level of CD123 expression than RS4-ll cells, which correlated with increased sensitivity to DART-A ed cytotoxicity in vitro in MOLMl3 cells.

Should it be necessary to replicate this example it will be appreciated that one of skill in the art may, within reasonable and acceptable , vary the above- described protocol in a manner appropriate for replicating the described results. Thus, the exempliﬁed ol is not intended to be adhered to in a precisely rigid manner.

Example 7 CD123 Surface Expression On Leukemic Blast Cells And Stem Cells In Primary Tissue Sample From AML Patient 1 To deﬁne the CD123 expression pattern in AML patient 1 primary samples, eserved primary AML patient bone marrow and PBMC samples were evaluated for CD123 surface expression on leukemic blast cells.

AML Bone Marrow Sample — al Report Age: 42 Gender: Female AML Subtype: M2 Cancer cell percentage based on morphology: 67.5% Bone marrow immunophenotyping: CDlS=l9%, CD33=98.5%, CD38=28.8%, CD45=81.8%, CD64=39.7%, CDl l7=42.9%, HLA-DR=l7%, CD2=l.8%, CD5=0.53%, 2%, CD10=0.4l%, CDl9=l.l%, CD20=l.4%, .7l% CD34=0.82% CD123 Expression in Leukemic Blast Cells in Bone Marrow Mononucleocytes (BM MNC) A total of 0.5x106 bone marrow mononucleocytes (BM MNC) and peripheral blood mononucleocytes (PBMC)) from AML patient 1 were evaluated for CD123 expression. The Kasumi-3 cell line was included as a control. Leukemic blast cells were identiﬁed using the d marker CD33. As shown in Figure 9, Panel A, 87% of the cells ﬁom AML bone marrow ﬁom patient 1 expressed CD123 and CD33.

CD123 expression levels were slightly lower than the CD123 high-expressing Kasumi-3 AML cell line (Figure 9, Panel B).

Example 8 Autologous CTL Killing Assay Using AML t Primary Specimens Cryopreserved primary AML specimen (bone marrow mononucleocytes (BMNC) and peripheral blood mononucleocytes (PBMC)) from AML patient 1 were thawed in RPMI 1640 with 10% FBS and allowed to recover overnight at 37°C in 5% C02. Cells were washed with assay medium (RPMI l640+lO%FBS) and viable cell count was determined by Trypan Blue exclusion. 150,000 cells / well in 150 uL assay medium were added to 96-well U-bottom plate (BD Biosciences). Sequence- zed CD123 X CD3 bi-speciﬁc diabody (DART-A) was d to 0.1, and 0.01 ng/mL and 50 uL of each dilution was added to each well (ﬁnal volume = 200 uL).

Control ciﬁc diabody (Control DART) was diluted to 0.1 ng/mL and 50 uL of each dilution was added to each well (ﬁnal volume = 200 uL). A te assay plate was set up for each time point (48, 72, 120 and 144 hours) and plates were incubated at 37 0C in a 5% C02 incubator. At each time point, cells were stained with CD4, CD8, CD25, CD45, CD33, and CD123 antibodies. Labeled cells were analyzed in FACS Calibur ﬂow cytometer equipped with CellQuest Pro acquisition software, Version 5.2.1 (BD Biosciences). Data analysis was performed using Flowjo v9.3.3 software (Treestar, Inc).T cell expansion was ed by gating on CD4+ and CD8+ populations and activation was determined by measuring CD25 mean cent intensity (MFI) on the CD4+ and CD8+ -gated populations. Leukemic blast cell population was identiﬁed by CD45+CD33+ gating. gous Tumor Cell Depletion, T Cell Expansion And Activation By Sequence-Optimized CD123 x CD3 Bi-Speciﬁc Diabody (DART-A) In Primary Specimens From AML Patient 1 To determine the sequence-optmized CD123 X CD3 bi-speciflc diabody (DART-A) mediated activity in AML patient 1, patient samples were incubated with 0.1 ng/mL or 0.01 ng/mL of DART-A and percentages of leukemic blast cells and T cells were measured at different time points following the treatment. Leukemic blast cells were identiﬁed by CD45+/CD33+ gating. tion of y AML bone marrow s with DART-A resulted in ion of the leukemic cell tion over time (Figure 10, Panel A), accompanied by a concomitant expansion of the residual T cells (Figure 10, Panel B) and the induction of T cell activation markers (Figure 10, Panel C). In DART-A treated samples, T cells were expanded from around 7 % to around 80% by 120 hours. T cell activation measured by CD25 expression on CD4 and CD8 cells peaked at 72 h and decreased by the 120 h timepoint.

Should it be necessary to replicate this example it will be appreciated that one of skill in the art may, within reasonable and acceptable limits, vary the above- described protocol in a manner appropriate for ating the described results. Thus, the iﬁed protocol is not intended to be d to in a precisely rigid manner.

Example 9 CD123 Surface Expression On Leukemic Blast Cells And Stem Cells In Primary Tissue Sample From ALL Patient To deﬁne the CD123 expression pattern in ALL patient primary samples, cryopreserved primary ALL patient PBMC sample was evaluated for CD123 surface expression on leukemic blast cells.

CD123 Expression in Leukemic Blast Cells in eral Blood Mononucleocytes (PBMC) A total of 0.5xlO6 peripheral blood mononucleocytes (PBMC)) from a healthy donor and an ALL patient were evaluated for CD123 sion. As shown in Figure 11, Panels E-H, the vast majority of the cells from ALL bone marrow expressed CD123. Conversely, as expected in the normal donor B cells are CD123 negative and pDCs and monocytes are CD123 positive e 11, Panel D).

The T cell population was identiﬁed in the ALL patent sample by staining the cells for CD4 and CD8. As shown in Figure 12, Panel B, only a small fraction of the total PBMCs in the ALL patient sample are T cells (approximately 0.5% are CD4 T cells and approximately 0.4% are CD8 T cells.

Example 10 Autologous CTL Killing Assay Using ALL Patient Primary Specimens Cryopreserved y ALL specimen (peripheral blood mononucleocytes (PBMC)) were thawed in RPM 11640 with 10% FBS and allowed to recover overnight at 37°C in 5% C02. Cells were washed with assay medium (RPMI 1640+10%FBS) and viable cell count was determined by Trypan Blue ion. 150,000 cells / well in 150 uL assay medium were added to 96-well U-bottom plate (BD Biosciences). Sequence-optimized CD123 X CD3 bi-specific y (DART-A) was diluted to 10, 1 ng/mL and 50 ML of each dilution was added to each well (ﬁnal volume = 200 uL). A separate assay plate was set up for each time point (48, 72, 120 and 144 hours) and plates were incubated at 37 0C in a 5% C02 incubator. At each time point, cells were d with CD4, CD8, CD25, CD45, CD33, and CD123 antibodies. Labeled cells were analyzed in FACS Calibur ﬂow cytometer ed with CellQuest Pro acquisition software, Version 5.2.1 (BD Biosciences). Data analysis was performed using Flowjo v9.3.3 software (Treestar, Inc).T cell expansion was measured by gating on CD4+ and CD8+ populations and activation was determined by measuring CD25 MFI on the CD4+ and CD8+ -gated populations.

Leukemic blast cell population was fied by CD45+CD33+ gating.

Autologous Tumor Cell Depletion, T Cell Expansion And Activation By Sequence-Optimized CD123 x CD3 Bi-Speciﬁc Diabody (DART-A) In Primary Specimens From ALL Patients To determine the sequence-optimized CD123 X CD3 bi-speciflc diabody (DART-A) mediated activity in ALL patient primary patient s, patient s were incubated with 1 ng/mL of DART-A and percentages of leukemic blast cells and T cells were measured at different time points following the treatment. Leukemic blast cells were identiﬁed by CD45+/CD33+ gating. Incubation of primary ALL bone marrow s with DART-A resulted in depletion of the leukemic cell population over time ed to untreated control or Control DART (Figure 13, Panel H versus Panels F and G). When the T cells were counted (CD8 and CD4 staining) and activation (CD25 staining) were assayed, the T cells expanded and were activated in the DART-A sample e 14, Panels I and L, respectively) compared to untreated or Control DART samples (Figure 14, Panels H, G, K and J, respectively).

Example 11 CD123 Surface Expression On Leukemic Blast Cells And Stem Cells In Primary Tissue Sample From AML Patient 2 To deﬁne the CD123 expression pattern in AML patient 2 primary s, cryopreserved primary AML patient bone marrow and PBMC samples were evaluated for CD123 e expression on leukemic blast cells.

CD123 Expression in Leukemic Blast Cells in Bone Marrow Mononucleocytes (BMNC) A total of 0.5x106 bone marrow mononucleocytes (BM MNC) and peripheral blood mononucleocytes (PBMC)) from an AML patient 2 were evaluated for leukemic blast cell identiﬁcation. Leukemic blast cells were identiﬁed using the d markers CD33 and CD45. As shown in Figure 15, Panel B, 94% of the cells from AML bone marrow are ic blast cells. The T cell population was identiﬁed by CD3 expression. As shown in Figure 15, Panel C, approximately 15% of the cell from the AML bone marrow and PBMC sample are T cells. e 12 Autologous CTL Killing Assay Using AML Patient 2 y Specimens eserved primary AML specimen (bone marrow mononucleocytes (BM MNC) and peripheral blood mononucleocytes (PBMC)) from AML patient 2 were thawed in RPMI 1640 with 10% FBS and allowed to recover overnight at 37°C in 5% C02. Cells were washed with assay medium (RPMI 1640+10%FBS) and viable cell count was determined by Trypan Blue exclusion. 150,000 cells / well in 150 uL assay medium were added to 96-well U-bottom plate (BD Biosciences).

Sequence-optimized CD123 X CD3 bi-speciﬁc diabody (DART-A) and control bi- c diabody (Control DART) were diluted to 0.1, and 0.01 ng/mL and 50 uL of each dilution was added to each well (ﬁnal volume = 200 uL). A separate assay plate was set up for each time point (48, 72, 120 and 144 hours) and plates were incubated at 37 0C in a 5% C02 tor. At each time point, cells were stained with CD4, CD8, CD25, CD45, CD33, and CD123 antibodies. Labeled cells were analyzed in FACS Calibur ﬂow cytometer equipped with CellQuest Pro acquisition software, Version 5.2.1 (BD Biosciences). Data analysis was med using Flowjo v9.3.3 re (Treestar, Inc). T cell expansion was ed by gating on CD4+ and CD8-- populations and activation was determined by ing CD25 MFI on the CD4-- and CD8+ -gated populations. Leukemic blast cell population was identified by CD45+CD33+ gating.

Autologous Tumor Cell Depletion, T Cell ion and Activation in Primary Specimens from AML Patient 2 To determine the sequence-optimized CD123 X CD3 bi-specific diabody (DART-A) mediated activity in AML patient primary patient 2 samples, patient samples were incubated with 0.1 or 0.01 ng/mL of DART-A and percentages of leukemic blast cells and T cells were measured at ent time points following the treatment. Incubation of primary AML bone marrow samples with DART-A resulted in depletion of the leukemic cell population over time (Figure 16, Panel A), accompanied by a concomitant expansion of the residual T cells (both CD4 and CD8) (Figure 16, Panel B and Figure 16, Panel C, respectively). To determine if the T cells were activated, cells were stained for CD25 or Ki-67, both markers of T cell activation. As shown in Figure 17, Panels A and B, incubation of primary AML bone marrow samples with DART-A resulted in T cell activation. These data ent the 144h time point.

Intracellular Staining for Granzyme B and Perforin To ine the intracellular levels of granzyme B and Perforin in T cells, CTL assay was setup. After approximately 18 h, cells from the assay plate were stained with anti-CD4 and anti-CD8 antibodies by incubating for 30 minutes at 4 °C. ing surface staining, cells were incubated in 100 ul Fixation and Permeabilization buffer for 20 min at 4 °C. Cells were washed with permeabilization/wash buffer and incubated in 50 ul of granzyme B and perforin antibody mixture prepared in 1X Perm/Wash buffer at 4 C for 30 s. Then cells were washed with 250 ul Perm/Wash buffer and resuspended in Perm/Wash buffer for FACS acquisition.

Upregulation Of Granzyme B And Perforin By Sequence-Optimized CD123 x CD3 Bi-Speciﬁc Diabody (DART-A) In T Cells During Redirected Killing.

To investigate the possible mechanism for sequence-optimized CD123 X CD3 bi-speciﬁc diabody A) mediated cytotoxicity by T cells, intracellular granzyme B and perforin levels were measured in T cells after the redirected killing.

There was no upregulation of granzyme B and in when T cells were incubated with control bi-speciﬁc diabody (Control DART). Upregulation of granzyme B and perforin levels in both CD8 and CD4 T cells was observed with sequence-optimized CD123 X CD3 bi-speciﬁc diabody (DART-A) (Figure 17, Panels C and D).

Interestingly, the upregulation was almost two-fold higher in CD8 T cells ed to CD4 T cells (Figure 17, Panel C and Figure 17, Panel D). These data indicate that DART-A-mediated target cell killing was mediated through granzyme B and perforin pathway.

Example 13 ce-Optimized CD123 x CD3 Bi-Speciﬁc Diabody Cross-Reacts With Non- Human Primate CD123 And CD3 ns.

In order to quantitate the extent of binding between sequence-optimized CD123 X CD3 bi-speciﬁc diabody (DART-A) and human or cynomolgus monkey CD3, BIACORETM analyses were conducted. BIACORETM analyses measure the dissociation te, kd. The binding afﬁnity (KD) n an dy and its target is a function of the kinetic constants for association (on rate, ka) and dissociation (off-rate, kd) ing to the formula: KD = [kd]/[ka]. The BIACORETM analysis uses surface plasmon resonance to directly measure these kinetic parameters.

Recombinant human or cynomolgus monkey CD3 was directly immobilized to a support. d human or cynomolgus monkey CD123 was captured and immolbilized to a support. The time course of dissociation was measured and a bivalent ﬁt of the data conducted. Binding constants and afﬁnity were obtained using a 1:1 binding ﬁt. The results of the BIACORETM analyses ing binding to human versus cynomologus monkey CD123 and CD3 proteins are shown in Figure 18. Binding afﬁnities to the cynomolgus monkey CD123 (Figure 18D) and CD3 (Figure 18B) proteins is able to binding ies for human CD123 (Figure 18C) and CD3 (Figure 18A) proteins.

Example 14 Autologus Monocyte Depletion In Vitro With Human And lgus Monkey PBMCs PBMCs from human or cynomolgus monkey whole blood samples were added to U-bottom plates at cell density of 200,000 cells/well in 150 uL of assay medium. Dilutions of sequence-optimized CD123 X CD3 bi-speciﬁc diabodies (DART-A or DART-A w/ABD) were prepared in assay medium. 50 [LL of each DART-A or DART-A w/ABD dilution was added to the plate containing PBMCs in duplicate wells. The plates were incubated for ~ 18 - 24 h at 37°C. Supematants were used to determine the cytotoxicity as described above. As shown in Figure 19 (Panels A and B), depletion of pDCs cells was observed in both human (Figure 19, Panel A) and cynomolgus monkey PBMCs (Figure 19, Panel B). These s indicate that circulating pDC can be used as a pharmacodynamic marker for preclinical toxicology studies in cynomolgus monkeys.

Should it be necessary to replicate this example it will be appreciated that one of skill in the art may, within reasonable and able limits, vary the above- bed protocol in a manner appropriate for replicating the described results. Thus, the exempliﬁed protocol is not intended to be adhered to in a precisely rigid manner. e 15 Plasmacytoid Dendritic Cell Depletion In Cynomolgus Monkeys Treated With Sequence-Optimized CD123 x CD3 Bi-Speciﬁc Diabody (DART-A) ] As part of a dose-range ﬁnding toxicology study, cynomolgus monkeys were stered sequence-optimized CD123 X CD3 bi-speciﬁc diabody (DART-A) as 4- day infusions at doses of 0.1, 1, 10, 30 100, 300, or 1000 ng/kg .

The Control DART was administered at 100 ng/kg. To identify pDCs and te populations in cynomolgus monkey PBMCs, cells were labeled with CD14-FITC antibody.

Monocytes were identiﬁed as the CD14+ tion and pDCs were identiﬁed as the CDl4'CD123+ population. As shown in Figure 20 Panels K and L, the pDCs were depleted as early as 4 days post infusion with as little as 10 ng/kg DART-A. No pDC depletion was seen in the control bi-specif1c diabody-(Control DART) treated s or the vehicle + r-treated monkeys at the 4d time point (Figure 20, Panels G, H, C and D, tively). Cytokine levels of interferon-gamma,TNF- alpha, 1L6, 1L5, 1L4 and IL2 were determined at 4 hours after inﬁlsion. There was little to no elevation in cytokine levels at the DART-A treated animals compared to Control DART or vehicle-treated animals.

Figure 21 and Figure 22 provide the results of the FACS analysis for B cells (CD20+) (Figure 21, Panel A), monocytes (CD14+) (Figure 21, Panel B), NK cells +CD16+) (Figure 21, Panel C), pDC (CD123HI, CD14) (Figure 21, Panel D), and T cells (total, CD4+, and CD8+) (Figure 22, Panel A, Figure 22, Panel B, and Figure 22, Panel D, respectively).

Treatment of monkeys with Control DART had no noticeable effects on T or B lymphocytes, NK cells, monocytes and pDCs. Treatment of monkeys with DART- A at doses of 10 ng/kg/d or higher resulted in the abrogation of pDCs (Figure 21, Panel D). The depletion of pDC was complete and durable, returning to pre-dose levels l weeks after completion of dosing. Circulating levels of T lymphocytes decreased upon DART-A administration, but returned to pre-dose level by the end of each weekly cycle, suggesting s in trafficking rather than true depletion. Both CD4 and CD8 T lymphocytes followed the same pattern. The T-lymphocyte activation , CD69 e 22, Panel C), was only marginally positive among circulating cells and did not track with DART-A dosing. B lymphocytes, monocytes and NK cells ted over the course of DART-A dosing with substantial variability ed among monkeys. A trend toward increased circulating levels of B lymphocytes and monocytes was ed in both monkeys at the highest doses.

In summary, the above results demonstrate the therapeutic efficacy of the sequence-optimized CD123 X CD3 bi-specif1c diabody (DART-A). The sequence- optimized CD123 X CD3 bi-specif1c diabody (DART-A) may be ed as a therapeutic agent for the treatment of multiple diseases and conditions, including: AML, ABL (ALL), CLL, MDS, pDCL, mantel cell leukemia, hairy cell leukemia, Ricter ormation of CLL, Blastic crisis of CML, BLL (subset are CD123+) (see Example 2); Autoimmune Lupus (SLE), allergy hils are CD123+), asthma, etc.

Example 16 Comparative Properties of Sequence-Optimized CD123 x CD3 Bi-Speciﬁc Diabody (DART-A) and Non-Sequence-Optimized CD123 xCD3 Bi-Speciﬁc Diabody (DART-B) Unexpected Advantage and Attributes of the Sequence-Optimized CD123 x CD3 Bi-Speciﬁc Diabodies As discussed above, DART-A and DART-B are similarly designed and the ﬁrst polypeptide of both constructs comprise, in the N—terminal to C-terminal direction, an N—terminus, a VL domain of a monoclonal antibody capable of binding to CD3 (VLCDg), an intervening linker peptide (Linker l), a VH domain of a monoclonal antibody capable of binding to CD123 (VHCDm), a Linker 2, an E-coil Domain, and a C-terminus. Likewise, the second polypeptide of both constructs se, in the N—terminal to inal direction, an N—terminus, a VL domain of a monoclonal antibody capable of binding to CD123 (VLCDm), an intervening linker peptide (Linker l), a VH domain of a monoclonal antibody e of binding to CD3 (VHCDg), a Linker 2, a K-coil Domain and a C-terminus.

As indicated in Example 1, both CD123 X CD3 bi-speciﬁc diabodies were found to be capable of simultaneously binding to CD3 and CD123. onally, as disclosed in e 3 and in Figure 4, Panels C and D, the two CD123 X CD3 bi- speciﬁc diabodies ted a potent redirected killing ability with concentrations required to achieve 50% of maximal activity (ECSOs) in sub-ng/mL range, regardless of CD3 epitope binding city (DART-A versus DART-B) in target cell lines with high CD123 expression. Thus, slight variations in the speciﬁc sequences of the CD123 X CD3 bi-speciﬁc diabodies do not completely te biological activity.

However, in all cell lines tested, DART-A was found to be more active and more potent at redirected killing than DART-B (see, e.g., Figure 4, Panels A, C, and D). Thus DART-A exhibited an unexpected age over similar DART-B.

Example 17 Non-Human Primate Pharmacology of DART-A for the Treatment of Hematological Malignancies The interleukin 3 (IL-3) receptor alpha chain, CD123, is overexpressed on malignant cells in a wide range of hematological malignancies (Munoz, L. et al. (2001) “Interleukin-3 Receptor Alpha Chain ) Is Widely Expressed In Hematologic Malignancies,” Haematologica 86:1261-1269; Testa, U. et al. (2014) “CD123 Is A Membrane Biomarker And A Therapeutic Target In Hematologic Malignancies,” Biomark. Res. 2:4) and is associated with poor prognosis (Vergez, F. et al. (2011) “High Levels 0fCD34+CD38low/—CDI23+ Blasts Are Predictive OfAn Adverse Outcome In Acute Myeloid ia: A Groupe Ouest-Est Des Leucemies Aigues Et Maladies Du Sang (GOELAMS) Study,” ologica 2-1798).

Moreover, CD123 has been reported to be expressed by leukemia stem cells (LSC) (Jordan, C.T. et al. (2000) “The Interleukin-3 Receptor Alpha Chain Is A Unique Marker For Human Acute Myelogenous ia Stem ” ia 14:1777- 1784; Jin, L. et al. (2009) lonal Antibody-Mediated Targeting 0fCD123, IL-3 Receptor Alpha Chain, Eliminates Human Acute Myeloid Leukemic Stem Cells,” Cell Stem Cell 5:31-42), which is an attractive feature that s targeting the root cause of such diseases. Consistent with this conclusion, CD123 also takes part in an lL-3 autocrine loop that sustains leukemogenesis, as shown by the ability of a CD123- blocking monoclonal antibody to reduce leukemic stem cell engraftment and improve survival in a mouse model of acute myelogenous leukemia (AML) (Jin, L. et al. (2009) “Monoclonal Antibody-Mediated Targeting 0f CD123, IL-3 Receptor Alpha Chain, Eliminates Human Acute Myeloid Leukemic Stem Cells,” Cell Stem Cell 5:31- 42). In a phase 1 study in high-risk AML ts, however, the monoclonal dy exhibited no anti-leukemic ty (Roberts, A. W. et al. (2010) “A Phase I Study Of Anti-CD123 Monoclonal Antibody (mAb) CSL360 Targeting Leukemia Stem Cells (LSC) In AML,” J. Clin. Oncol. 28(Suppl):e13012). Thus, alternate CD123-targeting ches, including depleting strategies are desired. Although CD123 is expressed by a subset of normal hematopoietic progenitor cells (HPC), hematopoietic stem cells (HSC) express little to no CD123 (Jordan, C.T. et al. (2000) “The Interleukin-3 Receptor Alpha Chain Is A Unique Marker For Human Acute Myelogenous Leukemia Stem Cells,” Leukemia 14:1777-1784; Jin, W. et al. (2009) “Regulation 0fThI7 Cell Diﬁ’erentiation And EAE Induction By MAP3K NI ,” Blood 113:6603-6610), indicating that CD123 epleting gies allow reconstitution via normal hematopoiesis. ] ng a patient’s own T lymphocytes to target leukemic cells represents a promising immunotherapeutic strategy for the treatment of hematological malignancies. The therapeutic potential of this approach has been attempted using blinatumomab (a bi-specific antibody-based BiTE having the ability to bond CD3 and the B cell CD19 antigen) in patients with B cell lymphomas and ursor acute lymphoblastic leukemia (Klinger, M. et al. (2012) opharmacologic Response 0fPatients With B-Lineage Acute Lymphoblastic Leukemia To Continuous Infusion Of T ngaging CD19/CD3-Bispeciﬁc BiTE Antibody Blinatumomab,” Blood 119:6226-6233; Topp, M.S. et al. (2012) “Long-Term Follow-Up 0f Hematologic Relapse-Free Survival In A Phase 2 Study OfBlinatumomab In Patients With MRD In B-Lineage ALL,” Blood 120:5185-5187; Topp, M.S. et al. (2011) “Targeted Therapy With The —Engaging Antibody Blinatumomab 0f Chemotherapy-Refractory Minimal Residual Disease In age Acute Lymphoblastic Leukemia Patients Results In High Response Rate And Prolonged Leukemia-Free Survival,” J. Clin.

Oncol. 29:2493-2498).

The CD123 X CD3 bi-specif1c diabody les of the t invention, such as DART-A, comprise an alternate bi-specific, antibody-based modality that offers improved stability and more robust manufacturability properties (Johnson, S. et al. (2010) “Eﬂector Cell Recruitment With Novel Fv-Based Dual-Aﬁinity Re- Targeting n Leads To Potent Tumor Cytolysis And In Vivo B-Cell Depletion,” J.

Mol. Biol. 399:436-449; Moore, RA. et al. (2011) “Application Of Dual Aﬁinity Retargeting Molecules To Achieve Optimal Redirected T—Cell g 0f B-Cell Lymphoma,” Blood 117:4542-4551).

In order to demonstrate the superiority and effectiveness of the CD123 X CD3 bi-specif1c diabody molecules of the present invention, the biological activity of the above-described DART-A in in vitro and preclinical models of leukemia was conﬁrmed, and its pharmacokinetics, pharmacodynamics and safety pharmacology in cynomolgus macaques (Macaca fascicularis) was assessed relative to either the above-described Control DART (bi-speciﬁc for CD3 and ﬂuorescein) or a “Control DART-2” that was bi-speciﬁc for CD123 and ﬂuorescein).

Amino Acid Sequence of First Polypeptide Chain of “Control DART-2” (CD123VL — Linker — 4-4420VH — Linker — E-coil; linkers are underlined) (SEQ ID NO:58): DFVMTQSPDS LAVSLGLRVT MSCKSSQSLL NSGNQKVYLT WYQQKPGQPP {LLT YWASTR LSGVPDRFSG SGSGTDFTTT SSTQAmDVA VYYCQWDYSY GT<L m {GGGSGGG GL'VKTDL'TGG RRMK LSCVASGFTF WV<Q LWVA Q_TRNKRYNY_E TYYSDSV<GR FTTSRDDSKS SVYLQMNWL< VﬁDMG YYCT GSYYG DYWG QGTSVTVSSG GCGGGLVAAL ﬁ<ﬁVAALﬁ<ﬁ VAALmeVAA LTK Amino Acid Sequence of Second Polypeptide Chain of ol DART-2” (4420VL — Linker— CD123VH — Linker — K—coil) (SEQ ID NO:59): DVVMTQTRFS LPVSLGDQAS T_SCRSSQSLV HSNGNTYLRW YLQKPGQSPK VLT_YKVSNRF SGVPDRFSGS GSGTDb'TTK LDTGV THVP WTFGGGT<Lm KGGGSGGGG LVQLVQSGA_E SVKV SC<ASGYTFT DYYMKWVRQA PGQGTL'W GD FY NQKh'KG{VT_ STAY mLSSLRSﬁD TAVYYCARSd LLRASWFAYW GQGTLVTVSS GGCGGGKVAA L<LKVAAL<L KVAAL<LKVA AL<L Bifunctional ELISA A MaXiSorp ELISA plate (Nunc) coated overnight with the soluble human or cynomolgus IL3R—alpha (0.5 ug/mL) in onate buffer was blocked with 0.5% BSA; 0.1% Tween-20 in PBS (PBST/BSA) for 30 minutes at room temperature.

DART-A molecules were applied, followed by the sequential addition of human CD388—biotin and Streptavidin HRP (Jackson ImmunoResearch). HRP activity was detected by conversion of tetramethylbenzidine (BioFX) as ate for 5 min; the reaction was terminated with 40uL/well of 1% H2S04 and the absorbance read at 450 Surface Plasmon Resonance Analysis The ability of DART-A to bind to human and cynomolgus monkey CD3 or CD123 proteins was analyzed using a BIAcore 3000 biosensor (GE, Healthcare) as described by Johnson, S. et al. (2010) (“Eﬁ’ector Cell tment With Novel Fv- Based Dual-Aﬁinity Re-Targeting Protein Leads T0 Patent Tumor Cytolysis And In Vivo B-Cell Depletion,” J. Mol. Biol. 399:436-449) and Moore, RA. et al. (2011) (“Application OfDual Aﬁinity Retargeting Molecules To Achieve Optimal Redirected T-Cell Killing OfB-Cell Lymphoma,” Blood 117:4542-4551). Brieﬂy, the carboxyl groups on the CM5 sensor chip were activated with an injection of 0.2M N-ethyl-N- (3dietylamino-propyl) carbodiimide and 0.05M N—hydroxy-succinimide. Soluble CD3 or CD123 (1 ug/ml) was injected over the activated CM5 surface in 10mM sodium-acetate, pH 5.0, at ﬂow rate 5 , ed by 1 M ethanolamine for deactivation. g experiments were performed in 10mM HEPES, pH 7.4, 150mM NaCl, 3mM EDTA and 0.005% P20 surfactant. Regeneration of the immobilized receptor surfaces was performed by pulse injection of 10mM glycine, pH 1.5. KD values were ined by a global fit of binding curves to the Langmuir 1:1 binding model (BIAevaluation software v4.1).

Cell Killing Assay Cell lines used for cell g assays were obtained from the American Type Culture tion (ATCC) (Manassas, VA). PBMCs were isolated from healthy donor blood using the Ficoll-Paque Plus kit (GE Healthcare); T cells were puriﬁed with a negative selection kit (Life Technologies). CD123 cell-surface density was determined using Quantum Simply Cellular beads (Bangs Laboratories, Inc., Fishers, IN). Cytotoxicity assays were performed as described by Moore, PA. et al. (2011) (“Application OfDual y eting Molecules To Achieve l Redirected T-Cell Killing 0f B-Cell Lymphoma,” Blood 42-4551). Brieﬂy, target cell lines (105 cells/mL) were treated with serial dilutions of DART-A or Control DART proteins in the ce of T cells at the indicated effector cells:target cells ratios and incubated at 37°C overnight. Cell killing was determined as the release of lactate dehydrogenase (LDH, Promega) in culture supernatant. For ﬂow-based killing, target cells were labeled with CMTMR (Life Technologies) and cell killing was monitored using a FACSCalibur ﬂow cytometer. Data were analyzed by using PRISM® 5 software (GraphPad) and presented as percent cytotoxicity.

Cynomolgus Monkey Pharmacology Non-human e ments were performed at Charles River Laboratories (Reno, NV), according to the guidelines of the local Institutional Animal Care and Use Committee (IACUC). Purpose-bred, na'ive cynomolgus monkeys (Macaca fascicularz’s) of e origin (age range 2.5-9 years, weight range of 2.7-5 kg) were provided with vehicle or DART-A via intravenous inﬁlsion through femoral and jugular ports using battery-powered programmable infusion pumps (CADD- ®, SIMS Deltec, Inc., St. Paul, MN). Peripheral blood or bone marrow samples were collected in agulant containing tubes at the indicated time points.

Cell-surface phenotype analyses were performed with an LSR sa analyzer (BD ences) equipped with 488nm, 640nm and 405nm lasers and the following antibodies: CD4-V450, CD8-V450, CD123-PE-Cy7, CD45-PerCP, C-H7, CD8-FITC, CD25-PE-Cy7, CD69-PerCP, PD-l-PE, TIM3-APC, CD3-Pacif1c Blue, CD95-APC, CD28-BV421, CDl6-FITC, CD3-Alexa488, CD38-PE, CD123-PE-Cy7, CDll7-PerCP-Cy5.5, CD34-APC, CD90-BV421, CD45RA-APC -H7 and CD33- APC (BD Biosciences). The absolute number of cells was ined using TruCOUNT (BD Biosciences). Serum levels of IL-2, IL-4, IL-5, IL-6, TNF-u, and IFN—y nes were measured with the Non-Human Primate Thl/Th2 Cytokine Cytometric Bead Array Kit (BD Bioscience). The concentration of DART-A in monkey serum samples was measured using a sandwich immunoassay with electrochemiluminescence detection (MesoScale Diagnostics, MSD, Rockville, MD).

Brieﬂy, the assay plate (MSD) was coated with recombinant human IL-3 Ra (R&D System) and blocked with 5 % BSA. Calibration standards or diluted test samples were applied, followed by the addition of a biotinylated monoclonal dy exhibiting specific binding for the above-described E-coil (SEQ ID NO:34) and K- coil (SEQ ID NO:35) domains of the molecule A SULFO-TAGTM labeled streptavidin conjugate (MSD) was added and the formation of complexes was analyzed in an MSD SECTOR® imager. DART-A concentrations were determined from standard curves generated by ﬁtting light intensity data in a ﬁve-parameter logistic model.

Physicochemical characterization of the puriﬁed DART-A trated a homogeneous heterodimer with a molecular mass of 58.9 kDa (Figure 23; Figures 24A-24B), which was stable at 2—8°C for up to 12 months in PBS. SPR analysis demonstrated nearly identical binding afﬁnities of DART-A to the corresponding soluble human and cynomolgus monkey CD3 and CD123 antigens (Figures 25A-25D and Table 7). Furthermore, DART-A simultaneously bound both ns in an ELISA format that ed human or monkey CD123 for capture and human CD3 for detection (Figures 26A-26B), and demonstrated similar binding to human and monkey T lymphocytes (Figures 26C-26E). The data in Table 7 are averages of 3 independent experiments each performed in duplicates.

Table 7 Equilibrium Dissociation nts (KD) for the Binding of DART-A to Human and C nomol_us Monke CD3 and CD123 ka (i SD) k d (:|: SD) K1) (1; SD) Antigens -1 -1 -1 (M S ) (s ) Human CD3s/5 5.7 :0.6 x105 5.0 :09 x10"3 CynomolgusCD38/5 . :05 x105 5.0 :09 x10"3 Human CD123-His 1.6 :04 x106 1.9 :04 x10"4 CynomolgusCD123-His 1.5 :03 x106 4.0 :07 x10"4 DART-A Mediates Redirected Killing by Human 0r Cynomolgus Monkey T Lymphocytes DART-A mediated redirected target cell killing by human or monkey effector cells t CD123+ Kasumi-3 ic cell lines (Figure 27A-27D), which was accompanied by induction of activation markers. No activity was observed against CD123-negative s (U937 cells) or with Control DART, indicating that T cell activation is strictly dependent upon target cell engagement and that monovalent ment of CD3 by DART-A was insufﬁcient to trigger T cell activation. Since CD123 is expressed by subsets of normal circulating leukocytes, including pDCs and monocytes (Figure 27E), the effect of DART-A were ﬁlrther investigated in normal human and monkey’s PBMCs.

A graded effect was observed among human PBMC, with a ependent rapid depletion of CD14'CD123high cells (pDC and basophils) observed as early as 3 hours ing initiation of treatment, while monocytes (CD14+ cells) remained unaffected at this time point (Figures 27F-27G). CD14'CD123high cells depletion increased over time across all DART-A molecule concentrations, while monocytes were slightly decreased by 6 hours and depleted only after 18 hours and at the trations higher than 1 ng/mL. Incubation of monkey PBMCs with DART-A resulted in a comparable dose-dependent depletion of D123high cells (Figure 27H), further supporting the relevance of this species for DART-A pharmacology (CDl4+ monkey cells express little to no CD123 and were not depleted). cokinetics of DART-A in Cynomolgus Monkeys The cynomolgus monkey was selected as an appropriate cological model for DART-A analysis based on the lent distribution of both target antigens in this species compared to humans based on immunohistochemistry with the precursor mAbs, consistent with published information (Munoz, L. et al. (2001) “Interleukin-3 Receptor Alpha Chain (CD123) Is Widely Expressed In Hematologic Malignancies,” Haematologica 86:1261-1269; Korpelainen, E.I. et al. (1996) “IL-3 Receptor Expression, Regulation And Function In Cells Of The Vasculatare,” Immunol. Cell Biol. 74:1-7).

The study conducted in accordance with the present invention included 6 treatment groups consisting of 8 cynomolgus monkeys per group (4 males, 4 females) (Table 8). All groups received vehicle control for the first infusion; then vehicle or DART-A were administered intravenously for 4 weekly . Group 1 animals received e control for all 4 subsequent infusions, whereas Groups 2-5 received weekly escalating doses of DART-A for 4 days a week for all subsequent infusions.

Group 6 animals were treated with 7-day uninterrupted weekly escalating doses of DART-A for all infusions. The 4-day-on/3-day-off and 7-day-on schedules were designed to guish between durable from transient effects associated with DART- A stration. Two males and 2 females per group were sacriﬁced at the end of the treatment phase (Day 36), while the remaining monkeys were necropsied after a 4- week recovery (Day 65). A subset of monkeys developed anti-drug antibodies (ADA) directed t the humanized Fv of both CD3 and CD123 and the data points following the appearance of ADA were excluded from the PK analysis. All s were exposed to DART-A during the study period.

DART-A Infusion (4-day-on/3-day-ofi) (7-day-on) Vehicle on ng/kg/day ng/kg/day N0. [n__/k/7da s] Group Group Vehicle Vehicle Vehicle Vehicle Vehicle e Vehicle

[400] [400] [400] [700] 300 300 Vehicle [1]200 [1]200 [1200] [2100] Vehicle

[13200]00 [2400] [4200] -____-ehicle [1200196400100300 00 1000 1000

[7000] 36-65 [_4000] A two-compartment model was used to estimate PK parameters (Table 9 and Figure 28). T1001 was short (4-5min), reﬂecting rapid binding to circulating targets; T1013 was also rapid, as expected for a molecule of this size, which is subject to renal clearance. Analysis of serum samples collected at the end of each infusion from group 6 monkeys showed a dose-dependent increase in DART-A Cmax. In Table 9, Vehicle was PBS, pH 6.0, containing 0.1 mg/mL recombinant human albumin, 0.1 mg/mL PS- 80, and 0.24 % benzyl alcohol was used for all vehicle infusions during the ﬁrst 4 days of each inﬁJsion week followed the same ation without benzyl alcohol for the remaining 3 days of each weekly on. DART-A was administered for the indicated times as a continuous IV inﬁlsion of a solution of PBS, pH 6.0, containing 0.1 mg/mL recombinant human albumin, 0.1 mg/mL PS-80, and 0.24 % benzyl alcohol at the ed concentration.

Table 9 Two-Compartment Analysis of PK Parameters of DART-A in C s Monke s . 300 ng/kg/d 600 ng/kg/d Cmax (pg/mL) 77.4 9.4 113.8 __ 33.5 AUC (11* . _/mL) 7465 913 11188 3282 V (L/kg) 1.078 0.511 2.098 1.846 tm, alpha (h) 0.07 0.018 0.067 0.023 t1/2, beta h 13.79 4.928 21.828 18.779 MRT (h) 6.73 3.327 9.604 8.891 Cytokine Release in -Treated Cynomolgus Monkeys Given the T cell activation properties of DART-A, an increase in circulating cytokines accompanying the inﬁasion was anticipated and a low starting dose was therefore used as a “desensitization” strategy, based on previous experience with r compounds (see, e.g., Topp, M.S. et al. (2011) “Targeted Therapy With The T- Cell—Engaging Antibody Blinatumomab 0f Chemotherapy-Refractory Minimal Residual Disease In B-Lineage Acute Lymphoblastic Leukemia Patients s In High Response Rate And Prolonged Leukemia-Free Survival,” J. Clin. Oncol. 29:2493-2498; Bargou, R. et al. (2008) “Tumor Regression In Cancer Patients By Very Low Doses OfA T Cell-Engaging Antibody,” e 321:974-977). Of the cytokine tested, lL-6 demonstrated the t changes upon infusion, albeit transient in nature, of minimal magnitude and with large inter-animal and inter-group variations (Figures 29A-29C). Small, transient increases in lL-6 were also observed after vehicle infusions (all Group 1 and all Day 1 infusions), indicating a sensitivity of this cytokine to manipulative stress. Nonetheless, DART-A-dependent increases (<80pg/mL) in serum lL-6 were seen in some monkeys following the ﬁrst DART-A inﬁlsion (lOOng/kg/day), which returned to baseline by 72 hours. Interestingly, the magnitude of lL-6 release decreased with each sive DART-A infusion, even when the dose level was sed to up to 1000 day. Minimal and transient DART-A-related increases in serum TNF-u (<lOpg/mL) were also observed; as with lL-6, the largest magnitude in TNF-u release was observed following the ﬁrst inﬁasion. There were no DART-A-related s in the levels of lL-S, lL-4, lL-2, or lFN—y hout the study when compared with controls. In conclusion, cytokine e in response to treatment of monkeys with DART-A was minimal, transient and represented a ﬁrst-dose effect manageable via intra-subject dose escalation.

DART-A-Mediated Depletion of Circulating CD14'/CD123Jr Leukocytes in vivo The circulating absolute levels of CDl4-/CD123+ cells were measured throughout the study as a pharmacodynamic endpoint. While the number of CD123+ cells in control Group I remained stable over time, DART-A treatment was associated with extensive ion of circulating CD123+ cells (94-lOO% from dy baseline) observable from the first time point measured (72 hours) following the start of the first DART-A inﬁlsion (100 ng/kg/day) in all animals across all active treatment groups (Figures 30A-30C). The depletion was durable, as it persisted during the 3-day weekly dosing holiday in Group 2-5, returning to baseline levels only during the prolonged recovery period. To eliminate the possibility of DART-A masking or modulating CD123 (an unlikely scenario, given the low circulating DART-A levels), pDCs were enumerated by the orthogonal marker, CD303.

Consistent with the CD123 data, CD303+ pDC were similarly depleted in monkeys treated with DART-A (Figures 30D-30F).

Circulating T-Lymphocyte Levels, Activation and Subset Analysis In contrast to the persistent depletion of ating CD123+ cells, DART-A administered on the 4-day-on/3-day-off schedule s 2-5) were associated with weekly ﬂuctuations in circulating T cells, while administration as uous 7-day inﬁlsions resulted in similarly decreased ating T cell levels ing the first administration that slowly recovered without ﬂuctuation even during the dosing period (Figures 31A-31C). The difference between the two dosing strategies indicates that the effect of DART-A on T cytes is consistent with trafficking and/or margination, rather than depletion. Following ion of dosing, T cells rebounded to levels approximately 2-fold higher than baseline for the duration of the recovery period. Infusion of DART-A was associated with an exposure-dependent, progressive increased frequency of T cells expressing the late activation marker, PD- I, ularly in CD4+ cells, with dose Group 6 displaying the highest overall levels (Figures 1 and Figures 32A-32F and Figures 33A-33F). Tim-3, a marker associated with T cell exhaustion, was not detected on CD4+ T cells and only at low frequency among CD8+ cells (5.5-9.7%) and comprising 20.5-35.5% of the CD8+/PD-l+ double-positive cells. There was no consistent change in the early T cell activation marker, CD69, and only modest variations in CD25 expression among ating cells.

To rule out exhaustion after in viva exposure, the ex vivo cytotoxic potential of effector cells isolated from cynomolgus monkeys receiving multiple infusions of DART-A was compared to that of cells from na'ive monkeys. As shown in Figure 34, PBMC isolated from DART-A-treated monkeys show cytotoxicity comparable to that of cells isolated from na'ive monkeys, indicating that in viva exposure to DART-A does not vely impact the y of T cells to kill target cells.

DART-A exposure increased the relative frequency of central memory CD4 cells and effector memory CD8+ cells at the expense of the corresponding naive T cell tion (Figures 35A-35F and Figures 32A-32F and Figures 33A-33F), indicating that DART-A exposure promoted expansion and/or mobilization of these cells.

Effects on Hematopoiesis and Bone Marrow Precursors DART-A was well tolerated in monkeys at all doses tested; however, reversible reductions in red cell ters were observed at the highest doses (Figures 36A-36C). Frequent blood sampling could have been a potential contributing factor, since e-treated animals showed a modest e in red cell mass. Reticulocyte response was observed in all animals; at the t exposure (Group 6), however, the response ed slightly less robust for r decrease in red cell mass (Figures 36D-36F). Morphological analysis of bone marrow smears throughout the study was unremarkable. Flow cytometry analysis, however, revealed that the frequency of CD123+ cells within the immature lineage-negative (Lin-) bone marrow populations decreased in DART-A-treated animals at the end of the dosing period, returning to baseline levels by the end of the recovery time (Figure 37A-37B).

HSC (defined as Lin-/CD34+/CD38-/CD45RA-/CD90+ cells (Pang, W.W. et al. (2011) “Human Bone Marrow Hematopoietic Stem Cells Are Increased In Frequency And Myeloz'd—Bz'ased With Age,” Proc. Natl. Acad. Sci. (U.S.A.) lO8:20012-20017)) showed large inter-group variability; Group 4-6 DART-A-treated monkeys show some apparent reduction compared to the ponding pre-dose , however, no decrease was seen in all treated groups compared to vehicle-treated animals. These data indicate that HSC are less susceptible to targeting by DART-A and are tent with the observed reversibility of the negative effects of DART-A treatment on hematopoiesis.

As demonstrated above, with respect to infusions for 4 weeks on a 4-day- on/3-day-off weekly schedule or a 7-day-on schedule at starting doses of lOOng/kg/day that were escalated stepwise weekly to 300, 600, and l,000ng/kg/day, the administration of DART-A to cynomolgus monkeys was well ted. Depletion of circulating CD123+ cells, including pDCs, was observed after the start of the ﬁrst administration and ted throughout the study at all doses and schedules.

Reversible ion in bone marrow CD123+ precursor was also ed. Cytokine release, as signiﬁcant safety concern with CD3-targeted ies, appeared manageable and consistent with a first-dose effect. Modest reversible anemia was noted at the highest doses, but no other (on- or rget) adverse events were noted.

The cynomolgus monkey is an appropriate animal model for the pharmacological assessment of DART-A, given the high homology between the orthologs and the y of DART-A to bind with similar affinity to the antigens and mediate redirected T cell killing in both s. Furthermore, both antigens are concordantly expressed in monkeys and humans, including similar expression by hematopoietic precursors and in the cytoplasm of the endothelium of multiple tissues.

Minor exceptions are the expression in testicular Leydig cells in humans but not monkeys and low-to-absent CD123 in monkey monocytes compared to humans.

A primary concern associated with therapeutic strategies that involve T cell activation includes the release of cytokines and off-target cytotoxic effects. A recent study with a 123 bi-specif1c scFv immunofusion construct with bivalent CD3 recognition demonstrated anti-leukemic activity in vitro, but caused eciﬁc activation of T cells and IFN—y secretion (Kuo, S.R. et al. (2012) “Engineering A CD123xCD3 Bispeciﬁc scFv Immunofusion For The Treatment Of Leukemia And Elimination 0f Leukemia Stem Cells,” Protein Eng. Des. Sel. 25:561-569). The monovalent nature of each binding arms and the highly homogeneous monomeric form of DART-A ensure that T cell activation depends exclusively upon target cell engagement: no T cell activation was observed in the absence of target cells or by using a Control DART molecule that included only the rgeting arm. rmore, high doses (up to 100ug/kg/day) of the l DART molecule did not trigger cytokine e in cynomolgus monkeys.

The DART-A molecule starting dose of 100ng/kg/day was well tolerated, with minimal cytokine release. Cytokine storm, however, did occur with a high starting dose (Sug/kg/day); however, such dose could be reached safely via stepwise weekly dose escalations, indicating that DART-A-mediated cytokine release appears to be primarily a first-dose effect. Depletion of the CD123+ target cells, thereby eliminating a source of CD3 ligation, may explain the dose effect: nearly complete CD123+ cell depletion was observed at doses as low as 3-10ng/kg/day, indicating that cytokine release in vivo follows a shifted dose-response compared to cytotoxicity. Dose-response profiles for cytotoxicity and ne release by human T cells were also consistent with this observation.

T cell itization, in which DART-A-induced PD1 upregulation may play a role, appears to also contribute to limit cytokine release after the ﬁrst inﬁlsion of DART-A. Recent studies show that sed PD-l expression after antigen- induced arrest of T cells at ation sites contributes, through interactions with PD-Ll, to terminating the stop signal, thus releasing and desensitizing the cells (Honda, T. et al. (2014) “Tuning 0fAntigen Sensitivity By T Cell Receptor-Dependent Negative Feedback Controls T Cell Eﬂector Function In Inflamed Tissues,” ty 40:235-247; Wei, F. et al. (2013) “Strength OfPD-I Signaling Diﬁ’erentially Aﬂects T-Cell Eﬁ’ector Functions,” Proc. Natl. Acad. Sci. (USA) 110:E2480-E2489). The PD-l countering of TCR signaling th is not m: while proliferation and cytokine production appear most sensitive to PD-l inhibition, cytotoxicity is the least affected (Wei, F. et al. (2013) “Strength OfPD-I Signaling Diﬁ’erentially Aﬂects T- Cell Eﬁ’ector Functions,” Proc. Natl. Acad. Sci. (U.S.A.) 110:E2480-E2489).

Consistently, the ex vivo cytotoxic potential of T cells from monkeys exposed to multiple infusions of DART-A was able to that of T cells from na'ive monkeys, e increased PD-l levels in the former. Furthermore, PD-l upregulation was not accompanied by TIM3 expression, a hallmark of T cell exhaustion, as shown for T cells exposed to protracted stimulation with CD3 antibodies or chronic infections (Gebel, H.M. et al. (1989) “T Cells From ts Successfully Treated With 0KT3 Do Not React With The T—Cell Receptor Antibody,” Hum. Immunol. 26:123-129; Wherry, E]. (2011) “T Cell Exhaustion,” Nat. Immunol. 12:492-499).

The depletion of circulating CD123+ cells in DART-A-treated s was rapid and persisted during the weekly dosing holidays in the 4-day-on/3-day-off le, consistent with target cell elimination. In contrast, the transient ﬂuctuations in circulating T cells were likely the result of trafﬁcking from/to tissues and lymphoid organs as a function of DART-A. DART-A re promotes the expansion and/or mobilization of antigen experienced T lymphocytes, cells that entially home to tissues and more readily exert cytotoxic or function (Mirenda, V. et al. (2007) “Physiologic And Aberrant Regulation Of Memory T-Cell Traﬁicking By The Costimulatory Molecule CD28,” Blood 109:2968-2977; Marelli-Berg, F.M. et al. (2010) “Memory T-Cell Traﬁicking.‘ New Directions For Busy Commuters,” Immunology 130:158-165).

Depletion of CD123+ normal cells may carry potential liabilities. pDCs and basophils express high levels of CD123, compared to lower levels in monocytes and eosinophils , A.F. et al. (1989) “Reciprocal Inhibition 0f Binding Between Interleukin 3 And ocyte-Macrophage Colony-Stimulating Factor To Human Eosinophils,” Proc. Natl. Acad. Sci. (U.S.A.) 86:7022-7026; Munoz, L. et al. (2001) “Interleukin-3 Receptor Alpha Chain (CD123) Is Widely Expressed In Hematologic Malignancies,” Haematologica 1-1269; , B.J. et al. (2006) “Characterization OfMyeloid And Plasmacytoid Dendritic Cells In Human Lung,” J.

Immunol. 177:7784-7793; Korpelainen, E.I. et al. (1995) “Interferon-Gamma Upregulates Interleukin-3 (IL-3) Receptor Expression In Human Endothelial Cells And Synergizes With IL-3 In Stimulating Major Histocompatibility x Class II Expression And Cytokine Production,” Blood 86:176-182). pDCs have been shown to play a role in the control of certain viruses in mouse or monkey models of infection, although they do not appear critical for controlling the immune se to ﬂu (Colonna, M. et al. (1997) “Speciﬁcity And Function 0f Immunoglobulin Superfamily NK Cell Inhibitory And Stimulatory Receptors,” Immunol. Rev. 155:127- 133; Smit, J.J. et al. (2006) “Plasmacytoid Dendritic Cells Inhibit Pulmonary Immunopathology And Promote Clearance 0f Respiratory Syncytial ” J. Exp.

Med. 203:1153-1159). In tumor models, pDCs may promote tumor growth and metastasis, while pDC depletion resulted in tumor inhibition (Sawant, A. et al. (2012) “Depletion 0f Plasmacytoid Dendritic Cells Inhibits Tumor Growth And Prevents Bone asis 0f Breast Cancer Cells,” J. Immunol. 189:4258-4265). Transient, , dose-independent facial swelling was observed in some monkeys treated with DART-A; however, no increased histamine levels were observed in these monkeys or when human basophils were lysed via DART-A-mediated T cell killing. Monocyte depletion may carry increased risks of infection; the consequence of pDC, basophil or eosinophils depletion in humans should thus be monitored.

Committed hematopoietic precursors that express CD123, such as the common myeloid precursor (CMP) n, C.T. et al. (2000) “The Interleukin-3 Receptor Alpha Chain Is A Unique Marker For Human Acute Myelogenous Leukemia Stem Cells,” ia 14:1777-1784; Rieger, MA. et al. (2012) “Hematopoiesis,” Cold Spring Harb. Perspect. Biol. 4:a008250), may be ed by DART-A, a possible explanation for the modest anemia observed following administration of DART-A at the highest dose. The erythropoietic reticulocyte response appeared to function at all DART-A dose levels; however, for commensurate drops in red cell ters, animals subjected to the greatest DART-A exposure (Group 6, 7-day-on inﬁJsion) showed a reduced reticulocyte response, suggesting a possible cytotoxic activity on sors (e.g., CMP). The effect was reversible following cessation of DART-A treatment, consistent with repopulation from spared CD123low/negative HSC.

Alternate approaches for depletion of CD123+ cells e a second- generation CD123-specif1c Fc-enhanced monoclonal antibody (Jin, L. et al. (2009) “Monoclonal dy-Mediated Targeting 0f CD123, IL-3 Receptor Alpha Chain, Eliminates Human Acute Myeloid Leukemic Stem Cells,” Cell Stem Cell 5:31-42; Roberts, A. W. et al. (2010) “A Phase I Study 0fAnti-CDI23 Monoclonal Antibody (mAb) CSL360 ing Leukemia Stem Cells (LSC) In AML,” J. Clin. Oncol. 28(Suppl):e13012), IL-3 bound diphtheria toxin el, A. et al. (2008) “Phase I Clinical Study Of Diphtheria Toxin-Interleukin 3 Fusion Protein In Patients With Acute Myeloid Leukemia And Myelodysplasia,” Leuk. Lymphoma 49:543-553), cytokine-induced killer (CIK) cells expressing CD123-speciﬁc ic antigen ors (CAR) (Tettamanti, S. et al. (2013) “Targeting 0fAcute Myeloid Leukaemia By ne-Induced Killer Cells Redirected With A Novel CD123-Speciﬁc Chimeric Antigen Receptor,” Br. J. Haematol. 9-401) and CD123 CAR T cells (Gill, S. et al. (2014) “Eﬁicacy Against Human Acute Myeloid Leukemia And Myeloablation 0f Normal Hematopoiesis In A Mouse Model Using Chimeric Antigen Receptor- Modiﬁed T Cells,” Blood 123(15): 2343-2354; Mardiros, A. et al. (2013) “T Cells Expressing CD123-Speciﬁc Chimeric Antigen Receptors Exhibit Speciﬁc Cytolytic Eﬂector Functions And mor Eﬂects Against Human Acute Myeloid ia,” Blood 122:3138-3148). CAR T cells exhibited potent leukemic blast cell killing in vitro and anti-leukemic activity in a xenogeneic model of disseminated AML (Mardiros, A. et al. (2013) “T Cells Expressing CD123-Speciﬁc Chimeric Antigen Receptors Exhibit Speciﬁc Cytolytic Eﬂector Functions And Antitumor Eﬂects Against Human Acute d Leukemia,” Blood 122:3138-3148). A recent study reported ablation of normal hematopoiesis in NSG mice engrafted with human CD34+ cells following CD123 CAR T cell transfer (Gill, S. et al. (2014) “Eﬁicacy Against Human Acute Myeloid Leukemia And Myeloablation 0fNormal Hematopoiesis In A Mouse Model Using ic Antigen Receptor-Modiﬁed T Cells,” Blood 123(15): 2343- 2354), although others have not observed similar effects in vitro or in vivo (Tettamanti, S. et al. (2013) “Targeting 0f Acute Myeloid Leukaemia By Cytokine- Induced Killer Cells cted With A Novel Speciﬁc Chimeric Antigen or,” Br. J. Haematol. 161:389-401; Pizzitola, I. et al. (2014) “Chimeric Antigen Receptors Against CD33/CDI23 Antigens Eﬁiciently Target Primary Acute Myeloid Leukemia Cells in vivo,” Leukemia doi:10.1038/leu.2014.62). In the above-discussed experiments, depletion of CD123+ bone marrow precursor populations was observed, but reversed during recovery; ﬁthhermore, depletion of this minority population resulted in no changes in bone marrow arity or erythroid to myeloid cell (E:M) ratio at all DART-A dose levels tested. These differences underscore the potential advantages of DART-A over cell therapies, as it provides a titratable system that relies on autologous T cells in contrast to “supercharged” ex vivo transduced cells that may be more difﬁcult to control. CD123 is overexpressed in l hematologic malignancies, including AML, hairy cell leukemia, blastic cytoid dendritic cell neoplasms ( BPDCNs), a subset of B-precursor acute blastic leukemia (B- ALL) and chronic cytic leukemia, Hodgkin’s disease Reed-Stemberg cells, as well as in myelodysplastic syndrome and systemic mastocytosis (Kharfan-Dabaja, MA. et al. (2013) “Diagnostic And Therapeutic Advances In Blastic Plasmacytoid Dendritic Cell Neoplasm: A Focus On Hematopoietic Cell lantation,” Biol.

Blood Marrow Transplant. 19:1006-1012; Florian, S. et al. (2006) “Detection 0f Molecular Targets On The Surface +/CD38-- Stem Cells In s d Malignancies,” Leuk. Lymphoma 47:207-222; Munoz, L. et al. (2001) “Interleukin-3 Receptor Alpha Chain ) Is Widely Expressed In Hematologic Malignancies,” Haematologica 86:1261-1269; Fromm, JR. (2011) “Flow Cytometric Analysis Of CD123 Is Useful For Immunophenotyping Classical Hodgkin Lymphoma,” Cytometry B Clin. Cytom. 80:91-99). The predictable pharmacodynamic activity and manageable safety profile ed in non-human primates r supports the clinical utility and efficacy of DART-A as therapy for these disorders.

In sum, DART-A is an antibody-based molecule engaging the CD38 subunit of the TCR to redirect T cytes against cells expressing CD123, an antigen up- regulated in several hematological malignancies. DART-A binds to both human and cynomolgus monkey’s antigens with similar afﬁnities and redirects T cells from both species to kill CD123+ cells. Monkeys inﬁJsed 4 or 7 days a week with weekly escalating doses of DART-A showed depletion of circulating CD123+ cells 72h after treatment initiation that persisted throughout the 4 weeks of treatment, irrespective of dosing schedules. A decrease in circulating T cells also occurred, but recovered to baseline before the subsequent infusion in monkeys on the 4-day dose schedule, consistent with DART-A-mediated mobilization. DART-A administration increased circulating PD1+, but not TIM-3+, T cells; ﬁarthermore, ex vivo analysis of T cells from treated monkeys exhibited unaltered redirected target cell lysis, indicating no exhaustion. Toxicity was d to a minimal transient release of cytokines following the DART-A ﬁrst infusion, but not after subsequent administrations even when the dose was escalated, and a minimal reversible decrease in red cell mass with concomitant reduction in CD123+ bone marrow progenitors. Clinical g of DART-A in hematological malignancies appears warranted.

All publications and patents mentioned in this speciﬁcation are herein incorporated by reference to the same extent as if each indiVidual publication or patent application was speciﬁcally and individually indicated to be incorporated by reference in its entirety. While the invention has been described in connection with speciﬁc embodiments thereof, it will be understood that it is e of ﬁarther modiﬁcations and this application is intended to cover any variations, uses, or tions of the invention ing, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary ce within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth.

What is d is: Claim 1. A sequence-optimized diabody capable of specific binding to an epitope of CD123 and to an epitope of CD3, wherein the diabody comprises a first polypeptide chain and a second polypeptide chain, covalently bonded to one another, n: A. the first polypeptide chain comprises, in the N-terminal to C-terminal direction: i. a Domain 1, comprising (1) a sub-Domain (1A), which comprises a VL Domain of a monoclonal antibody capable of binding to CD3 (VLCD3) (SEQ ID NO:21); and (2) a sub-Domain (1B), which comprises a VH Domain of a monoclonal antibody capable of binding to CD123 (VHCD123) (SEQ ID NO:26), wherein said sub-Domains 1A and 1B are separated from one another by a peptide linker (SEQ ID ; ii. a Domain 2, n said Domain 2 is an E-coil Domain (SEQ ID NO:34) or a K-coil Domain (SEQ ID , wherein said Domain 2 is separated from said Domain 1 by a peptide linker (SEQ ID NO:30); and B. the second polypeptide chain comprises, in the N-terminal to C-terminal direction: i. a Domain 1, comprising (1) a sub-Domain (1A), which comprises a VL Domain of a onal antibody capable of binding to CD123 (VLCD123) (SEQ ID NO:25); and (2) a sub-Domain (1B), which comprises a VH Domain of a monoclonal antibody capable of binding to CD3 (VHCD3) (SEQ ID NO:22), wherein said sub-Domains 1A and 1B are separated from one another by a peptide linker (SEQ ID NO:29); ii. a Domain 2, wherein said Domain 2 is a K-coil Domain (SEQ ID NO:35) or an E-coil Domain (SEQ ID NO:34), wherein said Domain 2 is separated from said Domain 1 by a e linker (SEQ ID NO:30); and wherein said Domain 2 of said first and said second polypeptide chains are not both E-coil Domains or both K-coil Domains; and n: (a) said VL Domain of said first polypeptide chain and said VH Domain of said second polypeptide chain form an Antigen Binding Domain capable of specifically g to an epitope of CD3; and (b) said VL Domain of said second polypeptide chain and said VH Domain of said first polypeptide chain form an Antigen Binding Domain capable of specifically binding to an epitope of CD123.

Claim 2. The diabody of claim 1, wherein said first polypeptide chain additionally ses an n-Binding Domain (SEQ ID NO:36) linked t o said Domain 2 via a peptide linker (SEQ ID NO:31).

Claim 3. The diabody of claim 1, wherein said second polypeptide chain additionally ses a Domain 3 comprising a CH2 and CH3 Domain of an immunoglobulin Fc Domain (SEQ ID NO:37), wherein said Domain 3 is linked to said Domain 1 via a peptide linker (SEQ ID NO:33).

Claim 4. The diabody of claim 1, wherein said first polypeptide chain additionally comprises a Domain 3 comprising a CH2 and CH3 Domain of an immunoglobulin Fc Domain (SEQ ID NO:37), wherein said Domain 3 is linked to said Domain 1 via a peptide linker (SEQ ID NO:33).

Claim 5. The diabody of claim 1, wherein said second polypeptide chain additionally comprises a Domain 3 sing a CH2 and CH3 Domain of an immunoglobulin Fc Domain (SEQ ID NO:37), wherein said Domain 3 is linked to said Domain 2 via a peptide linker (SEQ ID NO:32).

Claim 6. The diabody of claim 1, wherein said first polypeptide chain additionally comprises a Domain 3 comprising a CH2 and CH3 Domain of an immunoglobulin Fc Domain (SEQ ID NO:37), wherein said Domain 3 is linked to said Domain 2 via a e linker (SEQ ID NO:32).

Claim 7. The diabody of any one of claims 3 to 6, n said diabody further comprises a third polypeptide chain comprising a CH2 and CH3 Domain of an immunoglobulin Fc Domain (SEQ ID NO: 11).

Claim 8. The diabody of any one of claims 3 to 7, wherein said diabody further comprises a cysteine-containing peptide (SEQ ID NO:55) N -terminal to said CH2 and CH3 Domain of said immunoglobulin Fc Domain.

Claim 9. The diabody of any of claims 1 to 8, wherein said Domain 2 of said first polypeptide chain is a K-coil Domain (SEQ ID NO:35) and said Domain 2 of said second polypeptide chain is an E-coil Domain (SEQ ID NO:34).

Claim 10. The diabody of any of claims 1 to 8, wherein said Domain 2 of said first polypeptide chain is an E-coil Domain (SEQ ID NO:34) and said Domain 2 of said second polypeptide chain is a K-coil Domain (SEQ ID NO:35).

Claim 11. The diabody of any ing claim, wherein the diabody is capable of crossreacting with both human and primate CD123 and CD3 proteins.

Claim 12. The diabody of claim 1, wherein: A. said first polypeptide chain comprises the amino acid sequence of SEQ ID NO:1; and B. said second polypeptide chain comprises the amino acid sequence of SEQ ID NO:3.

Claim 13. The y of claim 1, wherein the diabody further comprises a third polypeptide chain, wherein: A. said first ptide chain ses the amino acid sequence of SEQ ID NO:15; B. said second polypeptide chain comprises the amino acid sequence of SEQ ID NO:13; and C. said third polypeptide chain comprises the amino acid sequence of SEQ ID NO:54.

Claim 14. The diabody of claim 1, wherein the y further comprises a third polypeptide chain, wherein: A. said first polypeptide chain comprises the amino acid sequence of SEQ ID NO:1; B. said second polypeptide chain comprises the amino acid sequence of SEQ ID NO:17; and C. said third polypeptide chain comprises the amino acid sequence of SEQ ID NO:54.

Claim 15. A pharmaceutical composition comprising the diabody of any one of claims 1 to 14 and a physiologically acceptable r.

Claim 16. Use of diabody of any one of claims 1 to 14, or the pharmaceutical composition of claim 15, in the preparation of a medicament for the ent of a disease or condition associated with or characterized by the expression of CD123.

Claim 17. The use of claim 16, wherein said disease or ion associated with or characterized by the expression of CD123 is cancer.

Claim 18. The use of claim 17, n said cancer is ed from the group consisting of: acute myeloid leukemia (AML), chronic myelogenous leukemia (CML), including blastic crisis of CML and Abelson oncogene associated with CML (Bcr-ABL translocation), myelodysplastic syndrome (MDS), acute B lymphoblastic leukemia (B-ALL), chronic lymphocytic leukemia (CLL), including Richter’s syndrome or r’s transformation of CLL, hairy cell leukemia (HCL), c plasmacytoid dendritic cell neoplasm (BPDCN), non- Hodgkin lymphomas (NHL), including mantel cell leukemia (MCL), and small cytic lymphoma (SLL), Hodgkin’s lymphoma, systemic mastocytosis, and Burkitt’s lymphoma.

Claim 19. The use of claim 16, wherein said disease or condition associated with or characterized by the expression of CD123 is an inflammatory condition.

Claim 20. The use of claim 19, wherein said inflammatory condition is selected from the group ting of: Autoimmune Lupus (SLE), allergy, asthma, and rheumatoid arthritis. 1/66 3:9 m=au NE wmlmm mm Ewum “Ergo? "DU Eu sN.h «0.. 4,, umuamoaaumeI #9sz Ea» m_ VMDU \ utuémau into: A word—.mofopao— 3.3MENOEDEm cor). 8 3 :0 Eek . :Ommmmaxm . «DFmOFHQ—PO—mcp Em_mI-,:n_ #mQU ,3 37¢ Em em... 5Z 3 + 31 a.

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E w DART-A DART-A wmen ~21 ......... .ﬁ “ 1G“ 1:1“2 Wei 1&2 Concentration (ngirrﬂ Figure 19 /66 le + Carrier 100 10‘ 101 103 10“ 10° 10" HF 103 104 CD14HAPC CDTf-LAPC 109 10‘ 102 103 104 1G“ 101 102 103 10“ CDI4WAPC CDT-’LAPC @ﬁﬁﬁ Figure 20 26/66 Control DART 1 OOpQ/kg/d 109 10‘ 102 103 10“ 100 1L”):t 102 10-3 1Q“ CD14WAPC CDi4mAPC ‘ 103 103 10“ 10" 10‘ 163 103 10“ PC CD'P-LAPC @ﬁﬁﬁ Figure 20 (Continued) 27/66 DART—A 10ng/kg/d " 10‘ 102 103 10“ 10” 101 103 103 104‘ CDHLAPC CDT4JXPC ° 19‘ 102 103 10“ 10‘) 101 102 103 10“ CDIILAPC PC 30ng/kg/d C0123 109 10‘ 102 103 10" 10" 10‘ 102 103 10“ CD14WAPC CDTILAPC C014 Figure 20 (Continued) 28/66 B Cells, CD20+ 10000 Femab 8000 .3260 6000 4000 2000 _ . _ L . . . _ _ _ . , ﬁn 6m 8 5N _om_ Sn 6m 5m a3 gm 5m m8 5m m3 5m wmo 50 men 9.; 9.2. 2. 9;9: 2. 9.8 9.8 o.n_ 9. 2: 952 0.. 9.8” 0.. 9.88 00M— 00m oom— tes, CD14+ N Female .3230 400 . . . . L _ x . . L _ _ .

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Cynomolgus MonkeyT Cells Figure 26E 37/66 100000 8 anti-CD123 E?) W 80000 Isotype I £3 60000 33 40000 $3 20000 Figure 27A U937 E:T=10:1 r? “QM DART-A '5 60 + Contra! DART '6 10-4 10'2 1O0 102 Concenﬂaﬁon(nghnL) Figure 273 38/66 Kasumi-3 + Human T cells E:T=10:1 -o- DART-A EC50=0.01 nglmL > 8° #5 + Control DART '6 10'4 10'2 10° 102 Concentration (nglm L) Figure 27C -3 + Cyno PBMCs E:T=15:1 EC50 = 0.02 nglmL a 80 3 6° + DART-A 3‘5 4o .. Control DART °\° 20 '6 10'4 10'2 10° 102 Concentration (nglmL) Figure 27D 39/66 Anti-CD123 -Isotype 150000 85 125000 100000 9.65m 0 «~50 0 \\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\ § \\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\ §§§§ Figure 27E 40/66 Monocytes (CD14+ I CD123"°) - 3 hours 3:8 11 20752 505050 61h8OhU0 rUsm :23.ng 352E: é §\ I §§§§N | I \\\\\\\\\\\\\\\\§N I I §§Q I Q Q.

Q G Dem§§§§N.TA. (Mum§§Qk)L Figure 27F (CD14'ICD123H')_ 28 11 IKE 361 hhoo 00hUUO mmu :030300 38:5 20752 505050 m DART-A (nglmL) Figure 276 41/66 Cynomolgus Monkey PBMC Alone (FACS) CD14-CD123+pDCs PNo O '.x 01 Q '_\ Q + l DART -|:I- DART-A 0.05 0.00 '8010'6 10'4 10'2 10° 102 104 Concentration (nglmL) Figure 27H (pg/mL) NDO.a U!D Concentration .a GO EOI o o o o No 4? «3° «9° Dose (nglkglday) Figure 28 42/66 Vehide oo O ) .h 00‘) 0 |L-6 N0 22 29 36 Study Day Figure 29A DART-A 00 O (pglmL) h CO5O |L-6 Study Day Figure 293 43/66 DART-A on O ) O) O.hC lL-6 NO 1 8 15 22 29 36 Study Day Figure 29C 44/66 Vehicle Emmncmaé 4 0 8 3o 4.52.8 1 0 Study Day Figure 30A DART-A Emmncmmev 4 0 +£52.38 30 452.8 1 0 Study Day Figure 303 45/66 DART-A mé 40 +£52-38 30 432.8 1 0 Study Day Figure 30C 46/66 E 15 {E 12 co 8 9 ”g_. 2 3 1 8 15 22 29 36 StudyDay Figure 30D DART-A CD303+ (meaniSEM) Cells/“L Study Day Figure 30E 47/66 DART-A oé 11 +880 432.8 529630 1 15 22 29 36 Study Day Figure 30F 48/66 + Group 1 cellslmL T (meaniSEM) 1 81522293643505764 Study Day Figure 31A DART-A 12500 10000 cells/“L 7500 T (meaniSEM) N01 016 CO DOD Study Day Figure 31 B 49/66 DART-A 1 2500 1 0000 uL T (meaniSEM) 7500 N01 010 CO COO 1 81522293643505764 Study Day Figure 31C Group 1 Vehicle Cells SEM) CD4 i % (Mean N-hmm 0000 815 22 29 36 43 50 57 64 Study Day Figure 31D 50/66 Group 5 DART-A Cells SEM) CD4 i % (Mean O COCO 1 815 22 29 36 43 50 57 64 Study Day Figure 31E Group 6 DART-A Cells SEM) CD4 i % (Mean whoaoo 60°C 815 22 29 36 43 50 57 64 Study Day Figure 31F 51/66 Group 1 Cells SEM) “#01 COO CD8 i(Mean NO 0 ﬂ ‘ 181522293643505764, a V Study Day Figure 316 Group 5 DART-A (D E 40 ﬁ «”3 U 30 8 A 8 20 Q A A °\ V A L '11..“ 181522293643505764 Study Day Figure 31H 52/66 Group 6 DART-A Cells SEM) CD8 i % (Mean l OOOOO Study Day Figure 31| 53/66 DART-A Group 2 Cells SEM) CD4 i % (Mean N-BOOO 000° 00 _\ 01 N2 29 36 43 50 57 64 Study Day Figure 32A DART-A Group 3 Cells SEM) 0500 CO CD4 1 % (Mean 20 1 815 22 29 36 43 50 57 64 Study Day Figure 32B 54/66 DARTnA Group 4 00 O Cells SEM) CD4 i % (Mean Study Day Figure 32C DARTnA Group 2 Cells SEM) CD8 % (Mean Study Day Figure 32D 55/66 Group 3 01 O Cells SEM) CD8 % (Mean 22 29 36 43 50 57 64 Study Day Figure 32E ﬂARTnA Group 4 2 E 40 3 a) :H 30 °\° E _\ O 181522293643505764 Study Day Figure 32F 56/66 ﬁARTnA Group 2 Cells ~ V' C 181522293643505764 Study Day Figure 33A SARTnA Group 3 Cells g 80m‘0 60 'v v CD4 jg at, {I' ' m 40 % E 20 181522293643505764 Study Day Figure 333 57/66 DARRA Group 4 OO O Cells SEM) O) O CD4 i % (Mean 4020 293643505764 Study Day Figure 33C ﬁARTaA Group2 1, 3 CD RV g a v v 181522293643505764 Study Day Figure 33D 58/66 Group 3 Cells SEM) CD8 .h D % (Mean N GO 1 815 22 29 36 43 50 57 64 Study Day Figure 33E ﬁARTmA Group 4 Cells SEM) CD8 (Mean hC 1 815 22 29 36 43 50 57 64 Study Day Figure 33F 59/66 d to NaiVe DART-A Cytotoxicity 03 O % NC Figure 34 60/66 Group 1 Vehicle 1’ g 8 «”f: v V ‘- +| D s: o\° E 293643505764 Study Day Figure 35A DART-A Group 5 Cells g 80SE CD4 i % (Mean 181522293643505764 Study Day Figure 353 61/66 DART-A Group 6 1’ g 3 0’ 60 a 2 -'-v o m 40 ° é’ v 20 293643505764 Study Day Figure 35C Group 1 Vehicle m E 80 a a: U 60 § § g? \ +| \. o 8 40 °\ VO E V V ' v 181522293643505764 Study Day Figure 35D 62/66 DART-A Group 5 a, ’2‘ 80 ﬁ 3: o 60 § § § 3 40 o\ v V v v v 293643505764 Study Day Figure 35E DART-A Group 6 Cells ’2‘ 80m”3 60 +| \ CD8 \ g 40 % v E 20 V 181522293643505764 Study Day Figure 35F 63/66 (106luL) 01 8 1522 293643 50 5764 Study Day Figure 36A DART-A (106/uL) 0105‘!“ -40 01 815 22 29 36 43 50 57 64 Study Day Figure 363 64/66 DART-A 01 815 22 29 36 43 50 57 64 Study Day Figure 36C Vehicle L) .h C O 03lu + Group1 o: O O Reticulocytes NOO_\ O O -40 01 Study day Figure 36D 65/66 DART-A L) hCO -A- Group 2 03lu -v- Group 3 00 O O (1 -®- Group 4 locytes + Group 5 NO O -40 01 815 22 29 36 43 50 57 64 Study day Figure 36E DART-A L) hCO 03lu 00 O O Reticulocytes N o o—x o o -40 01 815 22 29 36 43 50 57 64 Study day Figure 36F 66/66 n0 - Group 1 Vehicle m Group 2 DART-A 2.8 2253.8. A4 Group 3 DART-A +250 III Group 4 DART-A .5...6 at Group 5 DART-A Group 6 DART-A § UQLIVUUQLZIVUO mammammanmmnmmammnmmnmm_ §§§§§§§ III-II nﬂllyunﬂ gamma nLIVUQKITUOQKIYUD ammmmmnmmnmmmm mo ﬂu Figure 37A - Group 1 Vehicle ‘ Group 2 DART-A n4 N um... £23.38. Group 3 DART-A .53 mmmmmmmmmmm w§§§§§§§§§§ III Group 4 DART-A Group 5 DART-A \\\\\\\\\\\\\\ \\\\\\\\\\\\\\\ nﬂlvunﬂly §§§ $§§§§ nﬂllvon é “magnum Group 6 DART-A Figure 373 SEQUENCE LISTING <110> MacroGenics, Inc.

Bonvini, Ezio Johnson, Leslie Huang, Ling Moore, Paul Chichili, Gurunadh Alderson, Ralph <120> Bi-Specific lent Diabodies That Are Capable Of Binding CD123, And CD3 And Uses Thereof <130> 1301.0109PCT <150> US 61/869,510 <151> 201323 <150> US 61/907,749 <151> 201322 <150> EP 13198784 <151> 201329 <150> US 61/990,475 <151> 201408 <160> 59 <170> In version 3.5 <210> 1 <211> 272 <212> PRT <213> Artificial Sequence <220> <223> First Polypeptide Chain of DART-A <400> 1 Gln Ala Val Val Thr Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly 1 5 10 15 Thr Val Thr Leu Thr Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Ser 25 30 Asn Tyr Ala Asn Trp Val Gln Gln Lys Pro Gly Gln Ala Pro Arg Gly 40 45 Leu Ile Gly Gly Thr Asn Lys Arg Ala Pro Trp Thr Pro Ala Arg Phe 50 55 60 2546512v1 Ser Gly Ser Leu Leu Gly Gly Lys Ala Ala Leu Thr Ile Thr Gly Ala 65 70 75 80 Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn 85 90 95 Leu Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gly Gly 100 105 110 Gly Ser Gly Gly Gly Gly Glu Val Gln Leu Val Gln Ser Gly Ala Glu 115 120 125 Leu Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly 130 135 140 Tyr Thr Phe Thr Asp Tyr Tyr Met Lys Trp Val Arg Gln Ala Pro Gly 145 150 155 160 Gln Gly Leu Glu Trp Ile Gly Asp Ile Ile Pro Ser Asn Gly Ala Thr 165 170 175 Phe Tyr Asn Gln Lys Phe Lys Gly Arg Val Thr Ile Thr Val Asp Lys 180 185 190 Ser Thr Ser Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp 195 200 205 Thr Ala Val Tyr Tyr Cys Ala Arg Ser His Leu Leu Arg Ala Ser Trp 210 215 220 Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly 225 230 235 240 Cys Gly Gly Gly Glu Val Ala Ala Leu Glu Lys Glu Val Ala Ala Leu 245 250 255 Glu Lys Glu Val Ala Ala Leu Glu Lys Glu Val Ala Ala Leu Glu Lys 260 265 270 <210> 2 <211> 816 <212> DNA <213> Artificial Sequence <220> <223> Nucleic Acid Molecule Encoding First Polypeptide Chain of DART-A <400> 2 caggctgtgg tgactcagga gccttcactg accgtgtccc caggcggaac tgtgaccctg 60 agat ccagcacagg cgcagtgacc acatctaact acgccaattg ggtgcagcag 120 aagccaggac aggcaccaag gggcctgatc gggggtacaa acaaaagggc gacc 180 2546512v1 cctgcacggt tttctggaag tctgctgggc ggaaaggccg ctctgactat taccggggca 240 caggccgagg acgaagccga ttactattgt gctctgtggt atagcaatct gtgggtgttc 300 ggca caaaactgac tgtgctggga gggggtggat ccggcggcgg aggcgaggtg 360 cagctggtgc agtccggggc gaag aaacccggag cttccgtgaa ggtgtcttgc 420 aaagccagtg cctt cacagactac tatatgaagt ggca ggctccagga 480 cagggactgg aatggatcgg cgatatcatt ccttccaacg cttt ctacaatcag 540 aagtttaaag gcagggtgac tattaccgtg gacaaatcaa caagcactgc ttatatggag 600 ctgagctccc tgcgctctga agcc gtgtactatt gtgctcggtc acacctgctg 660 agagccagct ggtttgctta ttggggacag ggcaccctgg tgacagtgtc ttccggagga 720 tgtggcggtg gagaagtggc cgcactggag aaagaggttg ctgctttgga gaaggaggtc 780 gctgcacttg aaaaggaggt cgcagccctg gagaaa 816 <210> 3 <211> 280 <212> PRT <213> Artificial Sequence <220> <223> Second Polypeptide Chain of DART-A <400> 3 Asp Phe Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser 25 30 Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln 40 45 Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Asn 85 90 95 Asp Tyr Ser Tyr Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile 100 105 110 2546512v1 Lys Gly Gly Gly Ser Gly Gly Gly Gly Glu Val Gln Leu Val Glu Ser 115 120 125 Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala 130 135 140 Ala Ser Gly Phe Thr Phe Ser Thr Tyr Ala Met Asn Trp Val Arg Gln 145 150 155 160 Ala Pro Gly Lys Gly Leu Glu Trp Val Gly Arg Ile Arg Ser Lys Tyr 165 170 175 Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp Ser Val Lys Asp Arg Phe Thr 180 185 190 Ile Ser Arg Asp Asp Ser Lys Asn Ser Leu Tyr Leu Gln Met Asn Ser 195 200 205 Leu Lys Thr Glu Asp Thr Ala Val Tyr Tyr Cys Val Arg His Gly Asn 210 215 220 Phe Gly Asn Ser Tyr Val Ser Trp Phe Ala Tyr Trp Gly Gln Gly Thr 225 230 235 240 Leu Val Thr Val Ser Ser Gly Gly Cys Gly Gly Gly Lys Val Ala Ala 245 250 255 Leu Lys Glu Lys Val Ala Ala Leu Lys Glu Lys Val Ala Ala Leu Lys 260 265 270 Glu Lys Val Ala Ala Leu Lys Glu 275 280 <210> 4 <211> 840 <212> DNA <213> Artificial Sequence <220> <223> c Acid le Encoding Second Polypeptide Chain of DART-A <400> 4 gacttcgtga tgacacagtc tcctgatagt ctggccgtga gtctggggga gcgggtgact 60 atgtcttgca agagctccca gtcactgctg aacagcggaa atcagaaaaa ctatctgacc 120 tggtaccagc cagg ccagccccct aaactgctga tctattgggc ttccaccagg 180 gaatctggcg tgcccgacag attcagcggc agcggcagcg gcacagattt taccctgaca 240 atttctagtc tgcaggccga ggacgtggct gtgtactatt gtcagaatga ttacagctat 300 2546512v1 ccctacactt tcggccaggg gaccaagctg gaaattaaag gaggcggatc cggcggcgga 360 ggcgaggtgc agctggtgga gtctggggga ggcttggtcc agcctggagg gaga 420 ctctcctgtg cagcctctgg attcaccttc agcacatacg ctatgaattg ggtccgccag 480 ggga tgga gtgggttgga aggatcaggt ccaagtacaa caattatgca 540 acctactatg ccgactctgt gaaggataga ttcaccatct caagagatga ttcaaagaac 600 tcactgtatc tgcaaatgaa cagcctgaaa accgaggaca cggccgtgta ttactgtgtg 660 agacacggta acttcggcaa ttcttacgtg tcttggtttg cttattgggg acaggggaca 720 ctggtgactg tgtcttccgg aggatgtggc ggtggaaaag tggccgcact gaaa 780 gttgctgctt tgaaagagaa cgca cttaaggaaa aggtcgcagc cctgaaagag 840 <210> 5 <211> 268 <212> PRT <213> Artificial Sequence <220> <223> First Polypeptide Chain of DART-B <400> 5 Asp Ile Gln Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly 1 5 10 15 Glu Lys Val Thr Met Thr Cys Arg Ala Ser Ser Ser Val Ser Tyr Met 25 30 Asn Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro Lys Arg Trp Ile Tyr 40 45 Asp Thr Ser Lys Val Ala Ser Gly Val Pro Tyr Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu Ala Glu 65 70 75 80 Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Leu Thr 85 90 95 Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Gly Gly Gly Ser Gly Gly 100 105 110 Gly Gly Gln Val Gln Leu Val Gln Ser Gly Ala Glu Leu Lys Lys Pro 115 120 125 Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr 2546512v1 130 135 140 Asp Tyr Tyr Met Lys Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu 145 150 155 160 Trp Ile Gly Asp Ile Ile Pro Ser Asn Gly Ala Thr Phe Tyr Asn Gln 165 170 175 Lys Phe Lys Gly Arg Val Thr Ile Thr Val Asp Lys Ser Thr Ser Thr 180 185 190 Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr 195 200 205 Tyr Cys Ala Arg Ser His Leu Leu Arg Ala Ser Trp Phe Ala Tyr Trp 210 215 220 Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Cys Gly Gly Gly 225 230 235 240 Glu Val Ala Ala Leu Glu Lys Glu Val Ala Ala Leu Glu Lys Glu Val 245 250 255 Ala Ala Leu Glu Lys Glu Val Ala Ala Leu Glu Lys 260 265 <210> 6 <211> 804 <212> DNA <213> Artificial Sequence <220> <223> Nucleic Acid Molecule Encoding First ptide Chain of DART-B <400> 6 gacattcagc tgacccagtc tccagcaatc atgtctgcat ctccagggga cacc 60 atgacctgca gttc aagtgtaagt tacatgaact ggtaccagca gaagtcaggc 120 acctccccca aaagatggat ttatgacaca tccaaagtgg cttctggagt cccttatcgc 180 ttcagtggca gtgggtctgg gacctcatac tctctcacaa tcagcagcat tgaa 240 gatgctgcca cttattactg ccaacagtgg agtagtaacc cgctcacgtt cggtgctggg 300 accaagctgg agctgaaagg aggcggatcc ggcggcggag gccaggtgca gcag 360 tccggggctg agctgaagaa acccggagct tccgtgaagg tgtcttgcaa agccagtggc 420 tacaccttca cagactacta tatgaagtgg gtcaggcagg ctccaggaca gggactggaa 480 tggatcggcg atatcattcc cggg gccactttct acaatcagaa gtttaaaggc 540 2546512v1 agggtgacta ttaccgtgga caaatcaaca agcactgctt atatggagct gagctccctg 600 cgctctgaag atacagccgt gtactattgt gctcggtcac acctgctgag agccagctgg 660 tttgcttatt ggggacaggg ggtg acagtgtctt ccggaggatg tggcggtgga 720 gaagtggccg cactggagaa agaggttgct gaga aggaggtcgc tgcacttgaa 780 gtcg cagccctgga gaaa 804 <210> 7 <211> 274 <212> PRT <213> Artificial Sequence <220> <223> Second Polypeptide Chain of DART-B <400> 7 Asp Phe Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser 25 30 Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln 40 45 Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Asn 85 90 95 Asp Tyr Ser Tyr Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile 100 105 110 Lys Gly Gly Gly Ser Gly Gly Gly Gly Asp Ile Lys Leu Gln Gln Ser 115 120 125 Gly Ala Glu Leu Ala Arg Pro Gly Ala Ser Val Lys Met Ser Cys Lys 130 135 140 Thr Ser Gly Tyr Thr Phe Thr Arg Tyr Thr Met His Trp Val Lys Gln 145 150 155 160 Arg Pro Gly Gln Gly Leu Glu Trp Ile Gly Tyr Ile Asn Pro Ser Arg 165 170 175 2546512v1 Gly Tyr Thr Asn Tyr Asn Gln Lys Phe Lys Asp Lys Ala Thr Leu Thr 180 185 190 Thr Asp Lys Ser Ser Ser Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr 195 200 205 Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala Arg Tyr Tyr Asp Asp His 210 215 220 Tyr Cys Leu Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser 225 230 235 240 Gly Gly Cys Gly Gly Gly Lys Val Ala Ala Leu Lys Glu Lys Val Ala 245 250 255 Ala Leu Lys Glu Lys Val Ala Ala Leu Lys Glu Lys Val Ala Ala Leu 260 265 270 Lys Glu <210> 8 <211> 822 <212> DNA <213> Artificial Sequence <220> <223> Nucleic Acid Molecule Encoding Second Polypeptide Chain of DART-B <400> 8 gacttcgtga tgacacagtc tagt ctggccgtga gtctggggga gact 60 atgtcttgca agagctccca gtcactgctg aacagcggaa atcagaaaaa ctatctgacc 120 tggtaccagc agaagccagg ccagccccct aaactgctga tctattgggc ttccaccagg 180 gaatctggcg tgcccgacag attcagcggc agcggcagcg gcacagattt gaca 240 atttctagtc tgcaggccga ggacgtggct gtgtactatt gtcagaatga ttacagctat 300 ccctacactt tcggccaggg gaccaagctg aaag gaggcggatc cggcggcgga 360 ggcgatatca aactgcagca gtcaggggct gcaa gacctggggc ctcagtgaag 420 atgtcctgca agacttctgg ctacaccttt actaggtaca cgatgcactg ggtaaaacag 480 aggcctggac agggtctgga atggattgga tacattaatc gtgg ttatactaat 540 tacaatcaga agttcaagga caaggccaca ttgactacag acaaatcctc cagcacagcc 600 tacatgcaac tgagcagcct gacatctgag gactctgcag tctattactg tgcaagatat 660 tatgatgatc attactgcct tgactactgg ggca ccactctcac agtctcctcc 720 2546512v1 ggaggatgtg gcggtggaaa agtggccgca ctgaaggaga aagttgctgc tttgaaagag 780 gccg cacttaagga aaaggtcgca gccctgaaag ag 822 <210> 9 <211> 322 <212> PRT <213> Artificial Sequence <220> <223> CD123 x CD3 Diabody Polypeptide Chain Having Albumin Binding Site <400> 9 Gln Ala Val Val Thr Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly 1 5 10 15 Thr Val Thr Leu Thr Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Ser 25 30 Asn Tyr Ala Asn Trp Val Gln Gln Lys Pro Gly Gln Ala Pro Arg Gly 40 45 Leu Ile Gly Gly Thr Asn Lys Arg Ala Pro Trp Thr Pro Ala Arg Phe 50 55 60 Ser Gly Ser Leu Leu Gly Gly Lys Ala Ala Leu Thr Ile Thr Gly Ala 65 70 75 80 Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn 85 90 95 Leu Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gly Gly 100 105 110 Gly Ser Gly Gly Gly Gly Glu Val Gln Leu Val Gln Ser Gly Ala Glu 115 120 125 Leu Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly 130 135 140 Tyr Thr Phe Thr Asp Tyr Tyr Met Lys Trp Val Arg Gln Ala Pro Gly 145 150 155 160 Gln Gly Leu Glu Trp Ile Gly Asp Ile Ile Pro Ser Asn Gly Ala Thr 165 170 175 Phe Tyr Asn Gln Lys Phe Lys Gly Arg Val Thr Ile Thr Val Asp Lys 180 185 190 Ser Thr Ser Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp 195 200 205 2546512v1 Thr Ala Val Tyr Tyr Cys Ala Arg Ser His Leu Leu Arg Ala Ser Trp 210 215 220 Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly 225 230 235 240 Cys Gly Gly Gly Glu Val Ala Ala Leu Glu Lys Glu Val Ala Ala Leu 245 250 255 Glu Lys Glu Val Ala Ala Leu Glu Lys Glu Val Ala Ala Leu Glu Lys 260 265 270 Gly Gly Gly Ser Leu Ala Glu Ala Lys Val Leu Ala Asn Arg Glu Leu 275 280 285 Asp Lys Tyr Gly Val Ser Asp Tyr Tyr Lys Asn Leu Ile Asp Asn Ala 290 295 300 Lys Ser Ala Glu Gly Val Lys Ala Leu Ile Asp Glu Ile Leu Ala Ala 305 310 315 320 Leu Pro <210> 10 <211> 966 <212> DNA <213> Artificial Sequence <220> <223> cleotide Encoding CD123 x CD3 Diabody Polypeptide Chain Having Albumin Binding Site <400> 10 caggctgtgg tgactcagga gccttcactg accgtgtccc caggcggaac tgtgaccctg 60 acatgcagat ccagcacagg cgcagtgacc acatctaact acgccaattg ggtgcagcag 120 ggac aggcaccaag gggcctgatc gggggtacaa acaaaagggc tccctggacc 180 cctgcacggt gaag tctgctgggc ggaaaggccg ctctgactat taccggggca 240 caggccgagg acgaagccga ttactattgt gctctgtggt atagcaatct gtgggtgttc 300 gggggtggca caaaactgac ggga gggggtggat ccggcggcgg aggcgaggtg 360 cagctggtgc agtccggggc tgagctgaag aaacccggag cttccgtgaa ggtgtcttgc 420 aaagccagtg gctacacctt cacagactac aagt gggtcaggca ggctccagga 480 cagggactgg aatggatcgg cgatatcatt ccttccaacg gggccacttt ctacaatcag 540 2546512v1 aaag gcagggtgac cgtg gacaaatcaa caagcactgc ttatatggag 600 ctgagctccc tgcgctctga agatacagcc gtgtactatt gtgctcggtc acacctgctg 660 agagccagct ggtttgctta ttggggacag ggcaccctgg tgacagtgtc ttccggagga 720 tgtggcggtg gagaagtggc cgcactggag aaagaggttg ctgctttgga gaaggaggtc 780 gctgcacttg aaaaggaggt cgcagccctg gagaaaggcg gcgggtctct ggccgaagca 840 aaagtgctgg ccaaccgcga taaa tatggcgtga gcgattatta taagaacctg 900 attgacaacg ccgc ggaaggcgtg aaagcactga ttgatgaaat tctggccgcc 960 ctgcct 966 <210> 11 <211> 217 <212> PRT <213> Artificial Sequence <220> <223> CH2-CH3 Domains of a Modified Human Antibody Fc Region <400> 11 Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 1 5 10 15 Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 25 30 Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr 40 45 Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 50 55 60 Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His 65 70 75 80 Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 85 90 95 Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln 100 105 110 Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met 115 120 125 Thr Lys Asn Gln Val Ser Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro 130 135 140 2546512v1 Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn 145 150 155 160 Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu 165 170 175 Val Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val 180 185 190 Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn Arg Tyr Thr Gln 195 200 205 Lys Ser Leu Ser Leu Ser Pro Gly Lys 210 215 <210> 12 <211> 681 <212> DNA <213> Artificial Sequence <220> <223> Nucleic Acid le Encoding Peptide 1 and the CH2 and CH3 Domains of an IgG Fc region <400> 12 gacaaaactc acacatgccc accgtgccca gcacctgaag ccgcgggggg accgtcagtc 60 ttcctcttcc ccccaaaacc caaggacacc ctcatgatct cccggacccc tgaggtcaca 120 tgcgtggtgg tggacgtgag ccacgaagac cctgaggtca agttcaactg gtacgtggac 180 ggcgtggagg atgc caagacaaag gagg agcagtacaa cagcacgtac 240 cgtgtggtca gcgtcctcac cgtcctgcac caggactggc tgaatggcaa ggagtacaag 300 tgcaaggtct ccaacaaagc cctcccagcc cccatcgaga tctc caaagccaaa 360 gggcagcccc gagaaccaca ggtgtacacc ctgcccccat cccgggagga gatgaccaag 420 aaccaggtca gttg cgcagtcaaa ggcttctatc ccagcgacat cgccgtggag 480 tgggagagca atgggcagcc ggagaacaac tacaagacca cgcctcccgt ctcc 540 tcct tcttcctcgt cagcaagctc accgtggaca agagcaggtg gcagcagggg 600 aacgtcttct catgctccgt gatgcatgag gctctgcaca accgctacac gcagaagagc 660 ctctccctgt ctccgggtaa a 681 <210> 13 <211> 510 <212> PRT <213> Artificial Sequence 2546512v1 <220> <223> First ptide Chain of DART-A w/Fc Version 1 Construct <400> 13 Asp Phe Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser 25 30 Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln 40 45 Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Asn 85 90 95 Asp Tyr Ser Tyr Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile 100 105 110 Lys Gly Gly Gly Ser Gly Gly Gly Gly Glu Val Gln Leu Val Glu Ser 115 120 125 Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala 130 135 140 Ala Ser Gly Phe Thr Phe Ser Thr Tyr Ala Met Asn Trp Val Arg Gln 145 150 155 160 Ala Pro Gly Lys Gly Leu Glu Trp Val Gly Arg Ile Arg Ser Lys Tyr 165 170 175 Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp Ser Val Lys Asp Arg Phe Thr 180 185 190 Ile Ser Arg Asp Asp Ser Lys Asn Ser Leu Tyr Leu Gln Met Asn Ser 195 200 205 Leu Lys Thr Glu Asp Thr Ala Val Tyr Tyr Cys Val Arg His Gly Asn 210 215 220 Phe Gly Asn Ser Tyr Val Ser Trp Phe Ala Tyr Trp Gly Gln Gly Thr 225 230 235 240 Leu Val Thr Val Ser Ser Gly Gly Cys Gly Gly Gly Glu Val Ala Ala 245 250 255 2546512v1 Leu Glu Lys Glu Val Ala Ala Leu Glu Lys Glu Val Ala Ala Leu Glu 260 265 270 Lys Glu Val Ala Ala Leu Glu Lys Gly Gly Gly Asp Lys Thr His Thr 275 280 285 Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe 290 295 300 Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro 305 310 315 320 Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val 325 330 335 Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr 340 345 350 Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val 355 360 365 Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys 370 375 380 Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser 385 390 395 400 Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro 405 410 415 Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val 420 425 430 Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 435 440 445 Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp 450 455 460 Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp 465 470 475 480 Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His 485 490 495 Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 500 505 510 <210> 14 <211> 1530 <212> DNA <213> cial Sequence 2546512v1 <220> <223> Nucleic Acid Molecule Encoding First Polypeptide Chain of DART-A w/Fc Version 1 Construct <400> 14 gacttcgtga agtc tcctgatagt gtga gtctggggga gcgggtgact 60 atgtcttgca agagctccca gtcactgctg aacagcggaa atcagaaaaa ctatctgacc 120 tggtaccagc agaagccagg ccagccccct aaactgctga tctattgggc ttccaccagg 180 gaatctggcg tgcccgacag attcagcggc agcg attt taccctgaca 240 atttctagtc tgcaggccga ggct gtgtactatt gtcagaatga ttacagctat 300 actt tcggccaggg gctg gaaattaaag gaggcggatc cggcggcgga 360 ggcgaggtgc agctggtgga gtctggggga ggcttggtcc agcctggagg gtccctgaga 420 ctctcctgtg cagcctctgg attcaccttc agcacatacg ctatgaattg ggtccgccag 480 gctccaggga aggggctgga gtgggttgga aggatcaggt ccaagtacaa caattatgca 540 acctactatg ccgactctgt gaaggataga ttcaccatct caagagatga gaac 600 tcactgtatc tgcaaatgaa gaaa accgaggaca cggccgtgta ttactgtgtg 660 agacacggta acttcggcaa ttcttacgtg tcttggtttg gggg acaggggaca 720 ctggtgactg tgtcttccgg aggatgtggc ggtggagaag tggccgcact ggagaaagag 780 gttgctgctt tggagaagga tgca cttgaaaagg aggtcgcagc cctggagaaa 840 ggcggcgggg acaaaactca cacatgccca ccgtgcccag cacctgaagc cgcgggggga 900 ccgtcagtct tcctcttccc accc aaggacaccc tcatgatctc ccggacccct 960 gaggtcacat gcgtggtggt ggacgtgagc cacgaagacc ctgaggtcaa gttcaactgg 1020 gacg gcgtggaggt gcataatgcc aagacaaagc cgcgggagga gcagtacaac 1080 agcacgtacc gtgtggtcag cacc gtcctgcacc aggactggct gaatggcaag 1140 aagt gcaaggtctc caacaaagcc ctcccagccc ccatcgagaa aaccatctcc 1200 aaagccaaag ggcagccccg agaaccacag gtgtacaccc tgcccccatc ccgggaggag 1260 atgaccaaga accaggtcag cctgtggtgc ctggtcaaag gcttctatcc cagcgacatc 1320 gccgtggagt gggagagcaa tgggcagccg gagaacaact acaagaccac gcctcccgtg 1380 ctggactccg acggctcctt cttcctctac agcaagctca ccgtggacaa gagcaggtgg 1440 2546512v1 cagcagggga acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacacg 1500 cagaagagcc tctccctgtc tccgggtaaa 1530 <210> 15 <211> 272 <212> PRT <213> Artificial Sequence <220> <223> Second Polypeptide Chain of DART-A w/Fc Version 1 uct <400> 15 Gln Ala Val Val Thr Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly 1 5 10 15 Thr Val Thr Leu Thr Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Ser 25 30 Asn Tyr Ala Asn Trp Val Gln Gln Lys Pro Gly Gln Ala Pro Arg Gly 40 45 Leu Ile Gly Gly Thr Asn Lys Arg Ala Pro Trp Thr Pro Ala Arg Phe 50 55 60 Ser Gly Ser Leu Leu Gly Gly Lys Ala Ala Leu Thr Ile Thr Gly Ala 65 70 75 80 Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn 85 90 95 Leu Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gly Gly 100 105 110 Gly Ser Gly Gly Gly Gly Glu Val Gln Leu Val Gln Ser Gly Ala Glu 115 120 125 Leu Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly 130 135 140 Tyr Thr Phe Thr Asp Tyr Tyr Met Lys Trp Val Arg Gln Ala Pro Gly 145 150 155 160 Gln Gly Leu Glu Trp Ile Gly Asp Ile Ile Pro Ser Asn Gly Ala Thr 165 170 175 Phe Tyr Asn Gln Lys Phe Lys Gly Arg Val Thr Ile Thr Val Asp Lys 180 185 190 Ser Thr Ser Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp 195 200 205 2546512v1 Thr Ala Val Tyr Tyr Cys Ala Arg Ser His Leu Leu Arg Ala Ser Trp 210 215 220 Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly 225 230 235 240 Cys Gly Gly Gly Lys Val Ala Ala Leu Lys Glu Lys Val Ala Ala Leu 245 250 255 Lys Glu Lys Val Ala Ala Leu Lys Glu Lys Val Ala Ala Leu Lys Glu 260 265 270 <210> 16 <211> 816 <212> DNA <213> Artificial Sequence <220> <223> c Acid Molecule Encoding Second Polypeptide Chain of DART-A w/Fc Version 1 Construct <400> 16 caggctgtgg agga gccttcactg accgtgtccc caggcggaac tgtgaccctg 60 acatgcagat ccagcacagg cgcagtgacc acatctaact acgccaattg ggtgcagcag 120 ggac aggcaccaag gggcctgatc gggggtacaa acaaaagggc tccctggacc 180 cggt tttctggaag tctgctgggc gccg ctctgactat taccggggca 240 caggccgagg acgaagccga ttactattgt gctctgtggt atagcaatct gtgggtgttc 300 gggggtggca caaaactgac tgtgctggga gggggtggat ccggcggcgg aggcgaggtg 360 cagctggtgc agtccggggc tgagctgaag aaacccggag cttccgtgaa ggtgtcttgc 420 agtg cctt cacagactac tatatgaagt gggtcaggca ggctccagga 480 cagggactgg aatggatcgg cgatatcatt ccttccaacg gggccacttt ctacaatcag 540 aaag gcagggtgac tattaccgtg gacaaatcaa caagcactgc ttatatggag 600 ctgagctccc tgcgctctga agatacagcc gtgtactatt gtgctcggtc acacctgctg 660 agagccagct ggtttgctta ttggggacag ggcaccctgg tgacagtgtc ttccggagga 720 tgtggcggtg gaaaagtggc cgcactgaag gagaaagttg ctgctttgaa agagaaggtc 780 gccgcactta aggaaaaggt cgcagccctg aaagag 816 <210> 17 <211> 515 2546512v1 <212> PRT <213> Artificial Sequence <220> <223> First Polypeptide Chain of DART-A w/Fc Version 2 uct <400> 17 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser 130 135 140 Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys Ala Pro Ser Ser Ser Pro Met Glu Asp Phe Val Met Thr 225 230 235 240 2546512v1 Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly Glu Arg Val Thr Met 245 250 255 Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser Gly Asn Gln Lys Asn 260 265 270 Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu 275 280 285 Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val Pro Asp Arg Phe Ser 290 295 300 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln 305 310 315 320 Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Asn Asp Tyr Ser Tyr Pro 325 330 335 Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Gly Gly Gly Ser 340 345 350 Gly Gly Gly Gly Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val 355 360 365 Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr 370 375 380 Phe Ser Thr Tyr Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly 385 390 395 400 Leu Glu Trp Val Gly Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr 405 410 415 Tyr Tyr Ala Asp Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp 420 425 430 Ser Lys Asn Ser Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp 435 440 445 Thr Ala Val Tyr Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr 450 455 460 Val Ser Trp Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser 465 470 475 480 Ser Gly Gly Cys Gly Gly Gly Lys Val Ala Ala Leu Lys Glu Lys Val 485 490 495 Ala Ala Leu Lys Glu Lys Val Ala Ala Leu Lys Glu Lys Val Ala Ala 500 505 510 Leu Lys Glu <210> 18 <211> 1545 <212> DNA <213> Artificial ce <220> <223> Nucleic Acid le Encoding First Polypeptide Chain of DART-A w/Fc Version 2 Construct <400> 18 gacaaaactc acacatgccc accgtgccca gcacctgaag ccgcgggggg agtc 60 ttcctcttcc ccccaaaacc caaggacacc ctcatgatct cccggacccc tgaggtcaca 120 tgcgtggtgg tggacgtgag ccacgaagac cctgaggtca agttcaactg gtacgtggac 180 ggcgtggagg tgcataatgc caagacaaag ccgcgggagg acaa cagcacgtac 240 cgtgtggtca gcgtcctcac cgtcctgcac caggactggc gcaa ggagtacaag 300 tgcaaggtct ccaacaaagc cctcccagcc cccatcgaga aaaccatctc caaa 360 gggcagcccc gagaaccaca ggtgtacacc ctgcccccat cccgggagga gatgaccaag 420 aaccaggtca gcctgtggtg cctggtcaaa ggcttctatc ccagcgacat cgccgtggag 480 tgggagagca atgggcagcc ggagaacaac tacaagacca ccgt gctggactcc 540 gacggctcct tcta cagcaagctc gaca agagcaggtg gcagcagggg 600 aacgtcttct catgctccgt gatgcatgag gctctgcaca accactacac gcagaagagc 660 ctgt ctccgggtaa agccccttcc agctccccta tggaagactt cgtgatgaca 720 cagtctcctg atagtctggc cgtgagtctg ggggagcggg tgactatgtc ttgcaagagc 780 tcccagtcac acag cggaaatcag aaaaactatc tgacctggta ccagcagaag 840 ccaggccagc cccctaaact gctgatctat tgggcttcca ccagggaatc tggcgtgccc 900 gacagattca gcggcagcgg cagcggcaca gattttaccc tgacaatttc tagtctgcag 960 gccgaggacg tggctgtgta ctattgtcag aatgattaca gctatcccta cactttcggc 1020 caggggacca agctggaaat taaaggaggc ggatccggcg gcggaggcga ggtgcagctg 1080 gtggagtctg ggggaggctt ggtccagcct ggagggtccc tgagactctc ctgtgcagcc 1140 tctggattca ccttcagcac atacgctatg aattgggtcc gccaggctcc agggaagggg 1200 tggg ttggaaggat caggtccaag tacaacaatt atgcaaccta ctatgccgac 1260 2546512v1 tctgtgaagg atagattcac catctcaaga gatgattcaa agaactcact gtatctgcaa 1320 atgaacagcc tgaaaaccga ggacacggcc gtgtattact gtgtgagaca cttc 1380 ggcaattctt cttg gtttgcttat tggggacagg ggacactggt gtct 1440 tccggaggat gtggcggtgg aaaagtggcc gcactgaagg agaaagttgc tgctttgaaa 1500 gagaaggtcg ccgcacttaa ggaaaaggtc gcagccctga aagag 1545 <210> 19 <211> 279 <212> PRT <213> Artificial Sequence <220> <223> First Polypeptide Chain of Control DART <400> 19 Asp Val Val Met Thr Gln Thr Pro Phe Ser Leu Pro Val Ser Leu Gly 1 5 10 15 Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser 25 30 Asn Gly Asn Thr Tyr Leu Arg Trp Tyr Leu Gln Lys Pro Gly Gln Ser 40 45 Pro Lys Val Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys Ser Gln Ser 85 90 95 Thr His Val Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110 Gly Gly Gly Ser Gly Gly Gly Gly Glu Val Gln Leu Val Glu Ser Gly 115 120 125 Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala 130 135 140 Ser Gly Phe Thr Phe Asn Thr Tyr Ala Met Asn Trp Val Arg Gln Ala 145 150 155 160 Pro Gly Lys Gly Leu Glu Trp Val Ala Arg Ile Arg Ser Lys Tyr Asn 165 170 175 2546512v1 Asn Tyr Ala Thr Tyr Tyr Ala Asp Ser Val Lys Asp Arg Phe Thr Ile 180 185 190 Ser Arg Asp Asp Ser Lys Asn Ser Leu Tyr Leu Gln Met Asn Ser Leu 195 200 205 Lys Thr Glu Asp Thr Ala Val Tyr Tyr Cys Val Arg His Gly Asn Phe 210 215 220 Gly Asn Ser Tyr Val Ser Trp Phe Ala Tyr Trp Gly Gln Gly Thr Leu 225 230 235 240 Val Thr Val Ser Ser Gly Gly Cys Gly Gly Gly Glu Val Ala Ala Leu 245 250 255 Glu Lys Glu Val Ala Ala Leu Glu Lys Glu Val Ala Ala Leu Glu Lys 260 265 270 Glu Val Ala Ala Leu Glu Lys <210> 20 <211> 270 <212> PRT <213> Artificial Sequence <220> <223> Second ptide Chain of Control DART <400> 20 Gln Ala Val Val Thr Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly 1 5 10 15 Thr Val Thr Leu Thr Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Ser 25 30 Asn Tyr Ala Asn Trp Val Gln Gln Lys Pro Gly Gln Ala Pro Arg Gly 40 45 Leu Ile Gly Gly Thr Asn Lys Arg Ala Pro Trp Thr Pro Ala Arg Phe 50 55 60 Ser Gly Ser Leu Leu Gly Gly Lys Ala Ala Leu Thr Ile Thr Gly Ala 65 70 75 80 Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn 85 90 95 Leu Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gly Gly 100 105 110 Gly Ser Gly Gly Gly Gly Glu Val Lys Leu Asp Glu Thr Gly Gly Gly 2546512v1 115 120 125 Leu Val Gln Pro Gly Arg Pro Met Lys Leu Ser Cys Val Ala Ser Gly 130 135 140 Phe Thr Phe Ser Asp Tyr Trp Met Asn Trp Val Arg Gln Ser Pro Glu 145 150 155 160 Lys Gly Leu Glu Trp Val Ala Gln Ile Arg Asn Lys Pro Tyr Asn Tyr 165 170 175 Glu Thr Tyr Tyr Ser Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg 180 185 190 Asp Asp Ser Lys Ser Ser Val Tyr Leu Gln Met Asn Asn Leu Arg Val 195 200 205 Glu Asp Met Gly Ile Tyr Tyr Cys Thr Gly Ser Tyr Tyr Gly Met Asp 210 215 220 Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser Gly Gly Cys Gly 225 230 235 240 Gly Gly Lys Val Ala Ala Leu Lys Glu Lys Val Ala Ala Leu Lys Glu 245 250 255 Lys Val Ala Ala Leu Lys Glu Lys Val Ala Ala Leu Lys Glu 260 265 270 <210> 21 <211> 110 <212> PRT <213> cial Sequence <220> <223> Light Chain CD3-Binding Domain of DART-A <400> 21 Gln Ala Val Val Thr Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly 1 5 10 15 Thr Val Thr Leu Thr Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Ser 25 30 Asn Tyr Ala Asn Trp Val Gln Gln Lys Pro Gly Gln Ala Pro Arg Gly 40 45 Leu Ile Gly Gly Thr Asn Lys Arg Ala Pro Trp Thr Pro Ala Arg Phe 50 55 60 Ser Gly Ser Leu Leu Gly Gly Lys Ala Ala Leu Thr Ile Thr Gly Ala 65 70 75 80 2546512v1 Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn 85 90 95 Leu Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 100 105 110 <210> 22 <211> 125 <212> PRT <213> Artificial Sequence <220> <223> Heavy Chain CD3-Binding Domain of DART-A <400> 22 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Thr Tyr 25 30 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 40 45 Gly Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Ser 65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe 100 105 110 Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 125 <210> 23 <211> 106 <212> PRT <213> Artificial ce <220> <223> Light Chain CD3-Binding Domain of DART-B <400> 23 Asp Ile Gln Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly 1 5 10 15 2546512v1 Glu Lys Val Thr Met Thr Cys Arg Ala Ser Ser Ser Val Ser Tyr Met 25 30 Asn Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro Lys Arg Trp Ile Tyr 40 45 Asp Thr Ser Lys Val Ala Ser Gly Val Pro Tyr Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu Ala Glu 65 70 75 80 Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Leu Thr 85 90 95 Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys 100 105 <210> 24 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> Heavy Chain nding Domain of DART-B <400> 24 Asp Ile Lys Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Met Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Arg Tyr 25 30 Thr Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 40 45 Gly Tyr Ile Asn Pro Ser Arg Gly Tyr Thr Asn Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Lys Ala Thr Leu Thr Thr Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Tyr Tyr Asp Asp His Tyr Cys Leu Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Thr Leu Thr Val Ser Ser 2546512v1 <210> 25 <211> 113 <212> PRT <213> Artificial Sequence <220> <223> Light Chain CD123-Binding Domain of DART-A <400> 25 Asp Phe Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser 25 30 Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln 40 45 Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Asn 85 90 95 Asp Tyr Ser Tyr Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile 100 105 110 <210> 26 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> Heavy Chain Binding Domain of DART-A <400> 26 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Leu Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 25 30 Tyr Met Lys Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 40 45 2546512v1 Gly Asp Ile Ile Pro Ser Asn Gly Ala Thr Phe Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Arg Val Thr Ile Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser His Leu Leu Arg Ala Ser Trp Phe Ala Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 27 <211> 113 <212> PRT <213> Artificial ce <220> <223> Light Chain CD123-Binding Domain of DART-B <400> 27 Asp Phe Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser 25 30 Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln 40 45 Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Asn 85 90 95 Asp Tyr Ser Tyr Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile 100 105 110 <210> 28 <211> 120 <212> PRT <213> Artificial Sequence 2546512v1 <220> <223> Heavy Chain CD123-Binding Domain of DART-B <400> 28 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Leu Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 25 30 Tyr Met Lys Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 40 45 Gly Asp Ile Ile Pro Ser Asn Gly Ala Thr Phe Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Arg Val Thr Ile Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser His Leu Leu Arg Ala Ser Trp Phe Ala Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 29 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Linker 1 Polypeptide <400> 29 Gly Gly Gly Ser Gly Gly Gly Gly 1 5 <210> 30 <211> 6 <212> PRT <213> Artificial ce <220> <223> Linker 2 Polypeptide <400> 30 2546512v1 Gly Gly Cys Gly Gly Gly 1 5 <210> 31 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Linker 3 Polypetide <400> 31 Gly Gly Gly Ser <210> 32 <400> 32 <210> 33 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Linker 4 Polypeptide <400> 33 Ala Pro Ser Ser Ser Pro Met Glu 1 5 <210> 34 <211> 28 <212> PRT <213> cial Sequence <220> <223> E-Coil Domain <400> 34 Glu Val Ala Ala Leu Glu Lys Glu Val Ala Ala Leu Glu Lys Glu Val 1 5 10 15 Ala Ala Leu Glu Lys Glu Val Ala Ala Leu Glu Lys 25 <210> 35 <211> 28 <212> PRT 2546512v1 <213> Artificial Sequence <220> <223> K-Coil Domain <400> 35 Lys Val Ala Ala Leu Lys Glu Lys Val Ala Ala Leu Lys Glu Lys Val 1 5 10 15 Ala Ala Leu Lys Glu Lys Val Ala Ala Leu Lys Glu 25 <210> 36 <211> 46 <212> PRT <213> Artificial Sequence <220> <223> Preferred Albumin Binding Domain <400> 36 Leu Ala Glu Ala Lys Val Leu Ala Asn Arg Glu Leu Asp Lys Tyr Gly 1 5 10 15 Val Ser Asp Tyr Tyr Lys Asn Leu Ile Asp Asn Ala Lys Ser Ala Glu 25 30 Gly Val Lys Ala Leu Ile Asp Glu Ile Leu Ala Ala Leu Pro 40 45 <210> 37 <211> 217 <212> PRT <213> Homo sapiens <220> <221> MISC_FEATURE <222> (1)..(217) <223> 3 Domains of Human Fc Region <400> 37 Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 1 5 10 15 Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 25 30 Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr 40 45 2546512v1 Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 50 55 60 Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His 65 70 75 80 Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 85 90 95 Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln 100 105 110 Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met 115 120 125 Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro 130 135 140 Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn 145 150 155 160 Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu 165 170 175 Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val 180 185 190 Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln 195 200 205 Lys Ser Leu Ser Leu Ser Pro Gly Lys 210 215 <210> 38 <211> 14 <212> PRT <213> Mus musculus <220> <221> MISC_FEATURE <222> (1)..(14) <223> CDR1 of Light Chain le Domain of Anti-CD3 Antibody <400> 38 Arg Ser Ser Thr Gly Ala Val Thr Thr Ser Asn Tyr Ala Asn 1 5 10 <210> 39 <211> 7 <212> PRT <213> Mus musculus 2546512v1 <220> <221> MISC_FEATURE <222> (1)..(7) <223> CDR2 of Light Chain Variable Domain of Anti-CD3 Antibody <400> 39 Gly Thr Asn Lys Arg Ala Pro 1 5 <210> 40 <211> 9 <212> PRT <213> Mus musculus <220> <221> MISC_FEATURE <222> (1)..(9) <223> CDR3 of Light Chain Variable Domain of D3 Antibody <400> 40 Ala Leu Trp Tyr Ser Asn Leu Trp Val 1 5 <210> 41 <211> 5 <212> PRT <213> Mus musculus <220> <221> MISC_FEATURE <222> 5) <223> CDR1 of Heavy Chain Variable Domain of Anti-CD3 Antibody <400> 41 Thr Tyr Ala Met Asn 1 5 <210> 42 <211> 19 <212> PRT <213> Mus musculus <220> <221> MISC_FEATURE <222> (1)..(19) <223> CDR2 of Heavy Chain Variable Domain of Anti-CD3 Antibody <400> 42 2546512v1 Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp Ser 1 5 10 15 Val Lys Asp <210> 43 <211> 14 <212> PRT <213> Mus musculus <220> <221> MISC_FEATURE <222> (1)..(14) <223> CDR3 of Heavy Chain Variable Domain of Anti-CD3 Antibody <400> 43 His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe Ala Tyr 1 5 10 <210> 44 <211> 17 <212> PRT <213> Mus musculus <220> <221> MISC_FEATURE <222> (1)..(17) <223> CDR1 of Light Chain Variable Domain of Anti-CD123 dy <400> 44 Lys Ser Ser Gln Ser Leu Leu Asn Ser Gly Asn Gln Lys Asn Tyr Leu 1 5 10 15 <210> 45 <211> 7 <212> PRT <213> Mus musculus <220> <221> MISC_FEATURE <222> (1)..(7) <223> CDR2 of Light Chain Variable Domain of Anti-CD123 Antibody <400> 45 Trp Ala Ser Thr Arg Glu Ser 1 5 2546512v1 <210> 46 <211> 9 <212> PRT <213> Mus musculus <220> <221> MISC_FEATURE <222> 9) <223> CDR3 of Light Chain Variable Domain of Anti-CD123 Antibody <400> 46 Gln Asn Asp Tyr Ser Tyr Pro Tyr Thr 1 5 <210> 47 <211> 5 <212> PRT <213> Mus musculus <220> <221> MISC_FEATURE <222> (1)..(5) <223> CDR1 of Heavy Chain Variable Domain of Anti-CD123 Antibody <400> 47 Asp Tyr Tyr Met Lys 1 5 <210> 48 <211> 17 <212> PRT <213> Mus musculus <220> <221> MISC_FEATURE <222> (1)..(17) <223> CDR2 of Heavy Chain le Domain of Anti-CD123 Antibody <400> 48 Asp Ile Ile Pro Ser Asn Gly Ala Thr Phe Tyr Asn Gln Lys Phe Lys 1 5 10 15 <210> 49 <211> 7 <212> PRT <213> Mus musculus 2546512v1 <220> <221> MISC_FEATURE <222> (1)..(7) <223> CDR3 of Heavy Chain Variable Domain of Anti-CD123 Antibody <400> 49 Ser His Leu Leu Arg Ala Ser 1 5 <210> 50 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Heterodimerization Domain <400> 50 Gly Val Glu Pro Lys Ser Cys 1 5 <210> 51 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Heterodimerization Domain <400> 51 Val Glu Pro Lys Ser Cys 1 5 <210> 52 <211> 7 <212> PRT <213> Artificial ce <220> <223> Heterodimerization Domain <400> 52 Gly Phe Asn Arg Gly Glu Cys 1 5 <210> 53 <211> 6 <212> PRT 2546512v1 <213> Artificial Sequence <220> <223> dimerization Domain <400> 53 Phe Asn Arg Gly Glu Cys 1 5 <210> 54 <211> 227 <212> PRT <213> Artificial Sequence <220> <223> Third Polypeptide Chain of DART-A w/Fc Version 1 Construct <400> 54 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser 130 135 140 Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 2546512v1 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn Arg Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys <210> 55 <211> 10 <212> PRT <213> Artificial ce <220> <223> Peptide 1 <400> 55 Asp Lys Thr His Thr Cys Pro Pro Cys Pro 1 5 10 <210> 56 <211> 217 <212> PRT <213> Artificial Sequence <220> <223> Preferred CH2 and CH3 Domains of Fc Region <400> 56 Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 1 5 10 15 Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 25 30 Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr 40 45 Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 50 55 60 Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His 65 70 75 80 Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 85 90 95 2546512v1 Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln 100 105 110 Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met 115 120 125 Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro 130 135 140 Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn 145 150 155 160 Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu 165 170 175 Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val 180 185 190 Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln 195 200 205 Lys Ser Leu Ser Leu Ser Pro Gly Lys 210 215 <210> 57 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Linker 4 Polypeptide <400> 57 Ala Pro Ser Ser Ser 1 5 <210> 58 <211> 273 <212> PRT <213> Artificial Sequence <220> <223> Amino Acid ce of First Polypeptide Chain of "Control DART-2" <400> 58 Asp Phe Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser 25 30 2546512v1 Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln 40 45 Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Asn 85 90 95 Asp Tyr Ser Tyr Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile 100 105 110 Lys Gly Gly Gly Ser Gly Gly Gly Gly Glu Val Lys Leu Asp Glu Thr 115 120 125 Gly Gly Gly Leu Val Gln Pro Gly Arg Pro Met Lys Leu Ser Cys Val 130 135 140 Ala Ser Gly Phe Thr Phe Ser Asp Tyr Trp Met Asn Trp Val Arg Gln 145 150 155 160 Ser Pro Glu Lys Gly Leu Glu Trp Val Ala Gln Ile Arg Asn Lys Pro 165 170 175 Tyr Asn Tyr Glu Thr Tyr Tyr Ser Asp Ser Val Lys Gly Arg Phe Thr 180 185 190 Ile Ser Arg Asp Asp Ser Lys Ser Ser Val Tyr Leu Gln Met Asn Asn 195 200 205 Leu Arg Val Glu Asp Met Gly Ile Tyr Tyr Cys Thr Gly Ser Tyr Tyr 210 215 220 Gly Met Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser Gly 225 230 235 240 Gly Cys Gly Gly Gly Glu Val Ala Ala Leu Glu Lys Glu Val Ala Ala 245 250 255 Leu Glu Lys Glu Val Ala Ala Leu Glu Lys Glu Val Ala Ala Leu Glu 260 265 270 <210> 59 <211> 274 <212> PRT <213> cial Sequence 2546512v1 <220> <223> Amino Acid Sequence of Second ptide Chain of "Control DART-2" <400> 59 Asp Val Val Met Thr Gln Thr Pro Phe Ser Leu Pro Val Ser Leu Gly 1 5 10 15 Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser 25 30 Asn Gly Asn Thr Tyr Leu Arg Trp Tyr Leu Gln Lys Pro Gly Gln Ser 40 45 Pro Lys Val Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys Ser Gln Ser 85 90 95 Thr His Val Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110 Gly Gly Gly Ser Gly Gly Gly Gly Glu Val Gln Leu Val Gln Ser Gly 115 120 125 Ala Glu Leu Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala 130 135 140 Ser Gly Tyr Thr Phe Thr Asp Tyr Tyr Met Lys Trp Val Arg Gln Ala 145 150 155 160 Pro Gly Gln Gly Leu Glu Trp Ile Gly Asp Ile Ile Pro Ser Asn Gly 165 170 175 Ala Thr Phe Tyr Asn Gln Lys Phe Lys Gly Arg Val Thr Ile Thr Val 180 185 190 Asp Lys Ser Thr Ser Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser 195 200 205 Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Ser His Leu Leu Arg Ala 210 215 220 Ser Trp Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 225 230 235 240 2546512v1 Gly Gly Cys Gly Gly Gly Lys Val Ala Ala Leu Lys Glu Lys Val Ala 245 250 255 Ala Leu Lys Glu Lys Val Ala Ala Leu Lys Glu Lys Val Ala Ala Leu 260 265 270 Lys Glu