NZ716914B2 - Bi-specific monovalent diabodies that are capable of binding cd123 and cd3, and uses therof - Google Patents

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

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NZ716914B2
NZ716914B2 NZ716914A NZ71691414A NZ716914B2 NZ 716914 B2 NZ716914 B2 NZ 716914B2 NZ 716914 A NZ716914 A NZ 716914A NZ 71691414 A NZ71691414 A NZ 71691414A NZ 716914 B2 NZ716914 B2 NZ 716914B2
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seq
cells
dart
diabody
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NZ716914A
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Ralph Froman Alderson
Ezio Bonvini
Gurunadh Reddy Chichili
Ling Huang
Leslie S Johnson
Paul A Moore
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Macrogenics Inc
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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

Title of the Invention: Bi-Specific Monovalent Diabodies That Are Capable Of Binding CD123 And CD3, And Uses Thereof Cross-Reference to Related Applications 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 Application No. 13198784 (filed on December 20, 2013), each of which applications is herein incorporated by reference in its entirety. nce to Sequence Listing: This ation includes one or more Sequence gs 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 Invention: The present invention is directed to CD123 X CD3 cific monovalent diabodies that are capable of simultaneous binding to CD123 and CD3, and to the uses of such molecules in the ent of hematologic malignancies.
Description of Related Art: I. CD123 CD123 leukin 3 receptor alpha, IL-3Ra) is a 40 kDa le 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 ed For Receptor tion 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 expressed on CD34+ committed progenitors (Taussig, D.C. et al. (2005) “Hematopoietic Stem Cells Express Multiple Myeloid Markers.‘ Implications For The Origin And Targeted Therapy 0f Acute Myeloid ia,” Blood 106:4086-4092), but not by CD34+/CD38- normal hematopoietic 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 ryocytes. 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 precursor tions, CD123 is expressed by a subset of hematopoietic progenitor cells (HPC) but not by normal hematopoietic stem cells (HSC). CD123 is also expressed by plasmacytoid dendritic cells (pDC) and basophils, and, to a lesser extent, monocytes and eosinophils (Lopez, A.F. et al. (1989) “Reciprocal Inhibition 0fBinding Between eukin 3 And Granulocyte-Macrophage Colony-Stimulating Factor To Human Eosinophils,” Proc. Natl. Acad. Sci. (USA) 86:7022-7026; Sun, Q. et al. (1996) “Monoclonal Antibody 7G3 Recognizes The N-Terminal Domain Of The Human Interleukin-3 (IL-3) Receptor Alpha Chain And ons As A Specific IL-3 Receptor Antagonist,” Blood 87:83-92; Munoz, L. et al. (2001) “Interleukin-3 Receptor Alpha Chain (CD123) Is Widely Expressed In Hematologic Malignancies,” Haematologica 86(12):1261-1269; Masten, B]. et al. (2006) cterization 0f Myeloid And cytoid 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 ating Major ompatibility Complex Class II sion And Cytokine Production,” Blood 86:176-182).
CD123 has been reported to be overexpressed on malignant cells in a wide range of hematologic ancies including acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) (Munoz, L. et al. (2001) “Interleukin-3 Receptor Alpha Chain (CD123) Is Widely sed In Hematologic Malignancies,” Haematologica 86(12):1261-1269). Overexpression of CD123 is associated with poorer prognosis in AML (Tettamanti, M.S. et al. (2013) ting 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 lly dormant (i.e., not rapidly dividing cells) and therefore resist cell death (apoptosis) and tional chemotherapeutic agents. LSCs are characterized by high levels of CD123 expression, which is not present in the corresponding normal poietic 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; , 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). CD123 is expressed in 45%-95% of AML, 85% of Hairy cell leukemia (HCL), and 40% of acute B lymphoblastic leukemia (B-ALL). CD123 sion is also associated with multiple other malignancies/pre-malignancies: chronic myeloid leukemia (CML) itor cells (including blast crisis CML); n’s Reed rg (RS) cells; transformed non-Hodgkin’s lymphoma (NHL); some chronic lymphocytic leukemia (CLL) (CD11c+); a subset of acute T lymphoblastic leukemia (T-ALL) (16%, most immature, mostly adult), plasmacytoid dendritic cell (pDC) (DC2) malignancies and CD34+/CD38- myelodysplastic syndrome (MDS) marrow cell malignancies.
AML is a clonal disease terized by the proliferation and accumulation of transformed myeloid progenitor cells in the bone marrow, which ultimately leads to hematopoietic failure. The incidence of AML increases with age, and older patients typically have worse treatment outcomes than do younger patients (Robak, T. et al. (2009) “Current And Emerging Therapies For Acute Myeloid Leukemia,” Clin. Ther. 2:2349-2370). Unfortunately, at present, most adults with AML die from their disease.
Treatment for AML initially s in the induction of ion (induction therapy). Once remission is achieved, treatment shifts to focus on securing such ion (post-remission or consolidation therapy) and, in some instances, maintenance therapy. The standard ion induction paradigm for AML is chemotherapy with an anthracycline/cytarabine combination, followed by either consolidation herapy (usually with higher doses of the same drugs as were used during the induction period) or human stem cell transplantation, depending on the patient's ability to tolerate intensive treatment and the likelihood of cure with chemotherapy alone (see, e.g., Roboz, G.J. (2012) “Current Treatment Of Acute Myeloid Leukemia,” Curr. Opin. Oncol. -719).
Agents ntly 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 associated with AraC treatment include decreased ance to infection, a result of decreased white blood cell production; bleeding, as a result of decreased platelet production; 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 ing inhibition of DNA and RNA synthesis, disruption of higher order structures of DNA, and production of cell damaging free oxygen radicals. The most uential adverse effect of anthracyclines is cardiotoxicity, which considerably limits administered life-time dose and to some extent their ness.
Thus, unfortunately, e substantial progress 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 complete remission are expected to relapse within 3 years. The optimum strategy at the time of relapse, or for patients with the resistant disease, remains uncertain. Stem cell transplantation has been established as the most ive 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 or Assembly, Ligand Recognition, And tion 0f Signaling,” Cold Spring Harb. Perspect. Biol. 2(4):a005140; pages 1-14). In s, the complex contains a CD3}! chain, a CD38 chain, and two CD38 chains. These chains associate with a le known as the T cell receptor (TCR) in order to generate 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 eutic 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 E,” 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 105(7):913-923; Kuhns, M.S. et al. (2006) “Deconstructing The Form And Function Of The TCR/CD3 x,” 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 antigen depends upon the presence of variable domains on the immunoglobulin light and heavy chains (i.e., the VL and VH domains, respectively). The design of a diabody is based on the single chain Fv construct (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 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 mor ty,” J. Biol. Chem. 280(20):19665-19672; WO 02/02781 (Mertens et al.); n, T. et al. (2004) “Covalent Disulfide-Linked Anti- CEA Diabody Allows Site-Specific Conjugation And Radiolabeling For Tumor Targeting Applications,” Protein 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 h Variable Domain Exchange,” Protein Engineering 14(2):1025- 1033; Asano et al. (2004) “A Diabody For Cancer Immunotherapy And Its Functional Enhancement By Fusion Of Human Fc Domain,” Abstract 3P-683, J. Biochem. 76(8):992; Takemura, S. et al. (2000) “Construction Of A Diabody (Small inant Bispecific Antibody) Using A Refolding System,” Protein Eng. 13(8):.583-588; le, P.A. et al. (2009) “Bispecific T-Cell Engaging Antibodies For Cancer Therapy,” Cancer Res. 69(12):4941-4944). ction of an antibody light chain and an antibody heavy chain and, in particular, ction 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 antibody contained in a single polypeptide chain wherein the domains are separated by a flexible linker of sufficient length to allow ssembly 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 molecule in which the VL of one chain associates with the VH of the other wed in Marvin et al. (2005) “Recombinant Approaches To IgG- Like Bispecific Antibodies, ” Acta Pharmacol. Sin. 26:649-658).
Natural antibodies are capable of binding to only one epitope s (i.e., mono-specific), although they can bind multiple copies of that species (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 capable of binding two or more different epitope species (i. e., exhibiting bi-specif1city or multispecif1city in addition to bi-Valency or multi-valency) (see, e.g., er 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 ific dy 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 BIOMEDICAL USE, A.
VanBroekhoven et al. (Eds.), Kluwer Academic Publishers, cht, The Netherlands (2001), pages 195-208; Wu, A. et al. (2001) “Multimerization Of A Chimeric Anti-CD20 Single Chain Fv-Fv Fusion Protein Is ed Through Variable Domain ge,” Protein Engineering 14(2):1025-1033; Asano et al. (2004) “A Diabody For Cancer Immunotherapy And Its Functional Enhancement By Fusion OfHuman Fc Domain,” Abstract , J. Biochem. 992; Takemura, S. et al. (2000) ruction OfA Diabody (Small Recombinant ific Antibody) Using A Refolding System,” n Eng. 13(8):583-588; Baeuerle, P.A. et al. (2009) “Bispecific T-Cell Engaging Antibodies For Cancer Therapy,” Cancer Res. 69(12):4941-4944).
The provision of non-monospecific diabodies provides a significant advantage: the capacity to co-ligate and co-localize cells that express different epitopes. Bi-specific ies thus have wide-ranging applications including therapy and immunodiagnosis. Bi-specificity allows for great lity in the design and engineering of the diabody in various applications, providing enhanced avidity to multimeric antigens, the cross-linking of ing antigens, and directed targeting to specific cell types relying on the presence of both target antigens. Due to their increased valency, low dissociation 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 ular 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 8-631, and Holliger et al. (1996) “Specific Killing OfLymphoma Cells By Cytotoxic T-Cells Mediated By A Bispecific Diabody, ” Protein Eng. 9:299-305).
Diabody epitope g domains may also be directed to a surface 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 s, diabody binding to effector cell determinants, e. g., Fcy receptors (FcyR), was also found to activate the effector cell (Holliger et al. (1996) fic Killing 0fLymphoma Cells By Cytotoxic T-Cells Mediated By A Bispecific y, ” Protein Eng. 305; er et al. (1999) “Carcinoembryonic Antigen (CEA)- Specific T-cell Activation In Colon Carcinoma Induced By Anti-CD3 x EA Bispecific ies And B7 x Anti-CEA Bispecific Fusion Proteins, ” Cancer Res. 9-2916; 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 Ig-like fianctionality independent of whether they se an Fc Domain (e. g., as assayed in any or 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). r, 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 ptides (i. e., such formation requires that the diabodies be formed through the heterodimerization of different polypeptide chain species). This fact is in contrast to mono-specific diabodies, which are formed h the homodimerization of identical polypeptide chains. Because at least two dissimilar polypeptides (i.e., two polypeptide species) must be provided in order to form a nonmonospecific diabody, and because 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 prevent covalent bonding between polypeptides of the same species (i.e., so as to prevent homodimerization) (Takemura, S. et al. (2000) “Construction Of A Diabody (Small Recombinant ific Antibody) Using A Refolding System,” Protein Eng. l3(8):583-588). The art has ore taught the non-covalent association of such polypeptides (see, e.g., Olafsen et al. (2004) “Covalent Disulfide- Linked Anti-CEA Diabody Allows Site-Specific ation 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 Enhancement By Fusion OfHuman Fc Domain,” Abstract 3P-683, J. Biochem. 76(8):992; Takemura, S. et al. (2000) “Construction OfA y (Small Recombinant Bispecific Antibody) Using A Refolding System,” Protein Eng. l3(8):583-588; 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).
However, the art has recognized that bi-specific diabodies composed of non- ntly associated polypeptides are unstable and readily dissociate into non- functional monomers (see, e.g., Lu, D. et al. (2005) “A Fully Human Recombinant ke 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):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 Re-Targeting 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 tion Via Recruitment Ochgamma Receptor Hb (CD323) Inhibitory Function With A Novel ific Antibody Scaflold,” Arthritis Rheum. 62(7):l933-l943; Moore, P.A. et al. (2011) “Application Of Dual Afiinity eting les To Achieve Optimal Redirected T-Cell Killing 0f B-Cell Lymphoma,” Blood ll7(l7):4542-4551). Such approaches e engineering one or more cysteine residues into each of the employed polypeptide s. For example, the addition of a ne 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 s, the tion of stable, fianctional heterodimeric, non-monospecif1c diabodies can be fiarther optimized by the careful consideration and placement of cysteine residues in one or more of the employed polypeptide chains. Such optimized diabodies can be produced in higher yield and with greater activity than non-optimized diabodies. The present invention is thus directed to the problem of providing polypeptides that are particularly designed and optimized to form heterodimeric ies. The invention solves this problem through the provision of exemplary, zed CD123 X CD3 diabodies.
Summary of the ion: The t invention is directed to CD123 X CD3 bi-specif1c diabodies that are capable of simultaneous binding to CD123 and CD3, and to the uses of such molecules in the treatment of disease, in particular hematologic malignancies.
The CD123 X CD3 bi-specific diabodies of the ion comprise at least two different ptide chains that ate 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 diabody of the invention is thus lent in that it is capable of binding to only one copy of an epitope 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 covalently bonded to one another, 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- c 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 ion: 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 ; and (2) a main (1B), which comprises a VH Domain of a monoclonal dy capable of binding to CD123 (VHCDm) (SEQ ID NO:26); wherein the sub-Domains 1A and 1B are ted 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 (VLCDm) (SEQ ID NO:25); and (2) a sub-Domain (1B), which ses 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 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 separated 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; -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 cally 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 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, sing: (1) a sub-Domain (1A), which comprises a VL Domain of a monoclonal antibody capable 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 ) (SEQ ID NO:28); 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 , n the Domain 2 is separated from the Domain 1 by a peptide linker (SEQ ID NO:30); B. the second ptide 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 (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 ; 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 a K-coil Domain (SEQ ID NO:35) or an E-coil Domain (SEQ ID NO:34), 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 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 onally provides the embodiment of the above-described cific monovalent ies, wherein the first or second ptide chain additionally comprises an Albumin-Binding Domain (SEQ ID NO:36) , C- terminally to Domain 2 or N-terminally to Domain 1, via a peptide linker (SEQ ID NO:31).
The invention additionally provides the embodiment of the above-described bi-specific monovalent diabodies wherein the first or second polypeptide chain additionally comprises a Domain 3 sing a CH2 and CH3 Domain of an immunoglobulin 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 invention onally provides 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 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 es the embodiment of any of the abovedescribed bi-specific monovalent diabodies 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 n 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 e of CD123 and to an epitope of CD3, wherein the diabody comprises a first polypeptide chain and a second ptide chain, covalently bonded to one r, wherein: said bi-specific y comprises: A. a first polypeptide chain having the amino acid sequence of SEQ ID NO:1; and B. a second ptide chain having the amino acid sequence of SEQ ID NO:3; wherein said first and said second polypeptide chains are covalently bonded to one r by a disulfide bond.
The ies of the invention exhibit unexpectedly enhanced functional activities as further described below.
The diabodies 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 invention are preferably capable of depleting, in an in vitro cell-based assay, plasmacytoid 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 0.01ng/ml or less. Preferably, the IC50 is about 0.01ng/ml or less. In the above-described assay, the e of primary PBMCs may be from cynomolgus monkey in which case said depletion is of cynomolgus monkey plasmacytoid tic cells (pDC). Optionally the diabodies of the invention may be capable of depleting plasmacytoid dendritic cells (pDC) from a y culture of PBMCs as described above wherein the assay is conducted by or in accordance with the protocol of e 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 ordinary 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 invention may exhibit cytotoxicity as described above wherein the assay is conducted by or in ance with the protocol of Example 3 as herein described, or by modification of such assay as would be understood by those of ry skill, or by other means known to those of ordinary skill.
The diabodies of the invention preferably exhibit cytotoxicity in an in vitro 3 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 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 tood 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 invention 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 t growth of a MOLM-l3 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. ally, the ies 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 ry skill.
The diabodies of the invention are preferably 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 xenograft 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 inhibit growth of a RS4-ll tumor xenograft 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 . 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 e 6 as herein bed, 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 depleting leukemic blast cells in vitro in a primary culture of AML bone marrow cells. Preferably the diabodies of the ion may be capable of depleting leukemic blast cells in vitro in a primary culture of AML bone marrow cells at trations 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 depleting 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 following incubation of the primary e with diabody for about 120 hours. ably leukemic blast cells are depleted in vitro in a primary culture of AML bone marrow cells to less than 20% of the total tion of primary leukemic blast cells at diabody concentrations of about 0.01 ng/ml or 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 expansion of a T cell population in vitro in a primary culture of AML bone marrow cells. Preferably, such expansion may be to about 70% or more of the maximum T cell population which can be expanded in the assay. Preferably the ies of the ion may be capable of inducing an expansion of a T cell population in vitro in a y 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 primary culture with diabody for about 120 hours.
Preferably, a T cell population is expanded 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 about 0.01 ng/ml or about 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 activation of a T cell population in vitro in a primary culture of AML bone marrow cells. Such activation may occur at y 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 primary culture with diabody for about 72 hours. Such activation may be measured by the expression 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 following tion of the primary culture with diabody for about 72 hours.
The ies of the invention are preferably capable of depleting 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 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 incubation of the y culture with diabody 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 ic blast cells in vitro in a primary culture of AML bone marrow cells and/or inducing an ion 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 ation of such assay as would be tood 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 onal attributes described herein.
Thus the diabodies of the invention may exhibit any combination of the fianctional utes described herein.
The ies of the invention may be for use as a pharmaceutical.
Preferably, the diabodies are for use in the treatment of a disease or condition associated with or characterized by the expression of CD123. The invention also relates to the use of diabodies of the invention in the manufacture of a pharmaceutical composition, preferably for the treatment of a disease or condition ated 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), c myelogenous leukemia (CML), including blastic crisis of CML and Abelson oncogene associated with CML (Bcr-ABL translocation), myelodysplastic syndrome (MDS), acute B lymphoblastic leukemia ), 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), ing mantel cell leukemia (MCL), and small lymphocytic lymphoma (SLL), Hodgkin’s lymphoma, systemic mastocytosis, and Burkitt’s lymphoma.
The e or condition associated with or characterized by the sion of CD123 may be an atory condition. For e, the inflammatory condition may be selected from the group consisting of: mune 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 es a use of the above-described pharmaceutical composition in the treatment of a disease or condition ated with or characterized by the expression of CD123.
The invention is particularly directed to the embodiment of such use, wherein the disease or condition associated with or characterized by the expression of CD123 is cancer (especially a cancer selected from the group consisting of: acute myeloid leukemia (AML), chronic myelogenous leukemia (CML), including blastic crisis of CML and n oncogene associated with CML (Bcr-ABL translocation), myelodysplastic syndrome (MDS), acute B lymphoblastic leukemia (B-ALL), c lymphocytic leukemia (CLL), ing r’s syndrome or Richter’s transformation of CLL, hairy cell leukemia (HCL), blastic plasmacytoid tic 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 directed to the embodiment of such use, n the disease or condition associated with or characterized by the expression of CD123 is an inflammatory 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 ption 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 present invention.
Figures 3A and 3B illustrate the structures of two versions of the first, second and third polypeptide chains of a three chain CD123 X CD3 cific monovalent Fc diabody of the present invention (Version 1, Figure 3A; Version 2, Figure 3B).
Figure 4 (Panels A-E) shows the ability of different CD123 X CD3 bi- specific diabodies to mediate T cell cted 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 (“DART-B”) in le 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 y of the sequence-optimized CD123 X CD3 bi-specif1c diabody (DART-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 cted killing of target cells. The Figure ts esponse 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 purified CD8 T cells at an E:T tor 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 presence (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 (Panels A-B) shows the in viva antitumor activity of the sequence- zed CD123 X CD3 bi-specif1c diabody (DART-A) at nanogram per kilogram dosing levels. MOLM-l3 cells (intermediate 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 antitumor activity of the sequence-optimized CD123 X CD3 bi-specific diabody 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 concentrations of DART-A were compared to a control bi- specific diabody (Control DART).
Figure 9 (Panels A-B) shows CD123+ blasts in bone marrow cleocytes (BM MNC) and peripheral blood mononucleocytes (PBMCs) from AML patient 1 (Panel A) ed to Kasumi-3 AML cell line (Panel B).
Figure 10 (Panels A-C) shows the y of the sequence-optimized CD123 X CD3 bi-specific diabody (DART-A) to mediate blast reduction 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 cyte 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 tions of T cells present in the samples. The numbers indicate that CD4 T cells represent approximately 0.5% of the total cells and CD8 T cells ent approximately 0.4% of the total cells present in the ALL PBMC sample.
Figure 13 (Panels A-H) shows the y of the sequence-optimized CD123 X CD3 bi-specific diabody (DART-A) to mediate ALL blast depletion with autologous CTL. Panels A and E show the forward and side scatter of the input population 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 ted for 7 days followed by staining for CD34 and CD19.
Figure 14 (Panels A-L) shows the ability of the ce-optimized CD123 X CD3 bi-specific diabody A) to mediate T cell expansion (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 cific diabody (Control DART) (Panels B, E, H and K) or DART-A (Panels C, F, I and L) for 7 days.
Figure 15 (Panels A-C) shows the identification of the AML blast population and T cells in a y AML sample. Panel A shows the forward and side r of the input AML PBMCs. Panel B shows the identification of the AML blast tion in the AML sample. Panel C shows the identification of the T cell population in the AML sample.
Figure 16 (Panels 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 control 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 ol 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 t 2 following incubation with Control DART or DART-A with autologous PBMCs.
Figure 18 (Panels A-D) shows that the sequence-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 results 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) proteins. The KD values are ed.
Figure 19 (Panels A-B) shows the ability of the sequence-optimized CD123 X CD3 bi-specific diabody (DART-A) to mediate gous monocyte depletion in vitro with human and cynomolgus monkey PBMCs. The Panels present the s 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 bi-specific y A) to mediate the depletion ofpDC in cynomolgus monkeys without systemic cytokine induction. Panels A-D show control results obtained at 4h and 4d with vehicle and carrier. Panels E-H show control results ed at 4h and 4d with a control bi-specific diabody ol 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 cific diabody (DART-A) to mediate dose-dependent depletion ofpDC in cynomolgus s. Cynomolgus monkeys were dosed with DART-A at 0.1, 1, 10, 100, 300, or 1000 ng/kg. PBMCs were evaluated at the indicated 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 sequence-optimized CD123 X CD3 bi-specific diabody (DART-A) to intermittently modulate T cells in cynomolgus monkeys. Cynomolgus s 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 calibrated TSK G3000SWXL column. Figure 24B: Mass um of DART-A n.
Figures 25A-25D show SPR is 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 tional 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). Titrating DART-A and Control 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 e 26E). Binding was detected by FACS analysis using a monoclonal antibody specific to E-coil and K-coil region of the DART-A or Control DART molecule.
Figures H 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, trate the ability of the les to bind to subsets of normal circulating leukocytes, including pDCs and monocytes and demonstrate the y of the molecules to deplete CD14'CD123high cells (pDC and ils) without affecting tes (CD14+ cells). Figure 27A shows the relative anti-CD123-PE binding sites on U937 and Kasumi-3 leukemic cell lines as determined 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 sites). Figure 27C shows the percent cytotoxicity ed 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 binding sites on Kasumi-3 cells, human tes, human plasmacytoid dendritic cells (“pDC”), cynomolgus monkey monocytes and cynomolgus monkey plasmacytoid dendritic cells as determined by QFACS analysis. Figure 27F shows the y 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 lgus monkey CD14’ CD123Hi cells. xicity was determined by LDH release, with ECSO values ined using GraphPad PRISM® software.
Figure 28 shows the use of a two-compartment model to estimate pharmacokinetic parameters of . 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 29A-29C show the effect of DART-A ons 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 vehicle (Group 1) (Figure 29A) or DART-A stered as 4-day weekly infilsions ng on Days 8, , 22, and 29 s 2-5) (Figure 29B) or as a 7-day/week infusion 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 CDl4-/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, ed 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 infiJsion for 4 weeks starting on Days 8 (Group 6) (Figures 30C and 30F). Treatment intervals are indicated by the filled gray bars.
Figures 31A-311 show the observed changes in T cell populations es 31A-31C), CD4+ cell tions (Figures 31D-31F) and CD8+ cell populations (Figures 31G-311) ing 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. lgus s were treated with vehicle control on Day 1, followed by 4 weekly infiJsions of either vehicle (Group 1) or DART-A administered as 4-day weekly infilsions ng 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 percent :: SEM) of CD25+, CD69+, PD-1+ and Tim-3+ of CD4 (Figures 31D-31E) or CD8 T cells (Figures 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 uous 7-day infiasion of DART-A. The mean :: SEM percent of CD25+, CD69+, PD-1+ and Tim-3+ on CD4 (Figures C) or CD8 (Figures 32D-32F) T cells by Study Day for Groups 2, 3 and 4 are shown. Treatment intervals 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 (Figures C) 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 /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, ed by 4 weekly infilsions of or DART-A administered as 4- day weekly infusions starting on Days 8, 15, 22, and 29 (Groups 2-4). Treatment als are indicated by the filled gray bars. Legend: Naive (white triangles); CMT (black triangles), EMT (gray squares).
Figure 34 shows DART-A-mediated cytotoxicity against -3 cells with PBMCs from either naive monkeys or monkeys treated with multiple infilsions of DART-A.
Figures F show that DART-A exposure increased the relative frequency of central memory CD4 cells and or 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 control on Day 1, followed by 4 weekly infusions of either e (Group 1) or DART-A administered as 4-day weekly infilsions ng on Days 8, 15, 22, and 29 (Group 5) or as a 7-day/week lI‘lfiISlOIl 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 squares).
Figures 36A-36F show the effect of DART-A on red cell parameters in monkeys that had received infusions of the molecules. Circulating RBCs (Figures 36A-36C) or reticulocytes (Figures 36D-36F) levels (mean :: SEM) in samples ted at the indicated time points from monkeys treated with DART-A are shown.
Figures 37A-37B show that the frequency (mean percent :: 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. Cynomolgus monkeys were treated with vehicle control on Day 1, followed by 4 weekly infusions of either vehicle (Group 1) or DART-A administered as 4-day weekly infusions starting 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 t invention is directed to sequence-optimized CD123 X CD3 bi- specific lent diabodies that are capable of simultaneous binding to CD123 and CD3, and to the uses of such molecules in the treatment of hematologic malignancies.
Although non-optimized CD123 X CD3 bi-specific diabodies are fully functional, analogous to the improvements obtained in gene sion through codon optimization (see, e.g., Grosjean, H. et al. (1982) rential 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 on of CD123 X CD3 bi-specif1c diabodies by modifying or refining their sequences.
The preferred CD123 X CD3 cif1c ies of the present invention are composed of at least two polypeptide chains that 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 (Figure 2). The individual ptide chains of the diabody are covalently bonded to one another, for example by disulf1de g of cysteine residues located within each polypeptide chain. Each polypeptide chain contains 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 peptide (Linker 1) separates the n Binding Domain of the Light Chain Variable Domain from the Antigen Binding Domain of the Heavy Chain Variable 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 specific for the first antigen (i. e., either CD123 or CD3). Likewise, the n Binding Domain of the Light Chain Variable Domain of the second polypeptide chain cts with the Antigen Binding Domain of the Heavy Chain Variable Domain of the first ptide 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 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 Domains of light and Heavy Chain Variable Domains capable of g to CD123 and CD3.
The formation of heterodimers of the first and second polypeptide chains can be driven by the heterodimerization domains. Such domains 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 (US2007/0004909). atively, 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, arginine, histidine, etc. and/or the negatively charged amino acid may be glutamic acid, aspartic acid, etc. The positively charged amino acid is ably lysine and/or the negatively charged amino acid is ably 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 r via cysteine residues along their length. Such cysteine residues may be introduced into the intervening linker 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 the amino-terminus of the polypeptide chains or a position 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 ular 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 ion that comprise an immunoglobulin Fc Domain (2'.e., CD123 X CD3 bi-specific monovalent Fc ies) are composed of a first ptide chain, a second polypeptide chain and a third polypeptide chain. The first and second polypeptide chains associate with one another to form one g 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 disulfide g of cysteine residues located within each polypeptide chain. The first and second polypeptide chains each contain 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 intervening linker peptide r 1) separates the Antigen Binding Domain of the Light Chain le Domain from the Antigen g Domain of the Heavy Chain Variable 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 n binding site that is specific for the first n (2'.e., either CD123 or CD3).
Likewise, the Antigen 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 c for the second antigen (i.e., either CD3 or CD123, ing upon the identity of the first antigen). Thus, the selection of the n 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 Binding Domains of light and Heavy Chain le 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 te 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 present invention. The first and third polypeptide chains of the cific monovalent Fc diabodies of the present invention are ntly bonded to one another, for example by disulfide g 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 provides a sequence-optimized bi-specific diabody e 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 polypeptide 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 g 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 red sequence for such a VLCD3 Domain is SEQ ID NO:21: QAVVTQ?A PSLTVS LTCRS S TGAVTT SWYANWVQQKPGQAPRGL 22GG TNKRAPWT PARFSGSLLGGKAALT: TGAQA*12D *iADYYCALWYSNLWVFGGGT KLTVLG The Antigen g Domain of VLCD3 comprises CDRl SEQ ID NO:38: RSSTGAVTTSNYAN, CDRZ SEQ ID NO:39: P, and CDR3 SEQ ID NO:40: ALWYSNLWV.
A preferred sequence for such Linker l is SEQ ID NO:29: GGGSGGGG. A red ce for such a VHCDm Domain is SEQ ID NO:26: EVQLVQSGAELKKPGASVKVSCKASGYTFTDYYMKWVRQAPGQGT.*1W GD 9SNGATFYNQKFKGRVT 22TV2DKSTSTAYM'.T. SST.RS *iDTAVYYCA'RSl-ILLRA SWFAYWGQGTLVTVSS The Antigen Binding Domain ofVHCDm comprises 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 capable 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 preferred 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: '.VQT.V'...SGGGTVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGTWIVGR R ATYYADSVKDRFT SRDDSKNSLYLQMNSLKTEDTAVYYCVRHGNF GNSYVSWFAYWGQGTLVTVS S The Antigen Binding Domain of VHCD3 comprises CDRl SEQ ID NO:41: TYAMN, CDR2 SEQ ID NO:42: RIIRSKYNNYATYYADSVKD, and CDR3 SEQ ID N(h43:HGNFGNSYVSWFAY.
The ce-optimized CD123 X CD3 bi-specif1c diabodies of the present invention are engineered so that such first and second polypeptides covalently 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 polypeptide chain.
A red sequence for such Linker 2 is SEQ ID NO:30: GGCGGG.
The formation of heterodimers can be driven by further ering such polypeptide chains to contain polypeptide coils of opposing charge. Thus, in a preferred embodiment, 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 d domains promotes association n 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 preferred sequence-optimized CD123 X CD3 bi-specif1c diabody of the present invention -A”) has a first ptide chain having the sequence (SEQ ID NO:1): QAVVTQIEPSLTVSPGGTVTLTCRSSTGAVTTSWYANWVQQKPGQAPRGLIIGG TNKRAPWTPARFSGSLLGGKAALTI TGAQA*DfiADYYCALWYSWLWVFGGGT KLTVLGGGGSGGGGIEVQLVQSGAIELKKPGASV{VSC{ASGYTFTDYYM<WVR Tfi'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 acaaacaaaagggc:ccctggacccctgcacgg,,,,c,ggaag:ctgctgg gcggaaaggccgcchgaccacLaccggggcacaggccgaggacgaagccga ,Lac,aLLngchcgcgg,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 cL,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 composed 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 ng polynucleotide is SEQ ID NO:4: gac,ch,gaLgacacachLcc,gaLag,chgccgtgagtctgggggagc ggg,gac,a,g,cL,gcaagagc:cccag:cactgctgaacagcggaaatca gaaaaaccacccgaccEggcaccagcagaagccaggccagccccccaaaccg aLcgggc,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, cacca: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 activation and to cause the redirected killing of target cancer cells. 11. Comparative Non-Sequence-Optimized CD123 x CD3 Bi-Specific y, “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 . The first 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 CD3 (VLCDg), an intervening linker peptide (Linker l), a VH domain of a monoclonal antibody capable of g to CD123 (VHCDm), an intervening 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< VASGVPYQFSGSGSGTSYSTT SSMmAfiDAATYYCQQWSSNPLTFGAGTKLE The VHCDm Domain of the first polypeptide chain of DART-B has the ce (SEQ ID NO:28): SGAIELKKPGASVKVSCKASGYTFTDYYMKWVQQAPGQGT*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): QS?AIMSASPGEKVTMTCQASSSVSYMNWYQQKSGTSPKQW_ YDTS A VASGVPYQFSGSGSGTSYSTT SSM*A*DAATYYCQQWSSWPLTFGAGTKLLL-El LKGGGSGGGGQVQLVQSGAIELKK ?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 chngchggaccaagc2ggag ctgaaaggaggcgga:ccggcggcggaggccagg:gcagcngLgcachcg gggc:gagctgaagaaacccggagcLLcchgaagngLcLLgcaaagccag tggCCacaccttcacagacLacLaLa,gaagngchaggcaggctccagga cagggac,ggaa,ggachgcgaLa,caLLch,ccaacggggccaCLLLCL acaatcagaagLLLaaaggcaggngacLaLLacchggacaaatcaacaag caCLchLaLa,ggachgagc,cchgcchc,gaagatacagcchgLac chcgg,cacacctgc:gagagccagc,ggLL,chLaLngggac agggcacccngLgacangLcLLccggaggatgtggcggtggagaagtggc 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 dy capable of binding to CD123 ), an intervening linker peptide (Linker l) and a VH domain of a monoclonal antibody capable of g to CD3 (VHCDg), an intervening Linker 2, a K-coil Domain, and a C-terminus.
The VLCDm Domain of the second ptide 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 ce (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: ,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 gggaccaagc:ggaaa:taaaggaggcgga:ccggcggcggaggcg atatcaaaCCgcagcagtcaggggccgaaccggcaagacc:ggggcc:cagt gaagaLg,cc,gcaagacL,chgc,acach,,ac,agg,acacga:gcac tgggtaaaacagaggcc: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 Bi-Specific Diabody (DART-A) A. ce-Optimized CD123 x CD3 Bi-Specific Diabody Having An Albumin-Binding Domain A with ABD “w/ABD”) In a second embodiment of the invention, the sequence-optimized CD123 X CD3 bi-specific diabody (DART-A) will comprise one or more Albumin-Binding Domain (“ABD”) (DART-A with 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 ies, 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 y. 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. 277(10):8114-8120). Albumin is the most nt protein in plasma and has a half-life of 19 days in humans. Albumin possesses several small molecule binding sites that permit it to non-covalently bind to other ns and thereby extend their serum half-lives.
Thus, the first polypeptide chain of such a ce-optimized CD123 X CD3 bi-specific diabody having an Albumin-Binding Domain contains a third linker r 3), which separates the E-coil (or K-coil) of such polypeptide chain from the Albumin-Binding Domain. A preferred sequence for such Linker 3 is SEQ ID NO:31: GGGS. A preferred 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- specif1c 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 QAPGQGTfi'W GD 9SNGATbYNQKb<G<VT TVDKSTSTAYMTSST<SfiD TAVYYCARS{LLRASWFAYWGQGTLVTVSSGGCGGGWVAATfiKVAATfiKfiV VAAPm<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 mflmemmmngmmhasammweommnmflCIH23XCD3dfihflyhmflgan Albumin-Binding Domain derivative is SEQ ID NO:10: caggctgtggtgactcaggagccttcactgaccgtgtccccaggcggaactg tgaccctgacatgcagatccagcacaggcgcagtgaccaca:ctaactacgc caattgggtgcagcagaagccaggacaggcaccaaggggcc,gaecgggggt aaaagggc:ccctggacccctgcacgg,L,Lceggaagtctgctgg gcggaaaggccgcechaceaeLaccggggcacaggccgaggacgaagccga e,ac,aLLngc,cegeggea,agcaaech,ggg,geecgggggtggcaca acengcegggagggggeggaeccggcggcggaggcgaggtgcagc egngcag,ccggggcegagcegaagaaacccggagceecchgaaggtgtc eegcaaagccageggceacaccttcacagaceacLaeaegaageggchagg caggctccaggacagggaceggaa,ggachgcgaLaecaeLch,ccaacg c,L,cLacaa,cagaag,,Laaaggcaggg,gac,aLeaccgegga caaatcaacaagcac,ch,aea,ggachgagc,cchgcgcecegaagat acagccgegeacLaLegechcgg,cacacctgc:gagagccagc,gg,,,g CL,a,ngggacagggcacccegg,gacagech,Lccggaggatgtggcgg ag:ggccgcac:ggagaaagaggLechchLeggagaaggaggtc gcegcaceegaaaaggaggtcgcagccctggagaaaggcggcgggechegg ccgaagcaaaagtgctggccaaccgcgaaceggaLaaa,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 ce 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 embodiment, the invention provides a sequence-optimized CD123 X CD3 bi-specific diabody ed 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 n, in the N-terminal to C-terminal direction, a cysteine-containing peptide, (most preferably, Peptide 1 having the amino acid sequence (SEQ ID NO:55): 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- containing diabodies of the present invention. The first and second polypeptide chains of the bi-specific monovalent Fc diabodies of the present invention are covalently bonded to one another, for e 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 example, 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 ction with a similarly mutated domain and will obligate the d 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 ered 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 globulin-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 ling 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 entirety). 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 merizing 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 modifying a native IgG Fc Domain to contain the modification T366W. A preferred hole is created by modifying a native IgG Fc Domain to n the modification T366S, L368A and Y407V. To aid in purifying the third polypeptide chain homodimer from the final bi-specific monovalent Fc y comprising the first, second and third polypeptide chains, the protein A binding site of the CH2 and CH3 s of the third polypeptide chain is preferably mutated by amino acid substitution at position 435 (H435R). Thus, the third ptide chain homodimer will not bind to protein A, Whereas the bi-specific monovalent Fc diabody will retain its y to bind protein A via the protein A binding site on the first polypeptide chain.
] A preferred sequence for the CH2 and CH3 Domains of an antibody Fc Domain present in the first polypeptide chain is (SEQ ID NO:56): APLAAGGPSVFLFPPKPKDTL VTCVVVDVSH4DP*‘V<EWWYV3GV LUVTNAKTK9<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): GPSVFLFPPKPKDTT SRTPLVTCVVVDVSH4DP4V<EWWYVJGV 'UI—IILUVTNAKTK9<4'QYWSTYRVVSVLTVL{QDWLNG<LYKC<VSN<AL9A9 4K :SKAKGQW9{L9QVYTTP9SRL*MTKVQVSLSCAV<GEY9SD AV4W4SWGQ .3WNYKTT 99VLDSDGSFFLVS<LTVD<S9WQQGWVFSCSV HLALTV<YTQ <SLSLSPG< C. DART-A w/Fc Version 1 Construct 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 version 1 uct comprises, 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 e (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 preferred 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 sequence (SEQ ID NO:37): APEAAGGPSVFLFPPKPKDTP VTCVVVDVSH4DPL1V<EWWYVDGV 'Ul—ZIL-ljViNAKTK9&4L1QYNSTY9VVSVLTVL{QDWLNG<EYKC<VSN<AL9A9 4K :SKAKGQW9{_L9QVYTTP9SRLLMTKNQVSLWCLV<GEY9SD AV4W4SWGQ .3WNYKTT99VLDSDGSFFLYS<LTVD<S9WQQGWVFSCSV HEALdWiYTQ 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 sequence 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 SRDDSKNSLYLQ NS LKTEDTAVYYCVR{GWFGWSYVSWFAYWGQGTLVTVSSGGCGGGLVAATLK4 4VAAPLKLVAAT4<GGGD<TdTC99C9APLAAGG9SVFLFPPK9KD TR S<TP4VTCVVVDVS{4DP4V<EWWYVDGVEV{NAKT<9<4L1QYWSTY9 VVSVLTVL{QDWLWG<EY<C<VSW<AP9A9 4<T 9<L9QVYTLP9 SR4L1MTKWQVSLWCLV<GEY9SD AV4W4SWGQ 9EWNYKTT99VLDSDGSFF LYS<LTVD<S9WQQGWVFSCSV {EALdeYTQ<SLSLSPG< A preferred polynucleotide encoding 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, cgccaggc2ccagggaaggggcngagvgggvngaaggavcaggv ccaagtacaacaattetgcaachacLa,gccgactc:gtgaagga,agaLL caccaLCLcaagagatgattcaaagaacLcachLaLchcaaa:gaacagc ctgaaaaccgaggacecggcchgLaLLachLngagacacggLaacLch gcaa,LC,LachgLCLngLLLchLa,ngggacaggggacactggtgac LngLcLLccggagge:gtggcggtggagaagtggccgcac:ggagaaagag gLLchgCLLngageaggaggvcchgcacLLgaaaaggaggtcgcagccc :ggagaaaggcggcggggacaaaac:cacacatgcccaccgtgcccagcacc :gaagccgcggggggecchcachLLCCLCLLccccccaaaacccaaggac accctcatgatctcccggacccc,gagchacaLgchgngg,ggacgtga gccacgaagacchgegchaag,Lcaac,ggLachggacggcgtggaggt gcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccg'(I gtggtcagcgtcc:caccgtcctgcaccaggactggc:gaatggcaaggag'(I acaagtgcaaggtctccaacaaagccctcccagccccca:cgagaaaacca(I agccaaagggcagccccgagaaccacaggtg:acaccctgccccca tcccgggaggagatgaccaagaaccaggtcagccchggchcvggvcaaag ch,cLa,cccagcgacatcgccgtggag:gggagagcaatgggcagccgga gaacaac2acaagaccacgccLccchgccggac ,ccgacggc,cc,Lc,Lc agcaagctcaccg:ggacaagagcaggtggcagcaggggaacg:ct gc,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 e (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): EPSLTVSPGGTVTLTCRSSTGAVTTSWYANWVQQKPGQAPRGL_IGG TNKRAPWTPARFSGSLLGGKAALTI TGAQAfiDfiADYYCALWYSWLWVFGGGT KLTVLGGGGSGGGG_EVQLVQSGA_ELKKPGASV{VSCKASGYTFTDYYMKWVR Tfi'W GD FYNQKFKGRVT--TVDKSTSTAY *TSSTRSmD TAVYYCARSHLLRASWFAYWGQGTLVTVSSGGCGGGKVAATK:‘J <VAAT.Km<V AAPKTKVAATKm A preferred polynucleotide encoding such a polypeptide has the sequence (SEQ ID NO:16): caggctgtggtgactcaggagccttcactgaccgtgtccccaggcggaactg tgaccctgacatgcagatccagcacaggcgcag:gaccaca:ctaactacgc caattgggtgcagcagaagccaggacaggcaccaaggggcc “gaucgggggt acaaacaaaagggc:ccctggacccctgcacgg ,L,Lc,ggaag:ctgctgg gcggaaaggccgcchgaccavLaccggggcacaggccgaggacgaagccga ,,ac,aLLngc,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 Domains of an IgG Fc Domain. A preferred polypeptide that is ed 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 SRTPfi'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 chcag,c3Lcc,cLoccccccaaaacccaaggacaccctcatgatctcccg gacccc,gagchacaLgchggngngacgtgagccacgeagaccc:gag chaagoLcaac,gg,acgngacggcgtggaggtgcataatgccaagacaa agccgcgggaggagcagtacaacagcacgLaccg,gogchegcgtcc:cac cgtcctgcaccaggactggc:gaatggcaaggag:acaagtgcaaggtctcc aacaaagccctcccagcccccatcgagaaaaccacccccaaegccaaagggc agccccgagaaccacaggtg: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 y, “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 inal ion, an N—terminus, a peptide linker (Peptide l), a polypeptide that contains the CH2 and CH3 s of an Fc Domain linked (via a Linker 4) to the VL domain of a monoclonal dy capable 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, 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 r 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 W<AP9A9 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): ac:cacacatgcccaccgtgcccagcacctgaagccgcggggggac chcag,c4Lcc,cL,ccccccaaaacccaaggacaccctcatgatctcccg gacccc,gagchacaLgchggngngacgtgagccacgeagaccc:gag chaag,Lcaac,gg,acgngacggcgtggaggtgcataatgccaagacaa agccgcgggaggagcagtacaacagcacgLaccg,g,gchegcgtcc:cac cgtcctgcaccaggactggc:gaatggcaaggag:acaagtgcaaggtctcc aacaaagccctcccagcccccatcgagaaaaccacccccaaegccaaagggc agccccgagaaccacaggtg:acaccctgcccccatcccgggaggagatgac caagaaccaggtcagcc,ngg,gcc,gg,caaagch,cLe,cccagcgac atcgccgtggag:gggagagcaatgggcagccggagaacaac:acaagacca chgc,ggac,ccgacggc,cc,Lchchc,acegcaagctcac cg:ggacaagagcaggtggcagcaggggaacg“CLLC,caLgc,ccg,gaLg catgaggc4ccgcacaaccactacacgcagaagagcc4c“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: chogogagacacgguaac,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 ), an intervening linker e (Linker l) and a VH domain of a monoclonal antibody capable of binding to CDl23 (VHCDm).
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 version 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 ce 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 preferred ptide is ed 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 activity 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 diabody (Control DART) was produced. The Control DART is capable of simultaneously binding to FITC and CD3. Its two polypeptide chains have the ing respective sequences: Control DART Chain 1 (SEQ ID NO: 19).
DVVMTQTPFSL9VSLGDQAS: SCRSSQSLVHSNGWTYLRWYLQKPGQSPKVL :YKVSWRFSGV9DRFSGSGSGTDETTK S<VfiAfiDTGVYFCSQSTHVPWTFG GGTKT.*' KGGGSGGGGfi'VQLVmSGGGLVQ9GGSLRLSCAASGFTFNTYAMNW VRQAPGKGTfi'WVA{ RSKYNNYATYYADSVKDRFT SRDDSKNSLYLQMNSL VYYCVR{GNFGWSYVSWFAYWGQGTLVTVSSGGCGGGfiVAALfiKfiV AALfiKfiVAALfiKfiVAALfiK Control DART Chain 2 (SEQ ID NO:20): 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 itions of the invention include bulk drug itions 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 ation of unit dosage forms. Such compositions comprise a prophylactically or therapeutically effective amount of the sequence-optimized CD123 X CD3 bi-specific diabodies of the present invention, or a combination of such agents and a ceutically acceptable carrier. Preferably, compositions of the invention comprise a prophylactically or therapeutically effective amount of the ce-optimized CD123 X CD3 bi-specific diabody of the invention and a pharmaceutically acceptable carrier.
] The ion 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 specific for a particular cancer antigen, and a pharmaceutically acceptable carrier.
In a specific embodiment, the term “pharmaceutically acceptable” means ed by a regulatory agency of the Federal or a state government or listed in the US. Pharmacopeia or other generally recognized pharmacopeia 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, ing those of petroleum, animal, vegetable or synthetic origin, 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 glycerol solutions can also be employed as liquid carriers, particularly for able solutions. Suitable pharmaceutical ents include starch, e, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, ene, glycol, water, ethanol and the like. The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like. lly, 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 trate in a hermetically sealed container such as an ampoule or sachette ting the quantity of active agent. Where the composition is to be administered by infusion, it can be dispensed with an infiJsion bottle ning sterile pharmaceutical grade water or saline. Where the composition is administered by ion, an ampoule of sterile water for injection or saline can be ed so that the ingredients may be mixed prior to administration.
The compositions of the invention can be formulated as neutral or salt forms.
Pharmaceutically acceptable salts include, but are not d to those formed with anions such as those derived from hydrochloric, phosphoric, acetic, oxalic, ic 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 provides a pharmaceutical pack or kit comprising one or more containers filled with sequence-optimized CD123 X CD3 bi-specif1c diabodies of the invention alone or with such pharmaceutically acceptable carrier. Additionally, one or more other prophylactic or therapeutic agents useful for the treatment of a disease can also be included in the pharmaceutical pack or kit. The invention also provides a pharmaceutical pack or kit sing one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention.
Optionally associated with such ner(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice s approval by the agency of manufacture, use or sale for human administration.
The present invention provides kits that can be used in the above s. A kit can comprise sequence-optimized CD123 X CD3 bi-specif1c diabodies of the ion. The kit can fiarther comprise one or more other lactic 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 cytotoxic antibodies that bind one or more cancer antigens associated with cancer. In certain embodiments, 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 t invention may be provided for the treatment, prophylaxis, and amelioration of one or more symptoms associated 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 molecule of the invention. In a preferred aspect, such compositions are substantially purified (z'.e., ntially 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 ster the compositions of the invention, e.g, encapsulation in liposomes, microparticles, microcapsules, recombinant cells e of expressing the antibody or fusion protein, receptor-mediated endocytosis (See, e.g., Wu et al. (1987) “Receptor-Mediated In Vitro Gene Transformation By A e DNA Carrier System,” J. Biol. Chem. 262:4429-4432), uction of a nucleic acid as part of a retroviral or other vector, etc.
Methods of administering a molecule 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 routes). In a specific embodiment, the sequence-optimized CD123 X CD3 bi-speciflc diabodies of the invention are administered intramuscularly, intravenously, or subcutaneously. The compositions may be administered by any convenient route, for example, by on or bolus injection, by absorption through epithelial or mucocutaneous s (e.g., oral mucosa, rectal and inal mucosa, etc.) and may be administered er with other biologically active . Administration can be systemic or local. In addition, ary administration can also be employed, e.g., by use of an inhaler or zer, and formulation with an aerosolizing agent. See, e.g., US. Patent Nos. 6,019,968; 5,985, 320; 309; 5,934,272; 5,874,064; ,855,913; 540; and 4,880,078; and PCT Publication Nos. WO 92/19244; WO 97/32572; WO 97/44013; WO 98/31346; and WO 99/66903, each of which is incorporated herein by reference in its entirety.
The invention also provides that the sequence-optimized CD123 X CD3 bi- specific diabodies of the invention are packaged in a hermetically sealed container such as an e 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 lyophilized 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 reconstituted. In an alternative embodiment, sequence-optimized CD123 X CD3 bi-specific diabodies of the invention are ed 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 container in which the molecules are t 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 ive in the treatment, prevention or amelioration of one or more symptoms associated with a disorder can be determined by standard clinical techniques. The precise dose to be employed in the formulation 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 esponse curves derived from in vitro or animal model test systems.
For sequence-optimized CDl23 X CD3 bi-specific diabodies assed by the invention, the dosage administered to a patient is preferably determined based upon the body weight (kg) of the recipient subject. The dosage administered 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 another ment, the patient is administered a treatment regimen comprising one or more doses of such lactically or eutically 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 comprises intermittently administering doses of the prophylactically or therapeutically effective amount of the sequence-optimized CD123 X CD3 bi-specif1c ies 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 prophylactically or therapeutically effective amount of the sequence-optimized CD123 X CD3 bi-specific diabodies encompassed by the invention on day 5, day 6 and day 7 of the same week).
Typically, there are 1, 2, 3, 4, 5 or more courses of ent. Each course may be the same n or a different regimen.
In r 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 diabodies 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% D1abody Dosage (ng d1abody 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.) hN-h none none none none none The dosage and frequency of administration of sequence-optimized CD123 X CD3 bi-specif1c diabodies of the ion may be reduced or altered by enhancing uptake and tissue penetration of the sequence-optimized CD123 X CD3 bi-specif1c diabodies by modifications such as, for example, lipidation.
] The dosage of the sequence-optimized CD123 X CD3 bi-specif1c diabodies of the invention administered to a patient may be calculated for use as a single agent therapy. 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 pharmaceutical compositions of the ion 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 stering a molecule of the invention, care must be taken to use als to which the le does not absorb.
The compositions of the invention can be delivered in a vesicle, in particular a liposome (See Langer (1990) “New Methods OfDrag Delivery,” e 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 ibid.).
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 98; 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 ing 0f Long-Circulating Emulsions, ” PDA Journal of Pharmaceutical e & 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 Antibody 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 Pumps,” CRC Crit. Rev. Biomed. Eng. 14:201- 240; Buchwald et al. (1980) “Long-Term, Continuous Intravenous Heparin Administration By An Implantable Infusion Pump In Ambulatory Patients With Recurrent Venous osis,” Surgery 88:507-516; and Saudek et al. (1989) “A inary Trial Of The Programmable Implantable tion System For Insulin Delivery,” N. Engl. J. Med. 321:574-579). In another embodiment, polymeric materials can be used to achieve controlled-release of the molecules (see e. g., MEDICAL ATIONS 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 cation rosthetic Heart Valves By Local Controlled-Release Diphosphonate, ” Science 228:190-192; During et al. (1989) “Controlled Release 0fDopamine From A Polymeric 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 polymers used in sustained-release formulations e, but are not limited to, poly(2-hydroxy ethyl methacrylate), poly(methyl methacrylate), poly(acrylic acid), poly(ethylene-co- vinyl acetate), poly(methacrylic acid), polyglycolides (PLG), polyanhydrides, poly(N— vinyl pyrrolidone), poly(vinyl alcohol), rylamide, poly(ethylene glycol), polylactides (PLA), actide-co-glycolides) (PLGA), and polyorthoesters. A lled-release system can be placed in ity of the therapeutic target (e.g., the , thus requiring only a fraction of the systemic dose (see, e.g., n, in MEDICAL APPLICATIONS OF CONTROLLED RELEASE, supra, vol. 2, pp. 115-138 (1984)). Polymeric compositions useful as controlled-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 lled-release of the bioactive material from the polymer system. The implantation can generally occur anywhere within the body of the patient in need of therapeutic treatment. A non-polymeric sustained delivery system can be used, whereby a non-polymeric t in the body of the subject is used as a drug delivery system. Upon implantation in the body, the organic 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 ned-release formulations comprising one or more therapeutic agents of the invention. See, e.g., US. Patent No. 4,526,938; International Publication Nos. WO 91/05548 and WO 98; Ning et al. (1996) “Intratumoral Radioimmunotberapby Of A Human Colon Cancer Xenograft Using A Sustained-Release Gel,” Radiotherapy & Oncology 39:179-189, Song et al. (1995) “Antibody Mediated Lung Targeting 0f Long-Circulating Emulsions,” PDA Journal of Pharmaceutical Science & Technology 50:372-397; Cleek et al. (1997) “Biodegradable Polymeric rs 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 zed Monoclonal Antibody For Local Delivery, ” Proc. Int’l. Symp. Control Rel. Bioact.
Mater. 24:759-760, each of which is orated herein by nce in its entirety.
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 cif1c diabody, by ucting 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. 4,980,286), or by direct injection, or by use of microparticle bombardment (e.g., a gene gun; tic, Dupont), or coating with lipids or cell-surface ors or transfecting agents, or by administering it in linkage to a homeobox-like peptide which is known to enter the s (See e.g, Joliot et al. (1991) “Antennapedz'a H0me0b0x Peptide tes 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 effective amount of sequence-optimized CD123 X CD3 bi-specif1c diabodies of the ion can include a single treatment or, preferably, 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 ive dosage of the molecules used for ent may increase or decrease over the course of a particular treatment.
VI. Uses of the Compositions of the Invention The sequence-optimized CD123 X CD3 bi-specif1c diabodies of the present invention have the y 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 diagnosis or treatment of acute myeloid leukemia (AML), chronic myelogenous leukemia (CML), including blastic crisis of CML and Abelson oncogene associated with CML (Bcr-ABL translocation), myelodysplastic me (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), blastic plasmacytoid dendritic cell sm (BPDCN), non- Hodgkin lymphomas (NHL), including mantel cell leukemia (MCL), and small lymphocytic ma (SLL), Hodgkin’s lymphoma, systemic mastocytosis, and t’s lymphoma (see Example 2); Autoimmune Lupus (SLE), allergy, asthma and rheumatoid arthritis. The cific diabodies of the present invention may additionally be used in the manufacture of medicaments for the treatment of the 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 l Protein ] Table 2 contains a list of bi-specif1c diabodies that were expressed and purified. ce-optimized CD123 X CD3 bi-specif1c diabody (DART-A) and non- ce-optimized CD123 X CD3 bi-specif1c diabody (DART-B) are capable of simultaneously binding 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 recited amino acid sequences.
Methods for forming bi-specif1c ies are provided in WC 13665, WO 2008/157379, , , and WO 2012/162067.
Polypeptide Chain EncoNtiiflelgfggnces. .
Bi—Specific Diabodies Amino Acid g q Se u uences Sequence-Optimized CD123 x CD3 .
Bi—Specific Diabody A) Egg £3 £8;° Egg £3 £8:.
(Binds to CD3 at epitope 1) ° Non-Sequence-Optimized CD123 x . .
CD3 Bi-Specific 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 Albumin-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 Nucleic 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 n 2) SEQ ID N0:17 SEQ ID N0:18 (Binds to CD3 at epitope 1) SEQ ID N0:1 SEQ ID N0:2 ses an Fc Domain for extension of half-life in vivo Control Bi-Specific y (Control DART (or Control DART) SEQ ID N0:19 (Binds to CD3 at e 1) SEQ ID N0:20 (Binds to an irrelevant target - FITC) Example 2 dy Labeling Of Target Cells For Quantitative FACS (QFACS) ] A total of 106 target cells were harvested 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 Laboratories, Inc., Fishers, IN) and target cells were labeled 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 fiaging 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- specific instrument settings (PMT voltages and compensation). Using the same ment settings, geometric mean fluorescence values of microspheres and target cells were recorded. A standard curve of dy binding sites on microsphere populations was generated from geometric mean fluorescence of microsphere populations. Antibody binding sites on target cells were ated based on geometric mean fluorescence of target cells using the standard curve generated for microspheres in QuickCal spreadsheet (Bangs Laboratories).
To determine suitable target cell lines for evaluating CD123 X CD3 bi- specific diabodies, CD123 e 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 highest 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 sion Line Antibod _ 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 collected 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 ed by Trypan Blue exclusion using a Beckman Coulter Vi-Cell counter. The target cells were diluted to 4 x105 cells/mL in the assay . 50 uL of the diluted cell suspension was added to a 96-well U- bottom cell culture d 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; detergent added at the end of the experiment to determine the maximal LDH release. 2) AICC: 50 uL assay medium without 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 lly 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 containing 50 uL target cells/well.
Purified T cells were washed once with assay medium and resuspended in assay medium at cell y of 2 x 106 cells/mL. 2 x 105 T cells in 100 uL were added to each well, for a final or-to-target cell (E:T) ratio of 10:1. Plates were incubated 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 maximum release l 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 solution (Promega ) 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 t cytotoxicity was calculated as bed below and dose-response curves were ted 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 ent Levels of CD123 Surface Levels: The CD123 X CD3 bi-specif1c diabodies exhibited a potent redirected killing ability with concentrations required to achieve 50% of maximal activity (EC50s) in sub-ng/mL range, regardless of CD3 epitope g specificity (DART-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 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 sion, 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 killing was also observed with multiple target cell lines with T cells from different donors and no cted killing activity was observed in cell lines that do not express CD123. Results are summarized in Table 4.
Target cell line CD123 surface EC50 of Sequence- Max % g expression optimized CD123 x (antibody binding CD3 bi-specific sites) diabodies (ng/mL) E:T=10:1 -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 ol in a manner appropriate for replicating the bed results. Thus, the exemplified protocol is not intended to be adhered to in a ely rigid manner.
Example 4 T Cell Activation During Redirected Killing By ce-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 presence or absence of ife 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 sequence-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 ependent manner indicating that sequence-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. se, CD8 T cells are not activated when ted 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 Intracellular Staining for me B and Perforin To determine the intracellular levels of granzyme B and perforin 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 D8 antibodies by incubating for 30 minutes at 4°C. Following surface staining, cells were incubated in 100 uL fixation 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 granzyme B and a perforin antibody mixture (prepared in 1X permeabilization/wash buffer) at 4°C for 30 minutes. Then cells were washed with 250 uL permeabilization/wash buffer and resuspended in permeabilization/wash buffer for FACS acquisition.
Upregulation Of Granzyme B And in By Sequence-Optimized CD123 x CD3 Bi-Specific Diabody (DART-A) In T Cells During Redirected Killing To igate the possible ism for ce-optimized CD123 X CD3 bi-specific diabody (DART-A) mediated cytotoxicity by T cells, ellular 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 incubation 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 (Figure 6, Panel A). When the assay was performed in the presence of granzyme B and in inhibitors no cell killing was observed. There was no lation 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) (Figure 5, Panel B). These data indicate that DART-A mediated target cell killing may be mediated through granzyme B and perforin isms.
Example 6 in vivo Antitumor 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 nt 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 discarded 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 ine, 10mM HEPES, 100u/100u/mL penicillin/Streptomycin (P/S).
T cell isolation: hed T cells were isolated by negative selection from PBMCs from human whole blood using Dynabeads Untouched Human T Cell isolation kit (Life logies) according to manufacturer’s instructions. 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 e 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 enously (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 Control (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 l SDO, 1, 2, 3 (RS4-11 + 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 ART-A SDO, 1, 2, 3 ———-_DART-A SDO, 1, 2, 3 Data Collection and tical Analysis: Animal weights - Individual animal weights were recorded twice weekly until study completion beginning at the time of tumor cell ion.
Moribundity/Mortality - Animals were observed twice weekly for general moribundity and daily for mortality. Animal deaths were assessed as elated or technical based on factors including gross observation and weight loss; animal deaths were recorded 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 calculated 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 elated deaths were censored from the TGI calculations. The National Cancer Institute criteria for compound activity is TGI>58% (Corbett et al. (2004) Anticancer Drug Development Guide; Totowa, NJ: Humana 99-123).
Partial/Complete Tumor Response - Individual mice possessing tumors measuring less than 1mm3 on Day 1 were fied as having partial response (PR) and a percent 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 te response (CR).
Tumor Volume Statistics — Statistical analyses were carried out between treated and control groups ing tumor volumes. For these analyses, a y analyses of variance followed by a roni 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 l or complete responses were included in these calculations.
MOLM13 Results 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 e 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. ent 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 ). 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 ment on SD18, the average volume of the tumors d with DART-A at the 0.8 — 20 ug/kg) ranged from 713.60 :: 267.4 to 0 mm3, all of which were cantly smaller than the tumors in the vehicle-treated control group. The TGI values were 100, 94, and 49% for the 20, 4, and 0.8 [Lg/kg dose groups, respectively. In comparison to the vehicle-treated MOLM13 tumor cell group, the groups that received DART-A at the 20 and 4 [Lg/kg dose level d 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 = p) 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 aggressive 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 animals were treated with DART-A at 5 dose levels (0.5, 0.2, 0.1, 0.02, and 0.004 mg/kg). Results are shown in Figure 8.
Sequence-optimized CD123 X CD3 bi-specific diabody (DART-A) ively inhibited the growth of both MOLM13 AML and RS4-11ALL tumors implanted SC in NOD/SCID 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 mediated cytotoxicity in vitro in MOLMl3 cells.
Should it be necessary to ate 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 replicating the described results. Thus, the ified protocol is not ed 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 y samples, cryopreserved primary AML patient bone marrow and PBMC s were ted for CD123 surface expression on leukemic blast cells.
AML Bone Marrow Sample — Clinical 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%, CD7=0.2%, CD10=0.4l%, CDl9=l.l%, CD20=l.4%, CD22=0.7l% 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. ic blast cells were identified using the myeloid 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 Patient 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- optimized CD123 X CD3 bi-specific diabody (DART-A) was diluted to 0.1, and 0.01 ng/mL and 50 uL of each dilution was added to each well (final volume = 200 uL). l bi-specific 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 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. d cells were ed in FACS Calibur flow cytometer equipped with est Pro acquisition software, n 5.2.1 (BD Biosciences). Data analysis was performed using Flowjo v9.3.3 re (Treestar, Inc).T cell expansion was measured by gating on CD4+ and CD8+ populations and activation was determined by measuring CD25 mean fluorescent intensity (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 tion By Sequence-Optimized CD123 x CD3 Bi-Specific Diabody (DART-A) In Primary Specimens From AML Patient 1 To ine the sequence-optmized CD123 X CD3 bi-speciflc diabody (DART-A) mediated ty 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+ . Incubation of primary AML bone marrow samples with DART-A resulted in depletion of the leukemic cell population 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 able limits, vary the above- described ol in a manner riate for replicating the described results. Thus, the exemplified 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 y ALL patient PBMC sample was evaluated for CD123 surface expression on leukemic blast cells.
CD123 Expression in Leukemic Blast Cells in Peripheral Blood Mononucleocytes (PBMC) A total of 0.5xlO6 peripheral blood mononucleocytes (PBMC)) from a healthy donor and an ALL patient were ted for CD123 expression. 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 (Figure 11, Panel D).
The T cell population was fied 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 primary ALL en (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 ined 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) 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 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, n 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 tion was determined by measuring CD25 MFI on the CD4+ and CD8+ -gated populations.
Leukemic blast cell tion was identified by CD45+CD33+ .
Autologous Tumor Cell ion, 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 samples, patient samples were ted 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 samples with DART-A resulted in depletion of the leukemic cell population over time compared 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 ed and were activated in the DART-A sample (Figure 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 t 2 To define the CD123 expression pattern in AML t 2 primary samples, 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 ted for leukemic blast cell identification. Leukemic blast cells were identified using the myeloid s CD33 and CD45. As shown in Figure 15, Panel B, 94% of the cells from AML bone marrow are leukemic 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.
Example 12 Autologous CTL Killing Assay Using AML Patient 2 Primary Specimens Cryopreserved y AML specimen (bone marrow mononucleocytes (BM MNC) and peripheral blood mononucleocytes (PBMC)) from AML t 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 ences).
Sequence-optimized CD123 X CD3 cific diabody (DART-A) and control bi- specific 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 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 est Pro acquisition software, Version 5.2.1 (BD Biosciences). Data analysis was med using Flowjo v9.3.3 software (Treestar, Inc). T cell expansion was measured by gating on CD4+ and CD8-- tions and activation was determined by measuring 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 Expansion 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, t samples were incubated with 0.1 or 0.01 ng/mL of DART-A and percentages of ic blast cells and T cells were measured at different time points following the treatment. Incubation of primary AML bone marrow samples with DART-A resulted in ion 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 ted, 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 y AML bone marrow samples with DART-A resulted in T cell activation. These data represent the 144h time point.
Intracellular Staining for me B and Perforin To determine the ellular 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.
Following surface ng, 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 minutes. 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 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 cted killing.
There was no upregulation of me B and perforin 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 ce-optimized CD123 X CD3 bi-specific diabody (DART-A) e 17, Panels C and D).
Interestingly, the upregulation was almost two-fold higher in CD8 T cells compared to CD4 T cells e 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 Sequence-Optimized CD123 x CD3 Bi-Specific Diabody Cross-Reacts With Non- Human Primate CD123 And CD3 Proteins.
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 iation off-rate, kd. The binding affinity (KD) between an antibody and its target is a function of the kinetic constants for association (on rate, ka) and dissociation (off-rate, kd) according 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. Purified human or lgus monkey CD123 was ed and immolbilized to a support. The time course of dissociation was measured and a bivalent fit of the data ted. Binding constants and affinity were obtained using a 1:1 binding fit. The s of the BIACORETM es comparing 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) ns is comparable to binding affinities for human CD123 (Figure 18C) and CD3 (Figure 18A) proteins.
Example 14 Autologus Monocyte Depletion In Vitro With Human And Cynomolgus Monkey PBMCs PBMCs from human or cynomolgus monkey whole blood samples were added to U-bottom plates at cell y of 200,000 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), ion of pDCs cells was observed in both human (Figure 19, Panel A) and cynomolgus monkey PBMCs (Figure 19, Panel B). These results indicate that ating 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 acceptable limits, vary the above- described 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.
Example 15 cytoid 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 administered 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 fy pDCs and te populations in cynomolgus monkey PBMCs, cells were labeled with CD14-FITC antibody. tes were fied as the CD14+ population 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 monkeys or the vehicle + carrier-treated monkeys at the 4d time point (Figure 20, Panels G, H, C and D, respectively). Cytokine levels of interferon-gamma,TNF- alpha, 1L6, 1L5, 1L4 and IL2 were ined at 4 hours after infilsion. There was little to no elevation in cytokine levels at the DART-A treated animals compared to l 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 ) (Figure 21, Panel B), NK cells (CD159+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, tes and pDCs. ent 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 e, returning to pre-dose levels several 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 changes in trafficking rather than true depletion. Both CD4 and CD8 T lymphocytes followed the same pattern. The T-lymphocyte tion marker, CD69 (Figure 22, Panel C), was only marginally positive among circulating cells and did not track with DART-A . B lymphocytes, monocytes and NK cells fluctuated over the course of DART-A dosing with substantial variability observed among monkeys. A trend toward increased circulating levels of B cytes and monocytes was observed in both monkeys at the highest doses.
In summary, the above results trate the therapeutic efficacy of the sequence-optimized CD123 X CD3 cif1c diabody (DART-A). The sequence- optimized CD123 X CD3 bi-specif1c diabody (DART-A) may be employed 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 transformation of CLL, Blastic crisis of CML, BLL (subset are CD123+) (see Example 2); Autoimmune Lupus (SLE), allergy (basophils are CD123+), asthma, etc.
Example 16 Comparative ties of ce-Optimized CD123 x CD3 Bi-Specific Diabody (DART-A) and Non-Sequence-Optimized CD123 xCD3 cific Diabody (DART-B) Unexpected Advantage and Attributes of the Sequence-Optimized CD123 x CD3 cific 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 inal direction, an N—terminus, a VL domain of a monoclonal antibody capable of binding to CD3 ), an intervening linker e (Linker l), a VH domain of a monoclonal antibody capable of binding to CD123 ), a Linker 2, an E-coil Domain, and a C-terminus. Likewise, the second polypeptide of both constructs comprise, in the N—terminal to C-terminal direction, an inus, 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 capable 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. Additionally, as disclosed in Example 3 and in Figure 4, Panels C and D, the two CD123 X CD3 bi- specific diabodies exhibited a potent cted 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 abrogate 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 ted an unexpected advantage 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 (CD123) 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 Leukemia: A Groupe Ouest-Est Des Leucemies Aigues Et Maladies Du Sang (GOELAMS) Study,” Haematologica 96:1792-1798).
Moreover, CD123 has been reported to be expressed by leukemia stem cells (LSC) (Jordan, C.T. et al. (2000) “The eukin-3 Receptor Alpha Chain Is A Unique Marker For Human Acute Myelogenous Leukemia Stem Cells,” Leukemia 14:1777- 1784; Jin, L. et al. (2009) “Monoclonal Antibody-Mediated Targeting 0fCD123, IL-3 Receptor Alpha Chain, Eliminates Human Acute Myeloid Leukemic Stem ” Cell Stem Cell 2), which is an attractive feature that s targeting the root cause of such es. 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 ic stem cell tment 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 patients, however, the monoclonal antibody exhibited no anti-leukemic activity (Roberts, A. W. et al. (2010) “A Phase I Study Of Anti-CD123 Monoclonal dy (mAb) CSL360 ing ia Stem Cells (LSC) In AML,” J. Clin. Oncol. 28(Suppl):e13012). Thus, alternate CD123-targeting approaches, 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 eukin-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 cell-depleting strategies allow reconstitution via normal hematopoiesis. ] ng a patient’s own T lymphocytes to target leukemic cells ents a promising immunotherapeutic strategy for the treatment of hematological malignancies. The therapeutic potential of this approach has been ted using blinatumomab (a cific antibody-based BiTE having the ability to bond CD3 and the B cell CD19 antigen) in patients with B cell lymphomas and B-precursor acute lymphoblastic leukemia (Klinger, M. et al. (2012) “Immunopharmacologic Response 0fPatients With B-Lineage Acute blastic Leukemia To Continuous Infusion Of T Cell-Engaging CD19/CD3-Bispecific BiTE Antibody umomab,” Blood 119:6226-6233; Topp, M.S. et al. (2012) “Long-Term Follow-Up 0f Hematologic Relapse-Free al 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 T-Cell—Engaging Antibody Blinatumomab 0f Chemotherapy-Refractory Minimal Residual Disease In age Acute blastic Leukemia Patients Results In High Response Rate And ged Leukemia-Free Survival,” J. Clin.
Oncol. 29:2493-2498).
The CD123 X CD3 bi-specif1c diabody molecules of the present invention, such as , 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 Protein 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 les To Achieve Optimal Redirected T—Cell Killing 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 ularis) was assessed relative to either the above-described Control DART (bi-specific for CD3 and cein) or a “Control DART-2” that was bi-specific for CD123 and fluorescein).
Amino Acid Sequence of First Polypeptide Chain of ol DART-2” (CD123VL — Linker — 4-4420VH — Linker — E-coil; linkers are ined) (SEQ ID NO:58): SPDS LAVSLGLRVT MSCKSSQSLL NSGNQKVYLT WYQQKPGQPP {LLT YWASTR LSGVPDRFSG SGSGTDFTTT SSTQAmDVA VYYCQWDYSY RYTFGQGT<L m {GGGSGGG GL'VKTDL'TGG GLVQRGRRMK LSCVASGFTF SDYWMNWV<Q SRLKGTLWVA Q_TRNKRYNY_E TYYSDSV<GR FTTSRDDSKS SVYLQMNWL< VfiDMG YYCT GSYYG DYWG QGTSVTVSSG GCGGGLVAAL fi<fiVAALfi<fi VAALmeVAA LTK Amino Acid ce of Second Polypeptide Chain of “Control DART-2” (4420VL — Linker— CD123VH — Linker — ) (SEQ ID NO:59): DVVMTQTRFS LPVSLGDQAS T_SCRSSQSLV HSNGNTYLRW YLQKPGQSPK VLT_YKVSNRF SGVPDRFSGS GSGTDb'TTK SRVL'ALDTGV YFCSQSTHVP WTFGGGT<Lm KGGGSGGGG LVQLVQSGA_E LKKRGASVKV SC<ASGYTFT DYYMKWVRQA PGQGTL'W GD RSWGATFY NQKh'KG{VT_ TVDKSTSTAY mLSSLRSfiD TAVYYCARSd LLRASWFAYW GQGTLVTVSS GGCGGGKVAA L<LKVAAL<L KVAAL<LKVA AL<L Bifunctional ELISA A rp ELISA plate (Nunc) coated overnight with the soluble human or cynomolgus IL3R—alpha (0.5 ug/mL) in bicarbonate 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 on of human CD388—biotin and Streptavidin HRP (Jackson ImmunoResearch). HRP activity was detected by conversion of tetramethylbenzidine (BioFX) as substrate for 5 min; the reaction was ated 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) ctor Cell Recruitment With Novel Fv- Based Dual-Afiinity Re-Targeting n Leads T0 Patent Tumor Cytolysis And In Vivo B-Cell Depletion,” J. Mol. Biol. 6-449) and Moore, RA. et al. (2011) (“Application OfDual Afiinity Retargeting Molecules To Achieve Optimal Redirected T-Cell Killing OfB-Cell Lymphoma,” Blood 42-4551). 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 ted CM5 e in 10mM sodium-acetate, pH 5.0, at flow rate 5 uL/min, followed by 1 M ethanolamine for vation. Binding ments 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 determined 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 killing assays were obtained from the American Type Culture Collection (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 ion kit (Life Technologies). CD123 cell-surface density was determined using Quantum Simply Cellular beads (Bangs Laboratories, Inc., Fishers, IN). Cytotoxicity assays were performed as bed by Moore, PA. et al. (2011) (“Application OfDual Afiinity Retargeting Molecules To Achieve Optimal cted T-Cell Killing 0f B-Cell Lymphoma,” Blood 117:4542-4551). Briefly, target cell lines (105 cells/mL) were treated with serial dilutions of DART-A or Control DART proteins in the presence 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 libur flow cytometer. Data were analyzed by using PRISM® 5 software (GraphPad) and presented as percent cytotoxicity.
Cynomolgus Monkey Pharmacology Non-human primate 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 a fascicularz’s) of Chinese 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 r ports using battery-powered programmable infusion pumps (CADD- Legacy®, SIMS Deltec, Inc., St. Paul, MN). Peripheral blood or bone marrow samples were collected in anticoagulant containing tubes at the indicated time points.
Cell-surface phenotype es were performed with an LSR Fortessa analyzer (BD Biosciences) equipped with 488nm, 640nm and 405nm lasers and the following antibodies: CD4-V450, 50, CD123-PE-Cy7, erCP, CD4-APC-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 cytokines 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 ch immunoassay with electrochemiluminescence detection (MesoScale Diagnostics, MSD, Rockville, MD).
Briefly, the assay plate (MSD) was coated with recombinant human IL-3 Ra (R&D ) and blocked with 5 % BSA. Calibration standards or diluted test samples were applied, followed by the on of a biotinylated monoclonal antibody 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 trations were determined from standard curves generated by fitting light intensity data in a five-parameter logistic model.
Physicochemical characterization of the purified DART-A demonstrated 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 e human and cynomolgus monkey CD3 and CD123 antigens (Figures D and Table 7). Furthermore, DART-A simultaneously bound both antigens in an ELISA format that employed human or monkey CD123 for capture and human CD3 for detection (Figures 26A-26B), and trated 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 Constants (KD) for the g 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 lgus Monkey T Lymphocytes DART-A mediated redirected target cell killing by human or monkey effector cells against CD123+ Kasumi-3 leukemic cell lines (Figure 27A-27D), which was accompanied by induction of activation s. No activity was ed against CD123-negative targets (U937 cells) or with Control DART, indicating that T cell tion is strictly dependent upon target cell engagement and that monovalent engagement 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 dose-dependent 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 le concentrations, while monocytes were slightly decreased by 6 hours and depleted only after 18 hours and at the concentrations higher than 1 ng/mL. Incubation of monkey PBMCs with DART-A resulted in a comparable dose-dependent ion 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).
Pharmacokinetics of DART-A in Cynomolgus Monkeys ] The cynomolgus monkey was selected as an appropriate pharmacological model for DART-A analysis based on the equivalent 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) leukin-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 ion 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 cycles. Group 1 animals received vehicle control for all 4 subsequent infusions, whereas Groups 2-5 received weekly escalating doses of DART-A for 4 days a week for all uent ons.
Group 6 animals were treated with 7-day rrupted weekly escalating doses of DART-A for all infusions. The 4-day-on/3-day-off and 7-day-on schedules were designed to distinguish between durable from ent effects associated with DART- A administration. Two males and 2 s 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 dies (ADA) directed against the humanized Fv of both CD3 and CD123 and the data points following the appearance of ADA were excluded from the PK is. All monkeys were exposed to DART-A during the study period.
DART-A Infusion (4-day-on/3-day-ofi) (7-day-on) Vehicle Infusion ng/kg/day ng/kg/day N0. [n__/k/7da s] Group Group Vehicle Vehicle e Vehicle Vehicle 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 te 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 s 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 formulation 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 required concentration.
Table 9 Two-Compartment Analysis of PK Parameters of DART-A in C nomolus 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 DART-A-Treated Cynomolgus Monkeys Given the T cell tion 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 similar 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,” Science 321:974-977). Of the cytokine tested, lL-6 demonstrated the largest s upon infusion, albeit transient in nature, of l magnitude and with large inter-animal and inter-group variations (Figures 29A-29C). Small, transient increases in lL-6 were also observed after vehicle ons (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 successive DART-A infusion, even when the dose level was increased to up to 1000 ng/kg/day. l and transient DART-A-related ses in serum TNF-u (<lOpg/mL) were also ed; as with lL-6, the largest magnitude in TNF-u release was ed following the first infiasion. There were no DART-A-related changes in the levels of lL-S, lL-4, lL-2, or lFN—y throughout the study when compared with controls. In sion, cytokine release in response to treatment of monkeys with DART-A was l, 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 CD123+ cells were ed 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 depletion of circulating CDl4-/CD123+ cells (94-lOO% from dy baseline) observable from the first time point measured (72 hours) following the start of the first DART-A on (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 ate the possibility of DART-A g 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 d with DART-A (Figures 30D-30F).
Circulating T-Lymphocyte Levels, Activation and Subset Analysis In contrast to the persistent depletion of circulating CD123+ cells, DART-A administered on the on/3-day-off schedule s 2-5) were associated with weekly fluctuations in circulating T cells, while administration as continuous 7-day infilsions resulted in similarly decreased circulating T cell levels following 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 lymphocytes is consistent with trafficking and/or margination, rather than ion. Following cessation of , T cells rebounded to levels approximately 2-fold higher than baseline for the duration of the recovery . Infusion of DART-A was associated with an exposure-dependent, progressive increased frequency of T cells expressing the late activation marker, PD- I, particularly in CD4+ cells, with dose Group 6 displaying the highest overall levels (Figures 31D-311 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 ncy among CD8+ cells (5.5-9.7%) and comprising 20.5-35.5% of the D-l+ double-positive cells. There was no tent change in the early T cell activation , CD69, and only modest variations in CD25 expression among circulating cells.
To rule out exhaustion after in viva exposure, the ex vivo cytotoxic potential of effector cells ed 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 negatively impact the ability 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 population (Figures 35A-35F and Figures 32A-32F and Figures 33A-33F), ting 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 ions in red cell parameters were ed at the highest doses (Figures 36A-36C). Frequent blood sampling could have been a ial contributing factor, since vehicle-treated animals showed a modest e in red cell mass. Reticulocyte response was observed in all animals; at the highest exposure (Group 6), however, the response appeared slightly less robust for similar decrease in red cell mass es 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 -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 s show some apparent reduction ed to the corresponding pre-dose levels, 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 consistent 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 se weekly to 300, 600, and l,000ng/kg/day, the administration of DART-A to cynomolgus monkeys was well tolerated. Depletion of circulating CD123+ cells, including pDCs, was observed after the start of the first administration and persisted throughout the study at all doses and schedules.
Reversible reduction in bone marrow CD123+ precursor was also ed. Cytokine release, as significant safety concern with CD3-targeted therapies, ed manageable and consistent with a first-dose effect. Modest reversible anemia was noted at the highest doses, but no other (on- or off-target) adverse events were noted.
The cynomolgus monkey is an appropriate animal model for the cological assessment of DART-A, given the high homology n the orthologs and the ability of DART-A to bind with similar affinity to the ns and e redirected T cell killing in both species. Furthermore, both antigens are concordantly expressed in monkeys and , ing 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 e of cytokines and off-target cytotoxic effects. A recent study with a CD3xCD123 bi-specif1c scFv fusion construct with bivalent CD3 recognition demonstrated anti-leukemic activity in vitro, but caused non-specific tion of T cells and IFN—y secretion (Kuo, S.R. et al. (2012) “Engineering A CD123xCD3 Bispecific scFv Immunofusion For The Treatment Of ia And Elimination 0f Leukemia Stem Cells,” Protein Eng. Des. Sel. 25:561-569). The lent 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 e of target cells or by using a Control DART molecule that included only the CD3-targeting arm. rmore, high doses (up to 100ug/kg/day) of the Control DART molecule did not trigger cytokine release in cynomolgus monkeys.
The DART-A molecule starting dose of 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 s to be ily a first-dose effect. Depletion of the CD123+ target cells, thereby eliminating a source of CD3 ligation, may explain the first-dose effect: nearly complete CD123+ cell depletion was ed 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 cytokine release by human T cells were also consistent with this observation.
T cell desensitization, 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 increased PD-l expression after antigen- induced arrest of T cells at inflammation 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 or-Dependent Negative ck Controls T Cell r 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 ing strength is not uniform: 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 ons,” 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 comparable to that of T cells from na'ive monkeys, despite 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 cted stimulation with CD3 antibodies or chronic infections (Gebel, H.M. et al. (1989) “T Cells From Patients 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. l. -499).
The depletion of circulating CD123+ cells in DART-A-treated monkeys was rapid and persisted during the weekly dosing holidays in the 4-day-on/3-day-off schedule, consistent with target cell ation. In contrast, the transient fluctuations in circulating T cells were likely the result of trafficking o tissues and lymphoid organs as a function of DART-A. DART-A exposure promotes the expansion and/or zation of antigen experienced T lymphocytes, cells that preferentially home to tissues and more readily exert cytotoxic effector 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 king.‘ 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 (Lopez, A.F. et al. (1989) “Reciprocal tion 0f Binding Between Interleukin 3 And Granulocyte-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 sed In Hematologic Malignancies,” Haematologica 86:1261-1269; Masten, 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 Complex Class II Expression And Cytokine Production,” Blood 86:176-182). pDCs have been shown to play a role in the l of certain viruses in mouse or monkey models of infection, although they do not appear critical for controlling the immune response to flu (Colonna, M. et al. (1997) “Specificity And Function 0f Immunoglobulin Superfamily NK Cell tory And Stimulatory Receptors,” Immunol. Rev. 155:127- 133; Smit, J.J. et al. (2006) “Plasmacytoid Dendritic Cells Inhibit ary Immunopathology And Promote Clearance 0f Respiratory Syncytial Virus,” J. Exp.
Med. 203:1153-1159). In tumor models, pDCs may promote tumor growth and asis, while pDC depletion resulted in tumor inhibition (Sawant, A. et al. (2012) “Depletion 0f Plasmacytoid Dendritic Cells ts Tumor Growth And Prevents Bone asis 0f Breast Cancer Cells,” J. Immunol. 189:4258-4265). Transient, modest, 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 poietic precursors that express CD123, such as the common myeloid precursor (CMP) (Jordan, C.T. et al. (2000) “The Interleukin-3 Receptor Alpha Chain Is A Unique Marker For Human Acute enous Leukemia Stem Cells,” Leukemia 14:1777-1784; Rieger, MA. et al. (2012) “Hematopoiesis,” Cold Spring Harb. Perspect. Biol. 4:a008250), may be targeted 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 surate drops in red cell parameters, s subjected to the st DART-A exposure (Group 6, 7-day-on infiJsion) showed a reduced reticulocyte response, suggesting a possible cytotoxic activity on precursors (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 Antibody-Mediated Targeting 0f CD123, IL-3 Receptor Alpha Chain, Eliminates Human Acute Myeloid Leukemic Stem Cells,” Cell Stem Cell 5:31-42; s, A. W. et al. (2010) “A Phase I Study 0fAnti-CDI23 Monoclonal Antibody (mAb) CSL360 Targeting Leukemia Stem Cells (LSC) In AML,” J. Clin. Oncol. 28(Suppl):e13012), IL-3 bound diphtheria toxin (Frankel, A. et al. (2008) “Phase I al 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 chimeric antigen receptors (CAR) (Tettamanti, S. et al. (2013) “Targeting 0fAcute Myeloid Leukaemia By Cytokine-Induced Killer Cells Redirected With A Novel CD123-Specific Chimeric Antigen Receptor,” Br. J. Haematol. 161:389-401) 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 ): 2343-2354; Mardiros, A. et al. (2013) “T Cells Expressing CD123-Specific Chimeric Antigen Receptors Exhibit Specific Cytolytic Eflector Functions And Antitumor 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 ed ablation of normal hematopoiesis in NSG mice engrafted with human CD34+ cells following CD123 CAR T cell er (Gill, S. et al. (2014) “Efiicacy Against Human Acute Myeloid Leukemia And Myeloablation 0fNormal Hematopoiesis In A Mouse Model Using Chimeric Antigen or-Modified T Cells,” Blood ): 2343- 2354), gh 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 Receptor,” Br. J. ol. 161:389-401; Pizzitola, I. et al. (2014) ric Antigen Receptors Against CD33/CDI23 ns 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 cellularity 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 ia, blastic plasmacytoid dendritic cell neoplasms ( BPDCNs), a subset of ursor acute lymphoblastic leukemia (B- ALL) and chronic lymphocytic 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 poietic Cell lantation,” Biol.
Blood Marrow Transplant. 19:1006-1012; Florian, S. et al. (2006) “Detection 0f Molecular Targets On The Surface 0fCD34+/CD38-- Stem Cells In s d Malignancies,” Leuk. Lymphoma 47:207-222; Munoz, L. et al. (2001) “Interleukin-3 Receptor Alpha Chain (CD123) Is Widely Expressed In Hematologic Malignancies,” Haematologica 86:1261-1269; Fromm, JR. (2011) “Flow Cytometric is Of CD123 Is Useful For Immunophenotyping Classical Hodgkin Lymphoma,” Cytometry B Clin. Cytom. 80:91-99). The predictable pharmacodynamic activity and manageable safety profile observed in non-human primates further supports the clinical utility and efficacy of DART-A as immunotherapy for these disorders.
In sum, DART-A is an antibody-based le ng the CD38 subunit of the TCR to redirect T lymphocytes against cells expressing CD123, an antigen up- regulated in l 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 les. 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 is of T cells from treated monkeys exhibited unaltered cted target cell lysis, indicating no exhaustion. Toxicity was d to a minimal transient release of nes 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 ation 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 capable of fiarther ations and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the ion and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth.

Claims (20)

What is claimed is:
1. 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 ptide chain, covalently bonded to one another, wherein: 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 g to CD123 (VHCD123) (SEQ ID NO:26), wherein said sub-Domains 1A and 1B are ted from one another by a peptide linker (SEQ ID NO:29); ii. a Domain 2, wherein said Domain 2 is an E-coil Domain (SEQ ID NO:34) or a K-coil Domain (SEQ ID NO:35), 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 main (1B), which comprises a VH Domain of a monoclonal antibody capable of binding to CD3 ) (SEQ ID NO:22), wherein said sub-Domains 1A and 1B are ted 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 peptide 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 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 e of specifically binding to an e of CD123.
2. Claim 2. The diabody of claim 1, wherein said first polypeptide chain additionally comprises an Albumin-Binding Domain (SEQ ID NO:36) linked t o said Domain 2 via a peptide linker (SEQ ID NO:31).
3. Claim 3. The diabody of claim 1, wherein said second 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).
4. 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).
5. Claim 5. The y of claim 1, wherein said second ptide chain additionally comprises a Domain 3 comprising a CH2 and CH3 Domain of an immunoglobulin Fc Domain (SEQ ID , wherein said Domain 3 is linked to said Domain 2 via a e linker (SEQ ID NO:32).
6. Claim 6. The diabody of claim 1, wherein said first polypeptide chain onally 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 peptide linker (SEQ ID NO:32).
7. Claim 7. The diabody of any one of claims 3 to 6, wherein said diabody further comprises a third polypeptide chain sing a CH2 and CH3 Domain of an immunoglobulin Fc Domain (SEQ ID NO: 11).
8. Claim 8. The diabody of any one of claims 3 to 7, wherein said y further comprises a cysteine-containing peptide (SEQ ID NO:55) N -terminal to said CH2 and CH3 Domain of said immunoglobulin Fc Domain.
9. 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).
10. 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).
11. Claim 11. The diabody of any preceding claim, wherein the diabody is capable of crossreacting with both human and primate CD123 and CD3 proteins.
12. Claim 12. The y of claim 1, n: 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.
13. Claim 13. The diabody of claim 1, n the diabody further comprises a third polypeptide chain, wherein: A. said first polypeptide chain comprises 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 ptide chain comprises the amino acid sequence of SEQ ID NO:54.
14. Claim 14. The diabody of claim 1, wherein the diabody 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 ptide 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.
15. Claim 15. A ceutical composition comprising the diabody of any one of claims 1 to 14 and a physiologically acceptable carrier.
16. 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 treatment of a disease or condition associated with or characterized by the expression of CD123.
17. Claim 17. The use of claim 16, wherein said disease or ion associated with or characterized by the expression of CD123 is cancer.
18. Claim 18. The use of claim 17, wherein said cancer is selected from the group consisting of: acute myeloid leukemia (AML), c myelogenous leukemia (CML), ing blastic crisis of CML and Abelson oncogene associated with CML (Bcr-ABL translocation), ysplastic syndrome (MDS), acute B lymphoblastic leukemia (B-ALL), chronic lymphocytic leukemia (CLL), including Richter’s syndrome or Richter’s transformation of CLL, hairy cell ia (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 ytosis, and Burkitt’s ma.
19. 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.
20. Claim 20. The use of claim 19, n said inflammatory condition is selected from the group consisting of: Autoimmune Lupus (SLE), allergy, asthma, and rheumatoid arthritis.
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US201461990475P 2014-05-08 2014-05-08
US61/990,475 2014-05-08
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