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

Links to related applications

According to this application, priority is claimed in accordance with applications for the grant of US patents No. 61/869510 (filed August 23, 2013; under consideration), 61/907749 (filed November 22, 2013; under consideration) and 61 / 990,475 (filed May 8, 2014; under consideration), and the application for the grant of European patent No. 13198784 (filed December 20, 2013), each of which is fully incorporated into this document by reference.

Sequence List Link

This application includes one or more sequence listings in accordance with 37 CFR 1.821 et seq., Which are disclosed in both paper and computer-readable media, and whose paper and computer-readable disclosures are fully incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to CD123xCD3 bispecific monovalent diabodies that are capable of simultaneously binding to CD123 and CD3, and to the use of such molecules in the treatment of hematological malignancies.

BACKGROUND OF THE INVENTION

I. CD123

CD123 (inerleukin 3 alpha receptor, IL-3Ra) is a 40 kDa molecule and is part of the interleukin 3 receptor complex (Stomski, FC et al. (1996) Human Interleukin-3 (IL-3) Induces Disulfide-Linked IL-3 Receptor Alpha- And Beta-Chain Heterodimerization, Which Is Required For Receptor Activation But Not High-Affinity Binding, Mol. Cell. Biol. 16 (6): 3035-3046). Interleukin 3 (IL-3) controls the early differentiation of multipotent stem cells into erythroid, myeloid and lymphoid progenitor cells. CD123 is expressed on CD34 + committed progenitor cells (Taussig, DC et al. (2005) Hematopoietic Stem Cells Express Multiple Myeloid Markers: Implications For The Origin And Targeted Therapy Of Acute Myeloid Leukemia Blood 106: 4086-4092), but not on CD34 + / CD38 - normal hematopoietic stem cells. CD123 is expressed by basophils, mast cells, plasmacytoid dendritic cells, some expression is observed in monocytes, macrophages and eosinophils, and low expression or its absence is observed in neutrophils and megakaryocytes. Some non-hematopoietic tissues (placenta, Leydig cells in the testes, certain cellular elements of the brain and some endothelial cells) express CD123; however, expression is mainly cytoplasmic.

CD123 has been reported to be expressed by leukoblasts and leukemia stem cells (LSC) (Jordan, ST. Et al. (2000) The Interleukin-3 Receptor Alpha Chain Is A Unique Marker For Human Acute Myelogenous Leukemia Stem Cells Leukemia 14: 1777-1784; Jin , W. et al. (2009) Regulation Of Th17 Cell Differentiation And EAE Induction By MAP3K NIK Blood 113: 6603-6610) (Fig. 1). In normal populations of human progenitor cells, CD123 is expressed by a subclass of hematopoietic progenitor cells (LDCs), but not 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 AF et al. (1989) Reciprocal Inhibition Of Binding Between Interleukin 3 And Granulocyte-Macrophage Colony-Stimulating Factor To Human Eosinophils Proc. Natl. Acad Sci. (USA) 86: 7022-7026; Sun Q. et al. (1996) Monoclonal Antibody 7G3 Recognizes The N-Terminal Domain Of The Human Interleukin-3 (IL-3) Receptor Alpha Chain And Functions 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 BJ et al. (2006) Characterization Of Myeloid And Plasmacytoid Dendritic Cells In Human Lung J. Immunol. 177: 7784-7793; Korpelainen, EI 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).

It has been reported that CD123 is overexpressed on malignant cells in a wide range of hematologic malignancies, including acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) (Munoz L. et al. (2001) Interleukin-3 Receptor Alpha Chain (CD123) Is Widely Expressed In Hematologic Malignancies Haematologica 86 (12): 1261-1269). Excessive expression of CD123 is associated with a poorer prognosis for AML (Tettamanti MS et at. (2013) Targeting Of Acute Myeloid Leukaemia By Cytokine-Induced Killer Cells Redirected With A Novel CD123 Special Chimeric Antigen Receptor Br. J. Haematol. 161: 389-401).

AML and MDS are believed to arise and be supported by a small population of leukemic stem cells (LSC), which are usually dormant (i.e., not rapidly dividing cells) and, therefore, resistant to cell death (apoptosis) and traditional chemotherapeutic means. LSCs are characterized by high levels of CD123 expression, which is absent in the corresponding normal population of hematopoietic cells in the normal human bone marrow (Jin, W. et al. (2009) Regulation Of Th17 Cell Differentiation And EAE Induction By MAP3K NIK Blood

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113: 6603-6610; Jordan, CT 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% to 95% of cases of AML, 85% of cases of hairy cell leukemia (ON) and 40% of cases of acute lymphoblastic leukemia (B-ALL). Expression of CD123 is also associated with numerous other malignant tumors / precancerous conditions: cells - the precursors of chronic myeloid leukemia (CML) (including CML with blast crisis); Reed-Sternberg cells (RS) with Hodgkin lymphoma; transformed non-Hodgkin lymphoma (NHL); some types of chronic lymphocytic leukemia (CLL) (CDllc +); a subclass of acute T-lymphoblastic leukemia (T-ALL) (16%, the most immature subclass, mainly affecting adults), plasmacytoid dendritic cell (pDC) malignant tumors (DC2), and bone marrow CD34 + / CD38 malignant tumors in myelodysplastic syndrome (MDS).

AML is a clonal disease characterized by 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 tend to have poorer treatment outcomes than younger patients (Robak, T. et al. (2009) Current And Emerging Therapies For Acute Myeloid Leukemia Clin. Ther. 2: 2349-2370). Unfortunately, most adult patients with AML are currently dying due to their illness.

AML treatment initially focuses on the induction of remission (induction therapy). As soon as induction is achieved, the focus of treatment shifts to consolidating this remission (post-remission or consolidation therapy) and, in some cases, to maintenance therapy. The standard paradigm of remission induction for AML is chemotherapy using a combination of anthracycline and cytarabine followed by either consolidation chemotherapy (usually using elevated doses of the same drugs that were used during the remission induction period) or human stem cell transplantation depending on the patient’s ability to tolerate intensive treatment and the likelihood of recovery only with chemotherapy (see, for example, Roboz, GJ (2012) Current Treatment Of Acute Myeloid Leukemia Curr. Opin. Oncol. 24: 711719).

Agents often used in induction therapy include cytarabine and anthracycline. Cytarabine, also known as AraC, destroys malignant cells (and other rapidly dividing normal cells), interfering with DNA synthesis. Side effects associated with treatment with AraC include reduced resistance to infection as a result of reduced white blood cell production; bleeding as a result of decreased platelet production; and anemia due to a potential decrease in red blood cell count. Other side effects include nausea and vomiting. Anthracyclines (e.g., daunorubicin, doxorubicin, and idarubicin) are characterized by several mechanisms of action, including inhibition of DNA and RNA synthesis, destruction of higher-order DNA structures, and production of free oxygen radicals damaging cells. The most significant side effect of anthracyclines is cardiotoxicity, which significantly limits the lifetime dose and, to some extent, the applicability of anthracyclines.

Thus, unfortunately, despite significant progress in the treatment of newly diagnosed AML, 20-40% of patients do not achieve remission using standard induction chemotherapy, and in 50-70% of patients who achieved complete remission at the beginning, they are expected to relapse no later than than after 3 years. The optimal strategy during relapse or for patients with treatment-resistant disease remains unclear. Stem cell transplantation has been found to be the most effective form of anti-leukemia therapy in patients with AML during the first or subsequent remission (Roboz, GJ (2012) Current Treatment Of Acute Myeloid Leukemia Curr. Opin. Oncol. 24: 711-719).

II. Cd3

CD3 is a T - cell coreceptor consisting of four different chains (Wucherpfennig, KW et al. (2010) Structural Biology Of The T-Cell Receptor: Insights Into Receptor Assembly, Ligand Recognition, And Initiation Of Signaling Cold Spring Harb. Perspect. Biol. 2 (4): a005140; pages 1-14). In mammals, the CD3 complex contains a CD3y chain, a CD35 chain, and two CD3e chains. These chains bind to a molecule known as T cell receptor (TCR) to create an activation signal in T lymphocytes. In the absence of CD3, the correct TCR assembly does not occur and they decompose (Thomas, S. et al. (2010) Molecular Immunology Lessons From Therapeutic T-Cell Receptor Gene Transfer Immunology 129 (2): 170-177). CD3 was found bound to the membranes of all mature T cells and to virtually no other cell type (see Janeway, CA et al. (2005) In: Immunobiology: The Immune System In Health And Disease, 6th ed. Garland Science Publishing, NY, pp. 214-216; Sun ZJ et al. (2001) Mechanisms Contributing To T Cell Receptor Signaling And Assembly Revealed By The Solution Structure Of An Ectodomain Fragment Of The CD3e: y Heterodimer Cell 105 (7): 913-923; Kuhns MS et al. (2006) Decon

- 2 034142 structing The Form And Function Of The TCR / CD3 Complex Immunity. 2006 Feb; 24 (2): 133-139).

III. Bispecific diabodies

The ability of an intact, unmodified antibody (e.g., IgG) to bind an antigen epitope depends on the presence of variable domains on the light and heavy chains of immunoglobulin (i.e., VL and VH domains, respectively). The diabody design is based on the single-chain Fv (scFv) construct (see, e.g., Holliger et al. (1993) 'Diabodies': Small Bivalent And Bispecific Antibody Fragments, Proc. Natl. Acad. Sci. (USA) 90: 6444-6448; U.S. Patent Publication No. 2004/0058400 (Hollinger et al.); US 2004/0220388 (Mertens et al.); Alt et al. (1999) FEBS Lett. 454 (l-2): 90-94; Lu, D. et al. (2005) A Fully Human Recombinant IgG-Like Bispecific Antibody To Both The Epidermal Growth Factor Receptor And The Insulin-Like Growth Factor Receptor For Enhanced Antitumor Activity J. Biol. Chem. 280 (20): 1966519672; WO 02/02781 (Mertens et al); Olafsen T. et al. (2004) Covalent Disulfide-Linked Anti-CEA Diabody Allows Site-Specific Conjugation And Radiolabeling For Tumor Targeting Applications, Protein Eng. Des. Sel. 17 (l): 21-27; Wu A. et al. (2001) Multimerization Of A Chimeric Anti-CD20 Single Chain Fv-Fv Fusion Protein Is Mediated Through Variable Domain Exchange Protein 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 Recombinant Bispecific Antibody) Using A Refolding System, Protein Eng. 13 (8): 583-588; Baeuerle PA et al. (2009) Bispecific T-Cell Engaging Antibodies For Cancer Therapy, Cancer Res. 69 (12): 4941-4944).

The interaction of the light chain of the antibody and the heavy chain of the antibody and, in particular, the interaction of its VL and VH domains forms one of the binding sites of the antibody epitope. In contrast, the scFv construct contains the VL and VH domains of an antibody contained in a single polypeptide chain, and into domains separated by a flexible linker, the length of which is sufficient to allow the self-assembly of two domains into a functional epitope binding site. If self-assembly of the VL and VH domains becomes impossible due to insufficient linker length (less than about 12 amino acid residues), the two scFv constructs interact with each other to form a bivalent molecule in which the VL of one chain is associated with the VH of the other (discussed in Marvin et al. ( 2005) Recombinant Approaches To IgG-Like Bispecific Antibodies, Acta Pharmacol. Sin. 26: 649-658).

Natural antibodies can bind to only one type of epitope (i.e., are monospecific), although they can bind to multiple copies of this type (i.e., exhibiting bivalence or multivalence). The prior art has reported the ability to produce diabodies that differ from such natural antibodies in that they are capable of binding two or more different types of epitopes (i.e. exhibiting bispecificity or multispecificity in addition to bivalence or multivalence) (see, for example, 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 (l-2): 90-94; Lu, D. et al. (2005) A Fully Human Recombinant IgG-Like Bispecific Antibody To Both The Epidermal Growth Factor Receptor And The Insulin-Like Growth Factor Receptor For Enhanced Antitumor Activity J. Biol. Chem. 280 (20): 19665-19672; WO 02/02781 (Mertens et al); Mertens, N. et al, New Recombinant Bi- and Trispecific Antibody Derivatives In: Novel Frontiers In The Production Of Compounds For Biomedical Use, A. VanBroekhoven et al (Eds.), Kluwer Academic Publishers, Dordrecht, The Netherlands (2001), pages 195-208; Wu A. et al. (2001) Multimerization Of A Chimeric Anti-CD20 Single Chain Fv-Fv Fusion Protein Is Mediated Through Variable Domain Exchange, Protein Engineering 14 (2): 1025-1033; Asano et al. (2004) A Diabody For Cancer Immunotherapy And Its Functional Enhancement By Fusion Of Human Fc Domain Abstract 3P683, J. Biochem. 76 (8): 992; Takemura, S. et al. (2000) Construction Of A Diabody (Small Recombinant Bispecific Antibody) Using A Refolding System Protein Eng. 13 (8): 583-588; Baeuerle, PA et al. (2009) Bispecific T-Cell Engaging Antibodies For Cancer Therapy Cancer Res. 69 (12): 4941-4944).

Providing non-monospecific diabodies provides a significant advantage: the ability to coligate and colocalize cells that express various epitopes. Thus, bispecific diabodies are characterized by a wide range of applications, including therapy and immunodiagnostics. Bispecificity allows for great flexibility in the design and development of diabodies in various applications, providing increased avidity for multimeric antigens, cross-linking of different antigens and targeted effects on specific cell types based on the presence of both target antigens. Due to their increased valency, low dissociation rates and rapid elimination from circulation (for small dyatels of ~ 50 kDa or lower), the known dyatel molecules in the art have also shown specific applications in the field of tumor imaging (Fitzgerald et al. (1997 ) Improved Tumor Targeting By Disulphide Stabilized Diabodies Expressed In Pichia pastoris, Protein Eng. 10: 1221). Of particular importance is the coligoning of various cells, for example, cross-linking of cytotoxic T cells with tumor cells (Staerz et al. (1985) Hybrid Antibodies Can Target Sites For Attack By T Cells, Nature 314: 628-631, and Holliger et al. ( 1996) Specific Killing Of Lymphoma Cells By Cytotoxic T-Cells Mediated By A Bispecific Diabody, Protein Eng. 9: 299-305).

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Diatel epitope binding domains can also be targeted to the 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. Many studies have also found that the binding of diabodies to determinants of effector cells, for example, Fey receptors (FcyR), activates an effector cell (Holliger et al. (1996) Specific Killing Of Lymphoma Cells By Cytotoxic T-Cells Mediated By A Bispecific Diabody, Protein Eng. 9: 299-305; Holliger et al. (1999) Carcinoembryonic Antigen (CEA) -Specific T-cell Activation In Colon Carcinoma Induced By Anti-CD3xAnti-CEA Bispecific Diabodies And B7xAnti-CEA Bispecific Fusion Proteins, Cancer Res. 59: 2909-2916; international patent publications No. WO 2006/113665; WO 2008/157379; WO 2010/080538; WO 2012/018687; WO 2012/162068). Typically, activation of effector cells is triggered by the binding of an antibody bound antigen to an effector cell through the interaction of Fc-FcyR; thus, in this regard, the molecules of diabodies can exhibit Ig-like functionality, regardless of whether they contain the Fc domain (for example, according to the results of any analysis of the effector function known in the art or presented as an example in this document ( e.g. ADCC analysis)). By cross-linking the tumor and effector cells, the diatelo not only brings the effector cell into spatial proximity with respect to the tumor cells, but also leads to efficient lysis of the tumor cells (see, e.g., Cao et al. (2003) Bispecific Antibody Conjugates In Therapeutics, Adv. Drug. Deliv. Rev. 55: 171-197).

However, the above benefits come at a significant cost. The formation of such non-monospecific diabodies requires the successful assembly of two or more separate and different polypeptides (i.e., such formation requires that the diabodies are formed by heterodimerization of various types of polypeptide chains). This fact contrasts with monospecific diabodies, which are formed by homodimerization of identical polypeptide chains. Since it is necessary to provide at least two dissimilar polypeptides (i.e., two types of polypeptides) for the formation of a non-monospecific diatel, and since the homodimerization of such polypeptides leads to inactive molecules (Takemura S. et al. (2000) Construction Of A Diabody (Small Recombinant Bispecific Antibody) Using A Refolding System, Protein Eng. 13 (8): 583-588), the production of such polypeptides should be carried out in such a way that prevents the formation of covalent bonds between polypeptides of the same species (i.e., so as to prevent homodimerization) (Takemura, S. et al. ( 2000) Construction Of A Diabody (Small Recombinant Bispecific Antibody) Using A Refolding System, Protein Eng. 13 (8): 583-588). In this regard, the non-covalent binding of such polypeptides is disclosed in the art (see, for example, Olafsen et al. (2004) Covalent Disulfide-Linked Anti-CEA Diabody Allows Site-Specific Conjugation And Radiolabeling For Tumor Targeting Applications, Prot. Engr. Des. Sel. 17: 21-27; Asano et al. (2004) A Diabody For Cancer Immunotherapy And Its Functional 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 Recombinant Bispecific Antibody) Using A Refolding System, Protein Eng. 13 (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, it has been found in the art that bispecific diabodies consisting of non-covalently linked polypeptides are unstable and easily dissociate to non-functional monomers (see, e.g., Lu, D. et al. (2005) A Fully Human Recombinant IgG-Like Bispecific Antibody To Both The Epidermal Growth Factor Receptor And The Insulin-Like Growth Factor Receptor For Enhanced Antitumor Activity, J. Biol. Chem. 280 (20): 19665-19672).

In view of this problem, in the art they have succeeded in developing stable, covalently linked heterodimeric non-monospecific diabodies (see, for example, international patent publications No. WO 2006/113665; WO / 2008/157379; WO 2010/080538; WO 2012 / 018687; WO / 2012/162068; Johnson S. et al. (2010) Effector Cell Recruitment With Novel Fv-Based Dual-Affinity ReTargeting Protein Leads To Potent Tumor Cytolysis And In Vivo B-Cell Depletion J. Molec. Biol. 399 ( 3): 436449; Veri MC et al. (2010) Therapeutic Control Of Cell Activation Via Recruitment Of Fcgamma Receptor lib (CD32B) Inhibitory Function With A Novel Bispecific Antibody Scaffold Arthritis Rheum. 62 (7): 19331943; Moore PA et al. (2011) Application Of Dual Affinity Retargeting Molecules To Achieve Optimal Redirected T-Cell Killing Of B-Cell Lymphoma Blood 117 (17): 4542-4551). Such approaches include the construction of one or more cysteine ​​residues in each of the types of polypeptides used. For example, it was shown that the addition of a cysteine ​​residue to the C-terminus of such constructs ensures the formation of a disulfide bond between the polypeptide chains, stabilizing the resulting heterodimer, without interfering with the binding characteristics of the divalent molecule.

Despite this success, the production of stable, functional heterodimeric, non-monospecific diabodies can be further optimized by careful consideration and placement of cysteine ​​residues in one or more of the polypeptide chains used. Such optimized diabodies can be obtained with a higher yield and with increased activity than

- 4,034,142 non-optimized diabodies. Thus, the present invention relates to the problem of producing polypeptides that are specifically designed and optimized for the formation of heterodimeric diabodies. The present invention solves this problem by providing illustrative, optimized CD123xCD3 diabodies.

SUMMARY OF THE INVENTION

The present invention relates to CD123xCD3 bispecific diabodies that are capable of simultaneously binding to CD123 and CD3, and to the uses of such molecules in the treatment of a disease, in particular hematological malignancies.

The CD123xCD3 bispecific diabodies of the present invention contain at least two different polypeptide chains that are associated with each other in a heterodimeric manner to form one binding site specific for the CD123 epitope and one binding site specific for the CD3 epitope. Thus, the CD123xCD3 diabody according to the present invention is monovalent in the sense that it is able to bind to only one copy of the CD123 epitope and only one copy of the CD3 epitope, but is bispecific in the sense that a single diabody is able to bind simultaneously with the CD123 epitope and with epitope CD3. The individual diatel polypeptide chains are covalently linked to each other, for example, by disulfide linking of cysteine ​​residues located within each polypeptide chain. In particular embodiments, the diabodies of the present invention further comprise an immunoglobulin Fc domain or an albumin binding domain to increase the in vivo half-life.

More specifically, the present invention also provides a CD123xCD3 sequence-optimized bispecific monovalent diatel capable of specifically binding to a CD123 epitope and a CD3 epitope, wherein the diatel comprises a first polypeptide chain and a second polypeptide chain covalently linked to each other, wherein:

A) the first polypeptide chain contains, in the direction from the N-end to the C-end:

i) domain 1, containing:

(1) a subdomain (1A) that contains the VL domain of a monoclonal antibody capable of binding to CD3 (VLcds) (SEQ ID NO: 21); and (2) a subdomain (1B) that contains the VH domain of a monoclonal antibody capable of binding to CD123 (VHCD123) (SEQ ID NO: 26);

moreover, subdomains 1A and 1B are separated from each other by a peptide linker (SEQ ID NO: 29);

ii) domain 2, wherein domain 2 is an E-helical domain (SEQ ID NO: 34) or K is a helical domain (SEQ ID NO: 35), and domain 2 is separated from domain 1 by a peptide linker (SEQ ID NO: 30); and

B. the second polypeptide chain contains, in the direction from the N-terminus to the C-terminus:

i) domain 1, containing:

(1) a subdomain (1A) that contains the VL domain of a monoclonal antibody capable of binding to CD123 (VL_CD123) (SEQ ID NO: 25); and (2) a subdomain (1B) that contains the VH domain of a monoclonal antibody capable of binding to CD3 (VHcd3) (SEQ ID NO: 22);

moreover, subdomains 1A and 1B are separated from each other by a peptide linker (SEQ ID NO: 29);

ii) domain 2, wherein domain 2 is a K-helical domain (SEQ ID NO: 35) or an helical domain (SEQ ID NO: 34), and domain 2 is separated from domain 1 by a peptide linker (SEQ ID NO: 30); and wherein domain 2 of the first and second polypeptide chains do not represent both Espiral domains or both K-helical 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 a CD3 epitope; 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 the CD123 epitope.

The present invention also provides a CD123xCD3 sequence characterized by a non-optimized bispecific monovalent diatel capable of specifically binding to a CD123 epitope and a CD3 epitope, wherein the diabody contains a first polypeptide chain and a second polypeptide chain covalently linked to each other, wherein:

A) the first polypeptide chain contains in the direction from the N-end to the C-end:

i) domain 1, containing:

(1) a subdomain (1A) that contains the VL domain of a monoclonal antibody capable of binding to CD3 (VL_Cd3) (SEQ ID NO: 23); and (2) a subdomain (1B) that contains the VH domain of a monoclonal antibody capable of binding to CD123 (VH_CD123) (SEQ ID NO: 28);

- 5 034142 and subdomains 1A and 1B are separated from each other by a peptide linker (SEQ ID N0: 29);

ii) domain 2, wherein domain 2 is an E-helical domain (SEQ ID NO: 34) or K is a helical domain (SEQ ID NO: 35), and domain 2 is separated from domain 1 by a peptide linker (SEQ ID NO: 30); and

B) the second polypeptide chain contains, in the direction from the N-terminus to the C-terminus:

i) domain 1, containing:

(1) a subdomain (1A) that contains the VL domain of a monoclonal antibody capable of binding to CD123 (VL_CD123) (SEQ ID NO: 27); and (2) a subdomain (1B) that contains the VH domain of a monoclonal antibody capable of binding to CD3 (VHcds) (SEQ ID NO: 24);

moreover, subdomains 1A and 1B are separated from each other by a peptide linker (SEQ ID NO: 29);

ii) domain 2, wherein domain 2 is a K-helical domain (SEQ ID NO: 35) or an helical domain (SEQ ID NO: 34), and domain 2 is separated from domain 1 by a peptide linker (SEQ ID NO: 30); and wherein domain 2 of the first and second polypeptide chains do not represent both Espiral domains or both K-helical 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 a CD3 epitope; 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 the CD123 epitope.

In addition, according to the present invention, there is provided an embodiment of the bispecific monovalent diabodies described above, wherein the first or second polypeptide chain further comprises an albumin binding domain (SEQ ID NO: 36) connected at the C-terminus to domain 2 or at the N-terminus to domain 1 by means of a peptide linker (SEQ ID NO: 31).

In addition, according to the present invention, there is provided an embodiment of the bispecific monovalent diabodies described above, wherein the first or second polypeptide chain further comprises a domain 3 comprising a CH2 and CH3 domain of an IgG immunoglobulin Fc domain (SEQ ID NO: 37), wherein domain 3 is connected to N -terminal to domain 1A via a peptide linker (SEQ ID NO: 33).

In addition, according to the present invention, there is provided an embodiment of the bispecific monovalent diabodies described above, wherein the first or second polypeptide chain further comprises a domain 3 comprising a CH2 and CH3 domain of an IgG immunoglobulin Fc domain (SEQ ID NO: 37), wherein domain 3 is connected to C -terminal to domain 2 via a peptide linker (SEQ ID NO: 32).

In addition, according to the present invention, there is provided an embodiment of any of the bispecific monovalent diabodies described above, wherein the first polypeptide chain domain 2 is a K-helical domain (SEQ ID NO: 35) and the second polypeptide chain domain 2 is an E-helical domain (SEQ ID NO: 34).

In addition, according to the present invention, there is provided an embodiment of any of the bispecific monovalent diabodies described above, wherein the first polypeptide chain domain 2 is an E-helical domain (SEQ ID NO: 34) and the second polypeptide chain domain 2 is a K-helical domain (SEQ ID NO: 35).

In addition, according to the present invention, there is provided an embodiment of a bispecific monovalent diatel capable of specifically binding to a CD123 epitope and a CD3 epitope, wherein the diabody contains a first polypeptide chain and a second polypeptide chain covalently linked to each other, wherein said bispecific diabetic contains:

A) a first polypeptide chain characterized by the amino acid sequence of SEQ ID NO: 1; and

B) a second polypeptide chain characterized by the amino acid sequence of SEQ ID NO: 3;

wherein said first and said second polypeptide chains are covalently linked to each other by a disulfide bond.

The diabodies of the present invention exhibit unexpectedly enhanced functional activities, as further described below.

The diabodies of the present invention are preferably able to cross-react with both human and primate CD123 and CD3 proteins, preferably cynomolgus macaque CD123 and CD3 proteins.

The diabodies of the present invention are preferably capable of depleting in vitro cell analysis plasmacytoid dendritic cells (pDC) from a primary PBMC culture with an IC50 value of about 1 ng / ml or less, about 0.8 ng / ml or less, about 0.6 ng / ml or less, approximately 0.4 ng / ml or less, approximately 0.2 ng / ml

- 6,034,142 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 0.01 ng / ml or less. The IC50 value is preferably about 0.01 ng / ml or less. In the analysis described above, the culture of primary PBMCs can be derived from cynomolgus macaque, in which case said depletion is the depletion of plasmacytoid dendritic cells of cynomolgus macaque (pDC). Optionally, the diabodies of the present invention may be able to deplete plasmacytoid dendritic cells (pDC) from a primary PBMC culture as described above, the assay being carried out according to or in accordance with the protocol of Example 14, as described herein, or by modifying such an assay that will be understood by those skilled in the art, or by other methods known to those skilled in the art.

The diabodies of the present invention preferably exhibit cytotoxicity in an in vitro Kasumi-3 assay with an EC50 value of about 0.05 ng / ml or less. The EC50 value is preferably 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 present invention may exhibit cytotoxicity as described above, wherein the analysis is carried out according to or in accordance with the protocol of Example 3, as described herein, or by modifying such an analysis as will be understood by those skilled in the art, or by other methods, known to those skilled in the art.

The diabodies of the present invention preferably exhibit cytotoxicity in an in vitro Molm-13 assay with an EC50 value of about 5 ng / ml or less. The EC50 value is preferably 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 present invention may exhibit cytotoxicity as described above, wherein the analysis is carried out according to or in accordance with the protocol of Example 3, as described herein, or by modifying such an analysis as will be understood by those skilled in the art, or by other methods, known to those skilled in the art.

The diabodies of the present invention are preferably capable of inhibiting the growth of a MOLM-13 tumor xenograft in a mouse. The diabodies of the present invention may preferably be able to inhibit the growth of a MOLM-13 tumor xenograft in a mouse at a concentration of at least about 20 μg / kg, at least about 4 μg / kg, at least about 0.8 μg / kg, at least about 0.6 μg / kg; or at least about 0.4 μg / kg. Preferred antibodies of the present invention will inhibit the growth of a MOLM-13 tumor xenograft in a mouse by at least 25%, but possibly at least about 40% or more, at least about 50% or more, at least about 60 % or more, at least about 70% or more, at least about 80% or more, at least about 90% or more, or even completely inhibit the growth of the MOLM-13 tumor after a certain period of time or causing regression or disappearance of the tumor. Said inhibition is observed at least for the NSG mouse line. Optionally, the diabodies of the present invention may be able to inhibit the growth of a MOLM-13 tumor xenograft in a mouse as described above according to or in accordance with the protocol of Example 6, as described herein, or by modifying such an analysis as will be understood by those skilled in the art, or other methods known to those skilled in the art.

The diabodies of the present invention are preferably capable of inhibiting the growth of an RS4-11 tumor xenograft in a mouse. The diabodies of the present invention may preferably be able to inhibit the growth of an RS4-11 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 present invention will inhibit the growth of an RS4-11 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 completely inhibit the growth of the RS4-11 tumor after a certain period of time or causing regression or disappearance of the tumor. Said inhibition is observed at least for the NSG mouse line. Optionally, the diabodies of the present invention may be able to inhibit the growth of an RS4-11 tumor xenograft in a mouse as described above according to or in accordance with the protocol of Example 6, as described herein, or by modifying such an analysis as will be understood by those skilled in the art, or other methods known to

- 7,034,142 sheets in the art.

The diabodies of the present invention are preferably capable of depleting in vitro leukemic blast cells in a primary AML bone marrow cell culture. The diabodies of the present invention may preferably be able to deplete leukemic blast cells in vitro in primary culture of AML bone marrow cells at concentrations of at least about 0.01 ng / ml, at least about 0.02 ng / ml, at least 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. The diatels of the present invention may preferably be able to deplete in vitro leukemic blast cells in a primary bone marrow cell culture of AML by less than 20% of the total primary leukemic blast cell population at a diabetic concentration 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 after incubation of the primary culture with diabody for about 120 hours Leukemic blast cells are preferably subjected to in vitro depletion in a primary culture of bone marrow cells, AML is less than 20% of the total population of primary leukemic blast cells at diabetic concentrations of approximately 0.01 ng / ml or 0.1 ng / ml after primary incubation diatel cultures for approximately 120 hours

The diabodies of the present invention are preferably capable of inducing in vitro expansion of the Tcellular population in primary AML bone marrow cell culture. Such expansion may preferably be approximately 70% or more of the maximum T cell population that can be subjected to expansion in the assay. The diabodies of the present invention may preferably be able to induce the expansion of the T cell population in vitro in the primary bone marrow cell culture of AML by approximately 70% or more of the maximum T cell population that can be expanded in the assay at diael concentrations of at least approximately 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 after incubation of the primary culture with diabel for about 120 hours The T cell population preferably increases in vitro in primary AML bone marrow cell cultures by approximately 70% or more of the maximum T cell population that can be expanded in the assay at diabetic concentrations of about 0.01 ng / ml or about 0 , 1 ng / ml after incubation of the primary culture with diatel for approximately 120 hours

The diabodies of the present invention are preferably capable of inducing in vitro activation of the Tcellular population in primary AML bone marrow cell culture. Such activation may occur at diabetic 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 after incubation of the primary culture with diabody for about 72 hours. Such activation can be measured by expression of a T cell activation marker, such as CD25. Activation The in vitro T cell population in primary AML bone marrow cell culture, as measured by CD25 expression, preferably occurs at diabetic concentrations of approximately 0.01 ng / ml or approximately 0.1 ng / ml, after incubation of the primary culture with diabel in for approximately 72 hours

The diabodies of the present invention are preferably able to deplete in vitro leukemic blast cells in primary AML bone marrow cell culture by less than 20% of the total primary leukemic blast cell population and at the same time induce expansion of the In vitro Cellular population in primary AML bone marrow cell culture approximately 70% or more of the maximum T-cell population that can be expanded in the analysis, at diabetic 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 after incubation of the primary culture with diabody for about 120 hours The concentration of the diabody is preferably about 0.01 ng / ml or about 0.1 ng / ml, and the primary culture is incubated with the diabel for about 120 hours.

The diabodies of the present invention may be able to deplete in vitro leukemic blast cells in a primary culture of AML bone marrow cells and / or induce an expansion of the T cell population in vitro in primary culture of AML bone marrow cells and / or induce activation of a T cell population in vitro in the primary culture of bone marrow cells AML in the manner described above according to or in accordance with the protocol of example 8, as described herein, or by modifying such an analysis, as will be understood by those skilled in the art.

- 8,034,142 of these techniques, or by other methods known to those skilled in the art.

In order to avoid misinterpretation, the diabodies of the present invention may exhibit one, two, three, more than three, or all of the functional features described herein. Thus, the diabodies of the present invention can exhibit any combination of the functional features described herein.

The diabodies of the present invention are suitable for use as a pharmaceutical. The diabodies are preferably suitable for use in the treatment of a disease or condition associated with the expression of CD123 or characterized by the expression of CD123. The present invention also relates to the use of a diatel according to the present invention in the manufacture of a pharmaceutical composition, preferably for the treatment of a disease or condition associated with the expression of CD123 or characterized by the expression of CD123, as further defined herein.

The disease or condition associated with the expression of CD123 or characterized by the expression of CD123 may be a malignant tumor. For example, a malignant tumor may be selected from the group consisting of the following: acute myeloid leukemia (AML), chronic myelogenous leukemia (CML), including CML blast crisis and Abelson oncogen associated with CML (Bcr-ABL translocation), myelodysplastic syndrome (MDS), acute B-lymphoblastic leukemia (B-ALL) chronic lymphocytic leukemia (CLL), including CLL with Richter syndrome or Richter transformation, hairy cell leukemia (ON), a tumor from plasmacytoid dendritic cells (OBPDK), non-Hodgkin’s lymphomas (NHL), including mantle cell lymphoma (MLC) and small cell lymphocytic lymphoma (MLL), Hodgkin lymphoma, systemic mastocytosis, and Burkitt’s lymphoma.

A disease or condition associated with the expression of CD123 or characterized by the expression of CD123 may be an inflammatory condition. For example, an inflammatory condition may be selected from the group consisting of the following: systemic lupus erythematosus (SLE), allergy, bronchial asthma, and rheumatoid arthritis.

In addition, according to the present invention, there is provided a pharmaceutical composition comprising any of the above-described diabodies and a physiologically acceptable carrier.

In addition, the present invention provides the use of the pharmaceutical composition described above in the treatment of a disease or condition associated with the expression of CD123 or characterized by the expression of CD123.

In particular, according to the present invention, there is provided an embodiment of such an application in which the disease or condition associated with the expression of CD123 or characterized by the expression of CD123 is a malignant tumor (especially a malignant tumor selected from the group consisting of the following: acute myeloid leukemia (AML), chronic myelogenous leukemia (CML), including CML blast crisis and Abelson oncogen associated with CML (Bcr-ABL translocation), myelodysplastic syndrome (MDS), acute B-lymphoblastic leukemia (BOLOL), lymphocytic leukemia (CLL), including CLL with Richter syndrome or Richter transformation, hairy cell leukemia (ON), neoplasm of plasmacytoid dendritic cells (OBPDK), non-Hodgkin’s lymphomas (NHL), including mantle cell lymphoma (MLC) and small cell lymphocytic lymphoma (MLL), Hodgkin's lymphoma, systemic mastocytosis, and Burkitt’s lymphoma).

In particular, according to the present invention, there is provided an embodiment of such an application in which a disease or condition associated with expression of CD123 or characterized by expression of CD123 is an inflammatory condition (especially an inflammatory condition selected from the group consisting of the following: systemic lupus erythematosus (SLE) , allergies, bronchial asthma and rheumatoid arthritis).

Terms such as are approximately understood as mean within 10%, more preferably within 5% of the indicated value, unless the context otherwise provides.

A brief description of the graphic materials

In FIG. 1 shows that CD123 is known to be expressed on leukemia stem cells.

In FIG. 2 illustrates the structures of the first and second polypeptide chains of a double-stranded CD123xCD3 bispecific monovalent diatel according to the present invention.

In FIG. 3A and 3B illustrate the structures of two versions of the first, second, and third polypeptide chains of the three-chain CD123xCD3 bispecific monovalent Fc dieatel according to the present invention (version 1, FIG. 3A; version 2, FIG. 3B).

In FIG. 4 (panels AE) shows the ability of various CD123xCD3 bispecific diabodies to mediate T-cell redirected cytolysis of target cells showing a variable amount of CD123. In FIG. dose-dependent curves are presented, indicating that the bispecific diatel (DART-A) characterized by the optimized CD123xCD3 sequence containing the albumin binding domain (DART-A with ABD w / ABD) showed greater cytotoxicity than the control bispecific diatel (control DART) or a non-optimized CD123xCD3 sequence of bispecific diatel (DART-B) in numerous types of target cells: RS4-11 (panel A); TF-1 (panel B); Molm-13 (panel C); Kasumi-3 (panel D); and THP-1 (panel E) in a ratio of E: T (effector: target) of 10: 1.

In FIG. 5 (panel AD) shows the ability of an optimized sequence of CD123xCD3 bispecific diabetic (DART-A), characterized by an optimized sequence of CD123xCD3 bispecific diabetic containing an albumin binding domain (DART-A with ABD w / ABD) and characterized by an optimized sequence of CD123xCD3 immuno-specific bispecific domain IgG (DART-A with Fc w / Fc), to mediate T-cell activation in the process of redirected cytolysis of target cells. In FIG. dose-response curves showing cytotoxicity mediated by DART-A, DART-A w / ABD and DART-A w / Fc in Kasumi-3 cells (panel A) and THP-1 (panel B) and purified CD8 T cells are shown with a ratio of E: T (effector cell: target cell) of 10: 1 (18 h incubation). Panels C and D show dose-response curves of T cell activation using the CD25 marker on CD8 T cells in the presence (panel D) and in the absence (panel C) of target cells.

In FIG. 6 (panel AB) shows the contents of granzyme B and perforin in CD4 and CD8 T cells after treatment with a sequence-optimized CD123xCD3 bispecific diatel (DART-A) (panel A) or a control bispecific diabel (control DART) (panel B) c the presence of Kasumi-3 target cells and dormant T cells in an E: T ratio of 10: 1.

In FIG. 7 (Panel AB) shows the in vivo antitumor activity of a sequence-optimized CD123xCD3 bispecific diabetic (DART-A) sequence at dosage levels in ng / kg. MOLM-13 cells (intermediate expression level of CD123) were mixed with T cells and implanted subcutaneously (T: E 1: 1) to NSG mice. Intravenous treatment was carried out once a day for 8 days (QDx8), starting from the day of implantation. Different concentrations of DART-A were compared with a control bispecific diabetic (control DART). Panel A shows Molm-13 cells alone or with T cells, and the effect of different doses of DART-A on tumor volume, even at temporary points beyond 30 days. Panel B shows the effect of increasing doses of DART-A on tumor volume observed in NSG mice that received MOLM-13 cells and T cells (T: E 1: 1) over a period of 0-18 days.

In FIG. Figure 8 shows the in vivo antitumor activity of a sequence-optimized CD123xCD3 bispecific diabetic (DART-A) sequence on RS4-11 cells (ALL with signs of monocytes). Cells were mixed with T cells and implanted subcutaneously (T: E 1: 1) with NSG mice. Intravenous treatment was carried out once a day for 4 days (QDx4), starting from the day of implantation. Different concentrations of DART-A were compared with a control bispecific diabetic (control DART).

In FIG. Figure 9 (panel AB) shows CD123 + blast cells in bone marrow mononuclear cells (BM MNC) and peripheral blood mononuclear cells (PBMC) from patient 1 with AML (panel A) compared to the Kasumi-3 AML cell line (panel B).

In FIG. 10 (Panels A-C) shows the ability of a bispecific diabetic diabetic (DART-A) characterized by a sequence-optimized CD123xCD3 to mediate the reduction of blast cells in primary AML after 120 h (panel A), to control the expansion of the T-cell population in primary AML after 120 h (panel B ) and induce T-cell activation in AML after 48 and 72 hours (panel C).

In FIG. 11 (Panels AH) shows the identification of a population of CD123 + blast cells in a primary sample of PBMC ALL. Panels A and E show direct and lateral light scattering of the introduced population of normal PBMCs (panel A) and PBMC ALL (panel E). Panels B and F show the identification of a population of lymphocytes as mainly B cells (panel B) and leukemic blast cells (panel F). Panels C and G show the identification of a population of lymphocytes that are CD123 +. Panels D and H show the identification of CD19 + cells and CD123 + cells.

In FIG. 12 (Panel AB) shows the identification of CD4 and CD8 T cell populations in the primary sample of PBMC ALL. Panel A shows direct and lateral light scattering of the introduced population of PBMC ALL. Panel B shows the CD4 or CD8 T cell populations present in the samples. The amounts indicate that CD4 T cells represent approximately 0.5% of the total number of cells, and CD8 T cells represent approximately 0.4% of the total number of cells present in the PBMC ALL sample.

In FIG. 13 (panels AH) shows the ability, characterized by the optimized CD123xCD3 sequence of bispecific diatel (DART-A), to mediate blast cell deletion in ALL using autologous CTL (cytotoxic T lymphocytes). Panels A and E show direct and side light scattering of the introduced population of normal PBMCs (panel A) and PBMC ALL (pa

- 10 034142 nel E). PBMCs were left untreated (panels B and F), treated with a bispecific control diabel (control DART) (panels C and G) or treated with DART-A (panels D and H) and incubated for 7 days, after which they were stained in relation to CD34 and CD19.

In FIG. 14 (AL panels) shows the ability of a sequence-optimized CD123xCD3 bispecific diabetic (DART-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 RVMS (AF panels) and RVMS ALL (GL panels). Cells were left untreated (panels A, D, G, and J) or treated with a bispecific control dietel (control DART) (panels B, E, H, and K) or DART-A (panels C, F, I, and L) in within 7 days.

In FIG. 15 (panels A-C) shows the identification of a blast population of AML and T cells in a primary AML sample. Panel A shows direct and lateral light scattering of the introduced population of PBMC AML. Panel B shows the identification of the blast population of AML in the AML sample. Panel C shows the identification of the T cell population in the AML sample.

In FIG. 16 (panels A-C) shows the ability of a sequence-optimized CD123xCD3 bispecific diatel (DART-A) to mediate the depletion of attack cells in AML using autologous CTL and T cell expansion. Primary PBMC AMLs from patient 2 were incubated with PBS, a control bispecific diabel (control DART) or DART-A for 144 h. Blast cells (panel A), CD4 T cells (panel B) and CD8 T cells (panel C were counted )

In FIG. 17 (AD panels) shows the ability of a sequence-optimized CD123xCD3 bispecific diabetic (DART-A) to mediate T cell activation in AML. Expression of CD25 (panel A) and Ki-67 (panel B) was determined for CD4 and CD8 T cells from patient 2 with AML after incubation with a control bispecific diabel (control DART) or DART-A with autologous PBMCs. The content of perforin (panel C) and granzyme B (panel D) was determined for CD4 and CD8 T cells from patient 2 with AML after incubation with control DART or DART-A with autologous PBMCs.

In FIG. Figure 18 (AD panels) shows that a bispecific diatel (DART-A) characterized by an optimized CD123xCD3 sequence is able to cross-react with both human and primate CD123 and CD3 proteins. The panels show BIACORE ™ sensogram curves of assays performed to evaluate the ability of DART-A to bind to human CD3 (panels A and B) and CD123 (panels C and D) human (panels A and C) and non-human primates (panels B and D ) KD values ​​are presented.

In FIG. 19 (Panel AB) shows the ability of a sequence-optimized CD123xCD3 bispecific diabetic (DART-A) to mediate in vitro deletion of autologous monocytes from human and cynomolgus macaque PBMCs. The panels show the results of dose-response curves of DART-A mediated cytotoxicity with primary human PBMCs (panel A) or cynomolgus monkeys PBMCs (panel B).

In FIG. 20 (AN panels) shows the ability of a sequence-optimized CD123xCD3 bispecific diatel (DART-A) to mediate pDC depletion in cynomolgus monkeys without systemic cytokine induction. The AD panels show the control results obtained after 4 hours and 4 days with an inert carrier and a carrier. The EH panels show the control results obtained after 4 hours and 4 days with the control bispecific diatel (control DART). The IN panels show the results obtained after 4 hours and 4 days at a dose of DART-A of 10 ng / kg / day, and after 4 days at a dose of DART-A of 30 ng / kg / day.

In FIG. Figure 21 (AD panels) shows the ability of a sequence-optimized CD123xCD3 bispecific diabetic (DART-A) to mediate dose-dependent pDC depletion in cynomolgus monkeys. Java macaques were injected with doses of DART-A of 0.1, 1, 10, 30, 100, 300, or 1000 ng / kg. PBMCs were evaluated at the indicated time and the total number of B cells (panel A), monocytes (panel B), NK cells (panel C) and pDC (panel D) were determined.

In FIG. 22 (AD panels) shows the ability of a CD123xCD3 bispecific diabetic (DART-A) sequence optimized to periodically modulate T cells in cynomolgus monkeys. Java macaques were injected with doses of DART-A of 0.1, 1, 10, 30, 100, 300 or 1000 ng / kg. PBMCs were evaluated at the indicated time and the total number of T cells (panel A), CD4 T cells (panel B), CD69 cells (panel C) and CD8 T cells (panel D) were determined.

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

In FIG. 24A-24B show the physicochemical characteristics of purified DART-A. In FIG. 24A: SEC profile of DART-A protein on a calibrated TSK G3000SWxL column.

In FIG. 24B is a mass spectrum of DART-A protein.

In FIG. 25A-25D show an SPR assay of DART-A binding to immobilized human or Cynomolgus macaque CD123 and CD3. The dashed lines represent the global approximation to 1: 1.

- 11,034,142 Langmuir models of experimental binding curves obtained at DART-A concentrations of 0, 6.25, 12.5, 25, 50, or 100 nM (continuous lines). Data are representative of three independent experiments.

In FIG. 26A-26E show that DART-A is capable of simultaneously binding to both CD3 and CD123. In FIG. 26A-26B show the results of a bifunctional ELISA and demonstrate the simultaneous involvement of both DART-A target antigens. Human CD123 (Fig. 26A) or cynomolgus macaque CD123 (Fig. 26B) was applied to ELISA plates. Titration of DART-A and control DART was followed by detection with human CD3-biotin. In FIG. 26C26E demonstrated DART-A cell surface binding to CD123 + Molm-13 target cells (Figure 26C), human T cells (Figure 26D) and T-cells of cynomolgus macaque (Figure 26E). Binding was detected by FACS analysis using a monoclonal antibody specific for the E-helical and K-helical regions of the DART-A molecule or control DART.

In FIG. 27A-27H show the ability of DART-A to mediate redirected cytolysis of a target cell by human or monkey effector cells against CD123 + Kasumi-3 leukemia cell lines, shows the ability of molecules to bind to a subclass of normal leukocytes circulating in the bloodstream, including pDC and monocytes, and shows the ability of molecules deplete CD14^-CD123^high cells (pDC and basophils) without affecting monocytes (CD14 + cells). In FIG. 27A shows the relative binding sites for the anti-CD123-PE U937 antibody and Kasumi-3 leukemia cell lines determined by QFACS analysis. In FIG. 27B shows the relatively low percentage cytotoxicity mediated by DART-A or control DART on the AML cell line (U937 cells), which, as shown in FIG. 27A contain relatively few binding sites for CD123). In FIG. 27C shows percent cytotoxicity mediated by DART-A or control DART in the presence of purified human T cells (as effector cells) on the AML cell line (Kasumi-3 cells), which, as shown in FIG. 27A, contain a significant number of CD123 binding sites. In FIG. 27B-27C, the ratio of E: T is 10: 1. In FIG. 27D shows the percentage cytotoxicity mediated by DART-A or control DART in the presence of purified cynomolgus macavirus PBMCs (as effector cells) on Kasumi-3 cells (E: T ratio of 15: 1), and it was shown that DART-A can bind to T cells of cynomolgus macaque. In FIG. 27E shows the relative binding sites for the anti-CD123-PE antibody on Kasumi-3 cells, human monocytes, human plasmacytoid dendritic cells (pDC), cynomolgus macaque monocytes and cynomolgus plasmacytoid dendritic cells, as determined by QFACS analysis. In FIG. 27F shows the ability of DART-A to deplete CD14^- CD123¹⁰ cells. In FIG. 27G shows the ability of DART-A to deplete CD14^- CD123^H1 human cells. In FIG. 27H shows the ability of DART-A to deplete CD14^- CD123^Hi cynomolgus macaque cells. Cytotoxicity was determined by the release of LDH, with EC50 values ​​determined using GraphPad PRISM® software.

In FIG. Figure 28 shows the use of a two-compartment model for evaluating the pharmacokinetic parameters of DART-A. Data show serum DART-A concentrations at the end of the infusion (EOI) in cynomolgus monkeys after receiving a 96-hour infusion at a dose of 100 ng / kg / day, 300 ng / kg / day, 600 ng / kg / day and 1000 ng / kg / day. Each dot represents a separate animal; horizontal lines represent the average value for the dosing group.

In FIG. 29A-29C show the effect of DART-A infusion on the production of cytokine, IL-6. Serum IL-6 levels (mean ± SEM) in monkeys who were given DART-A infusions are shown by treatment group. An inert carrier was introduced to the Macaque macaques - control at 1 day, after which 4 weekly infusions or an inert carrier (group 1) were administered (Fig. 29A), or DART-A, administered as 4-day infusions every week, starting from day 8, at 15 , 22 and 29 days (groups 2-5) (Fig. 29B) or as an infusion 7 days a week for 4 weeks starting on day 8 (group 6) (Fig. 29C). Intervals of treatment are indicated by gray bars.

In FIG. 30A-30F show the effect of DART-A infusion on the depletion of CD14- / CD123 + cells (FIG. 30A30C) and CD303 + cells (FIG. 30D-30F). Shown is the mean ± SEM of circulating CD14- / CD123 + (Fig. 30A-300) or CD303 + (Fig. 30D-30F) circulating blood levels over the study days and groups. An inert carrier was introduced to the Macaque macaques - control at 1 day, after which 4 weekly infusions or an inert carrier were introduced (group 1) (Fig. 30A and 30D), or DART-A, administered as a 4-day infusion every week starting on day 8, on days 15, 22, and 29 (groups 2-5) (FIGS. 30A and 30E) or as an infusion 7 days a week for 4 weeks starting on day 8 (group 6) (Fig. 30C and 30F). Intervals of treatment are indicated by gray bars.

In FIG. 31A-31I show the observed changes in T-cell populations (Figures 31A-31C), CD4 + cell populations (Figures 31D-31F) and CD8 + cell populations (Figures 31G-31I) receiving DART-A administered as a 4-day infusion starting on day 8, at 15, 22 and 29 days. Legend: CD25 + (gray squares); CD69 + (gray triangles), PD-1 + (white triangles); Tim-3 + (white

- 12,034,142 squares). T cells were counted using CD4 and CD8 markers, rather than canonical CD3, to eliminate potential interference from DART-A. Java monkeys were administered an inert control vehicle at 1 day, followed by 4 weekly infusions or an inert carrier (group 1), or DART-A, administered as 4-day infusions every week, starting from day 8, at 15, 22 and 29 day (group 5) or in the form of infusions 7 days a week for 4 weeks, starting on day 8 (group 6). Intervals of treatment are indicated by gray bars. Shown is the mean ± SEM of the absolute number of T cells circulating in the bloodstream by study days and groups (Fig. 31A-31C). Relative values ​​(mean percent ± SEM) of CD25 +, CD69 +, PD-1 + and Tim-3 + are shown on CD4 (Fig. 31D-31E) or CD8 T cells (Fig. 31F-31H) by study days and groups.

In FIG. 32A-32F show the observed changes in CD4 + T cell populations (Figures 32A32C) and CD8 + T cell populations (Figures 32D-32F) during and after a 7-day continuous DART-A infusion. Shown is the mean ± SEM as a percentage of CD25 +, CD69 +, PD-1 + and Tim-3 + on CD4 (Fig. 32A-32C) or CD8 (Fig. 32D-32F) T cells by study days and groups 2, 3 and 4. Treatment intervals are indicated by gray bars. Legend: CD25 + (gray squares); CD69 + (gray triangles), PD-1 + (white triangles); Tim-3 + (white squares).

In FIG. 33A-33F show the observed changes in CD4 + T cell populations (Fig. 33A33C) and CD8 + T cell populations (Fig. 33D-33F) during and after a continuous 7-day DART-A infusion. Shown are the mean ± SEM as a percentage of CD4 + naive cells (CD95- / CD28 +), CMT (CD95 + / CD28-) and EMT (CD95 + / CD28-) T cells in the CD4 + population (Fig. 33A-33O) or CD8 population (Fig. 33D-33F) by study days and groups 2, 3, and 4. Macaque monkeys were injected with an inert control vehicle at 1 day, after which 4 weekly infusions of either an inert carrier or DART-A, administered as 4-day infusions every week, starting from day 8, at 15, 22 and 29 days (groups 2- 4). Intervals of treatment are indicated by gray bars. Legend: naive cells (white triangles); SMT (black triangles), EMT (gray squares).

In FIG. 34 shows DART-A mediated cytotoxicity against Kasumi-3 cells with PBMCs from either unexposed monkeys or monkeys treated with multiple DART-A infusions.

In FIG. 35A-35F showed that exposure to DART-A increased the relative frequency of central CD4 memory cells and effector CD8 + memory cells due to the corresponding previously unexposed T cell population. Shown is the mean ± SEM as a percentage of CD4 + naive cells (CD95- / CD28 +), CMT (CD95 + / CD28 +) and EMT (CD95 + / CD28-) T cells in the CD4 + population (Fig. 35A-35C) or in the CD8 + population (Fig. .35D-35F by study day and group. An inert carrier was introduced to the Macaque macaques - control at 1 day, after which 4 weekly infusions of either an inert carrier (group 1) or DART-A, administered as 4-day infusions every week, starting from day 8, at 15, 22 and 29 days (group 5) or as an infusion 7 days a week for 4 weeks starting on day 8 (group 6). Intervals of treatment are indicated by gray bars. Legend: naive (white triangles); SMT (black triangles), EMT (gray squares).

In FIG. 36A-36F show the effect of DART-A on red blood cell parameters in monkeys that received infusion of molecules. The contents of circulating red blood cells (Fig. 36A-36C) or reticulocytes (Fig. 36D-36F) (mean ± SEM) in samples collected at indicated time points from monkeys treated with DART-A are shown.

In figures 37A-37B shows the frequency of occurrence (average percentage ± SEM) of CD123 + cells (Fig. 37A) or HSC (CD34 + / CD38- / CD45- / CD90 + cells) (Fig. 37B) in the Lin-cell population in bone marrow samples, collected at indicated time points from monkeys treated with DART-A. An inert carrier was introduced to the Macaque macaques - control at 1 day, after which 4 weekly infusions of either an inert carrier (group 1) or DART-A, administered as 4-day infusions every week, starting from day 8, at 15, 22 and 29 days (groups 2-5) or as an infusion 7 days a week for 4 weeks, starting on day 8 (group 6).

Detailed disclosure of the present invention

The present invention relates to bispecific monovalent diabodies characterized by the optimized sequence of CD123xCD3 that are capable of simultaneously binding to CD123 and CD3, and to the use of such molecules in the treatment of hematological malignancies. Despite the fact that non-optimized CD123xCD3 bispecific diabodies are fully functional, similar to the improvements obtained in gene expression through codon optimization (see, for example, Grosjean, H. et al. (1982) Preferential Codon Usage In Prokaryotic Genes: The Optimal Codon-Anticodon Interaction Energy And The Selective Codon Usage In Efficiently Expressed Genes Gene 18 (3): 199-209), it is possible to further enhance the stability and / or function of the CD123xCD3 bispecific diabodies by modifying or optimizing their sequences.

Preferred CD123xCD3 bispecific diabodies of the present invention consist of at least two polypeptide chains that associate with each other to form one binding site specific for the CD123 epitope and one binding site,

- 13 034142 specific for the epitope CD3 (Fig. 2). The individual diatel polypeptide chains are covalently linked to each other, for example, by disulfide linking of cysteine ​​residues located within each polypeptide chain. Each polypeptide chain contains an antigen binding domain of a light chain variable domain, an antigen binding domain of a heavy chain variable domain, and a heterodimerization domain. An intermediate linker peptide (linker 1) separates the antigen 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 to form a first functional antigen binding site that is specific for the first antigen (i.e. or CD123 or CD3). Similarly, 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 to form a second functional antigen binding site that is specific for the second antigen (i.e., either CD123 or CD3, depending on identity of the first antigen). Thus, the selection of the antigen binding domain of the variable domain of the light chain and the antigen binding domain of the variable domain of the heavy chain of the first and second polypeptide chains is coordinated so that the two polypeptide chains together contain the antigen binding domains of the variable domains of the light and heavy chains capable of binding to CD123 and CD3.

The formation of heterodimers of the first and second polypeptide chains can be controlled by 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 another polypeptide chain (US2007 / 0004909). Alternatively, such domains can be designed to contain helices of opposite charges. The heterodimerization domain of one of the polypeptide chains contains a sequence of at least six, at least seven or at least eight positively charged amino acids, and the heterodimerization domain of the other polypeptide chain contains a sequence of at least six, at least seven or of at least eight negatively charged amino acids. For example, the first or second heterodimerization domain will preferably contain a sequence of eight positively charged amino acids, and the other of the heterodimerization domains will preferably contain a sequence of eight negatively charged amino acids. A positively charged amino acid may be lysine, arginine, histidine, etc. and / or a negatively charged amino acid may be glutamic acid, aspartic acid, etc. The positively charged amino acid is preferably lysine and / or the negatively charged amino acid is preferably glutamic acid.

The CD123xCD3 bispecific diabodies of the present invention are designed so that such first and second polypeptide chains are covalently linked to each other via cysteine ​​residues along their length. Such cysteine ​​residues can be introduced into an intermediate linker that separates the VL and VH domains of the polypeptides. Alternatively and more preferably, a second peptide (linker 2) is introduced at each of the polypeptide chains, for example, at the amino terminus of the polypeptide chains or at 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 particular embodiments, the CD123xCD3 sequence optimized bispecific monovalent diabodies of the present invention further comprise an immunoglobulin Fc domain or an albumin binding domain to increase the in vivo half-life.

The CD123xCD3 bispecific monovalent diabodies of the present invention which contain an immunoglobulin Fc domain (i.e., CD123xCD3 bispecific monovalent Fc diabodies) are composed of a first polypeptide chain, a second polypeptide chain and a third polypeptide chain. The first and second polypeptide chains associate with each other to form one binding site specific for the CD123 epitope and one binding site specific for the CD3 epitope. The first polypeptide chain and the third polypeptide chain are associated with each other to form an immunoglobulin Fc domain (FIGS. 3A and 3B). The first and second polypeptide chains of a bispecific monovalent Fc-diatel are covalently linked to each other, for example, by disulfide linking of cysteine ​​residues located within each polypeptide chain.

The first and third polypeptide chains are covalently linked to each other, for example, by disulfide linking of cysteine ​​residues located within each polypeptide chain. Each of the first and second polypeptide chains contains the antigen binding domain of the variable domain of the light chain, the antigen binding domain of the variable domain of the heavy chain and the heterodimerization domain. An intermediate linker peptide (linker 1) separates the antigen binding domain

- 14,034,142 the variable domain of the light chain from the antigen binding domain of the variable domain of the heavy chain. 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 to form a first functional antigen binding site that is specific for the first antigen (i.e., either CD123 or CD3). Similarly, 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 to form a second functional antigen binding site that is specific for the second antigen (i.e., either CD3 or CD123, depending on identity of the first antigen). 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 so that the two polypeptide chains together contain the antigen binding domain of the light and heavy chain variable domains capable of binding to CD123 and CD3. Each of the first and third polypeptide chains contain some or all of the CH2 domain and / or some or all of the CH3 domain of the complete immunoglobulin F c domain and a cysteine-containing peptide. Some or all of the CH2 domain and / or some or all of the CH3 domain is associated with the formation of the immunoglobulin Fc domain of the bispecific monovalent Fc dielectric according to the present invention. The first and third polypeptide chains of the bispecific monovalent Fc dieatles of the present invention are covalently linked to each other, for example, by disulfide linking of cysteine ​​residues located within the cysteine-containing peptide of the polypeptide chains.

I. Characterized by the optimized sequence of CD123xCD3 bispecific diatel, DART-A

The present invention provides a sequence-optimized bispecific diatel capable of simultaneously and specifically binding to the CD123 epitope and the CD3 epitope (CD123xCD3 bispecific diatelo or DART-A). As discussed below, it has been found that DART-A exhibits increased functional activity with respect to other bispecific diabetics of a similar composition characterized by an unoptimized CD123xCD3 sequence and, therefore, is called a bispecific diatelo characterized by an optimized sequence of CD123xCD3.

The bispecific diatel (DART-A) characterized by the optimized sequence of CD123xCD3 contains a first polypeptide chain and a second polypeptide chain. The first polypeptide chain of the bispecific diabetic will contain, from the N-terminus to the C-terminus, the N-terminus, the light chain variable domain (VL domain) of a monoclonal antibody capable of binding to CD3 (VL_Cd3), an intermediate linker peptide (linker 1), the heavy chain variable domain (VH domain) of a monoclonal antibody capable of binding to CD123 (VH_CD123), and the C-terminus. A preferred sequence of such a VL domain_Cd₃ represents SEQ ID NO: 21:

QAVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRA PWTPARFSGSLLGGKAALTITGAQAEDEADYYCALWYSNLWVFGGGTKLTVLG

VL antigen binding domain_Cd3 contains CDR1 SEQ ID NO: 38: RSSTGAVTTSNYAN, CDR2 SEQ ID NO: 39: GTNKRAP and CDR3 SEQ ID NO: 40: ALWYSNLWV.

A preferred sequence of such linker 1 is SEQ ID NO: 29: GGGSGGGG. A preferred sequence for such a VH domain_CD123 represents SEQ ID NO: 26:

EVQLVQSGAELKKPGASVKVSCKASGYTFTDYYMKWVRQAPGQGLEWIGDIIPSNGA TFYNQKFKGRVTITVDKSTSTAYMELSSLRSEDTAVYYCARSHLLRASWFAYWGQGT LVTVSS

The VHCD123 antigen binding domain contains CDR1 SEQ ID NO: 47: DYYMK, CDR2 SEQ ID NO: 48: DIIPSNGATFYNQKFKG and CDR3 SEQ ID NO: 49: SHLLRAS.

The second polypeptide chain will contain, from the N-terminus to the C-terminus, the N-terminus, the VL domain of a monoclonal antibody capable of binding to CD123 (VL_CD123), an intermediate linker peptide (e.g., linker 1), the VH domain of a monoclonal antibody capable of binding to CD3 (VH_Cd3), and the C-terminus. A preferred sequence of such a VL domain_CD123 represents SEQ ID NO: 25:

DFVMTQSPDSLAVSLGERVTMSCKSSQSLLNSGNQKNYLTWYQQKPGQPPKLLIYWA STRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQNDYSYPYTFGQGTKLEIK

VL antigen binding domain_CD123 contains CDR1 SEQ ID NO: 44: KSSQSLLNSGNQKNYLT, CDR2 SEQ ID NO: 45: WASTRES and CDR3 SEQ ID NO: 46: QNDYSYPYT.

A preferred sequence for such a VH domain_CD3 represents SEQ ID NO: 22:

EVQLVESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVGRIRSKYNN YATYYADSVKDRFTISRDDSKNSLYLQMNSLKTEDTAVYYCVRHGNFGNSYVSWFAY WGQGTLV

VH antigen binding domain_Cd3 contains CDR1 SEQ ID NO: 41: TYAMN, CDR2 SEQ ID NO: 42:

- 15 034142

RIRSKYNNYATYYADSVKD and CDR3 SEQ ID NO: 43: HGNFGNSYVSWFAY.

The bispecific diabodies characterized by the optimized CD123xCD3 sequence of the present invention are designed so that the first and second polypeptides are covalently linked to each other via cysteine ​​residues along their length. Such cysteine ​​residues may be introduced into an intermediate 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 the N-terminal position relative to the VL domain or at the C-terminal relative to the VH domain of such a polypeptide chain. A preferred sequence of such linker 2 is SEQ ID NO: 30: GGCGGG.

The formation of heterodimers can be controlled by the additional engineering of such polypeptide chains so that they contain polypeptide helices of opposite charges. Thus, in a preferred embodiment, one of the polypeptide chains will be designed to contain an E-helical domain (SEQ ID NO: 34: EVAALEKEVAALEKEVAALEKEVAALEK), whose residues form a negative charge at pH 7, while the other of the two polypeptide chains will be constructed so as to contain a K-helical domain (SEQ ID NO: 35: KVAALKEKVAALKEKVAALKEKVAALKE), whose residues form a positive charge at pH 7. The presence of such charged domains provides an association between the first and second polypeptides and, thus, promotes heterodimerization.

Not essential, the helix is ​​provided by the first or second polypeptide chains. However, the preferred CD123xCD3 sequence-optimized bispecific diatel according to the present invention (DART-A) contains a first polypeptide chain characterized by the following sequence (SEQ ID NO: 1):

QAVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRA PWTPARFSGSLLGGKAALTITGAQAEDEADYYCALWYSNLWVFGGGTKLTVLGGGGS GGGGEVQLVQSGAELKKPGASVKVSCKASGYTFTDYYMKWVRQAPGQGLEWIGDIIP SNGATFYNQKFKGRVTITVDKSTSTAYMELSSLRSEDTAVYYCARSHLLRASWFAYW GQGTLVTVSSGGCGGGEVAALEKEVAALEKEVAALEKEVAALEK

Chain 1 of DART-A consists of the following: SEQ ID NO: 21 - SEQ ID NO: 29 - SEQ ID NO: 26 - SEQ ID

NO: 30 - SEQ ID NO: 34. The DART-A polynucleotide coding chain 1 is SEQ ID NO: 2:

caggctgtggtgactcaggagccttcactgaccgtgtccccaggcggaactgtgacc ctgacatgcagatccagcacaggcgcagtgaccacatctaactacgccaattgggtg cagcagaagccaggacaggcaccaaggggcctgatcgggggtacaaacaaaagggct ccctggacccctgcacggttttctggaagtctgctgggcggaaaggccgctctgact attaccggggcacaggccgaggacgaagccgattactattgtgctctgtggtatagc aatctgtgggtgttcgggggtggcacaaaactgactgtgctgggagggggtggatcc ggcggcggaggcgaggtgcagctggtgcagtccggggctgagctgaagaaacccgga gcttccgtgaaggtgtcttgcaaagccagtggctacaccttcacagactactatatg aagtgggtcaggcaggctccaggacagggactggaatggatcggcgatatcattcct tccaacggggccactttctacaatcagaagtttaaaggcagggtgactattaccgtg gacaaatcaacaagcactgcttatatggagctgagctccctgcgctctgaagataca gccgtgtactattgtgctcggtcacacctgctgagagccagctggtttgcttattgg ggacagggcaccctggtgacagtgtcttccggaggatgtggcggtggagaagtggcc gcactggagaaagaggttgctgctttggagaaggaggtcgctgcacttgaaaaggag gtcgcagccctggagaaa

The second DART-A polypeptide chain is characterized by the following sequence (SEQ ID NO: 3):

DFVMTQSPDSLAVSLGERVTMSCKSSQSLLNSGNQKNYLTWYQQKPGQPPKLLIYWA STRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQNDYSYPYTFGQGTKLEIKG GGSGGGGEVQLVESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVGR IRSKYNNYATYYADSVKDRFTISRDDSKNSLYLQMNSLKTEDTAVYYCVRHGNFGNS YVSWFAYWGQGTLVTVSSGGCGGGKVAALKEKVAALKEKVAALKEKVAALKE

Chain 2 of DART-A consists of the following: SEQ ID NO: 25 - SEQ ID NO: 29 - SEQ ID NO: 22 - SEQ ID

NO: 30 - SEQ ID NO: 35. The DART-A polynucleotide encoding chain 2 is SEQ ID NO: 4:

- 16 034142 gacttcgtgatgacacagtctcctgatagtctggccgtgagtctgggggagcgggtg actatgtcttgcaagagctcccagtcactgctgaacagcggaaatcagaaaaactat ctgacctggtaccagcagaagccaggccagccccctaaactgctgatctattgggct tccaccagggaatctggcgtgcccgacagattcagcggcagcggcagcggcacagat tttaccctgacaatttctagtctgcaggccgaggacgtggctgtgtactattgtcag aatgattacagctatccctacactttcggccaggggaccaagctggaaattaaagga ggcggatccggcggcggaggcgaggtgcagctggtggagtctgggggaggcttggtc cagcctggagggtccctgagactctcctgtgcagcctctggattcaccttcagcaca tacgctatgaattgggtccgccaggctccagggaaggggctggagtgggttggaagg atcaggtccaagtacaacaattatgcaacctactatgccgactctgtgaaggataga ttcaccatctcaagagatgattcaaagaactcactgtatctgcaaatgaacagcctg aaaaccgaggacacggccgtgtattactgtgtgagacacggtaacttcggcaattct tacgtgtcttggtttgcttattggggacaggggacactggtgactgtgtcttccgga ggatgtggcggtggaaaagtggccgcactgaaggagaaagttgctgctttgaaagag aaggtcgccgcacttaaggaaaaggtcgcagccctgaaagag

As discussed below, it has been found that a bispecific diatel (DART-A) characterized by the optimized sequence of CD123xCD3 is characterized by the ability to simultaneously bind CD123 and CD3 cells of both a human and a monkey. It was found that the provision of DART-A induced T-cell activation, mediated a decrease in blast cells, controlled the expansion of the T cell population, induced T-cell activation and caused redirected cytolysis of the target malignant cells.

II. Characterized by a non-optimized sequence of CD123xCD3 bispecific comparison diatel, DART-B

DART-B is a non-optimized CD123xCD3 sequence bispecific diatel characterized by a macrostructure similar to that of DART-A. The first DART-B polypeptide chain will contain, from the N-terminus to the C-terminus, the N-terminus, the VL domain of a monoclonal antibody capable of binding to CD3 (VL_Cd3), an intermediate linker peptide (linker 1), the VH domain of a monoclonal antibody capable of binding to CD123 (VH_CD123), intermediate linker 2, E-helical domain and C-terminus. VL Domain_Cd3 the first DART-B polypeptide chain is characterized by the following sequence (SEQ ID NO: 23):

DIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGV PYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELK

The VHCD123 domain of the first DART-B polypeptide chain is characterized by the following sequence (SEQ ID NO: 28):

QVQLVQSGAELKKPGASVKVSCKASGYTFTDYYMKWVRQAPGQGLEWIGDIIPSNGA TFYNQKFKGRVTITVDKSTSTAYMELSSLRSEDTAVYYCARSHLLRASWFAYWGQGT LVTVSS

Thus, DART-B chain 1 consists of the following: 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 DART-B polypeptide chain is as follows (SEQ ID NO: 5):

DIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGV PYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELKGGGSGGGG QVQLVQS GAELKKPGASVKVS OKAS GYT FTDYYMKWVRQAPGQGLEWIGD11P SNGA TFYNQKFKGRVTITVDKSTSTAYMELSSLRSEDTAVYYCARSHLLRASWFAYWGQGT LVTVS S GGCGGGEVAALEKEVAALEKEVAALEKEVAALEK

The DART-B polynucleotide encoding chain 1 is SEQ ID NO: 6: gacattcagctgacccagtctccagcaatcatgtctgcatctccaggggagaaggtc accatgacctgcagagccagttcaagtgtaagttacatgaactggtaccagcagaag tcaggcacctcccccaaaagatggatttatgacacatccaaagtggcttctggagtc ccttatcgcttcagtggcagtgggtctgggacctcatactctctcacaatcagcagc atggaggctgaagatgctgccacttattactgccaacagtggagtagtaacccgctc acgttcggtgctgggaccaagctggagctgaaaggaggcggatccggcggcggaggc caggtgcagctggtgcagtccggggctgagctgaagaaacccggagcttccgtgaag gtgtcttgcaaagccagtggctacaccttcacagactactatatgaagtgggtcagg caggctccaggacagggactggaatggatcggcgatatcattccttccaacggggcc actttctacaatcagaagtttaaaggcagggtgactattaccgtggacaaatcaaca agcactgcttatatggagctgagctccctgcgctctgaagatacagccgtgtactat tgtgctcggtcacacctgctgagagccagctggtttgcttattggggacagggcacc ctggtgacagtgtcttccggaggatgtggcggtggagaagtggccgcactggagaaa gaggttgctgctttggagaaggaggtcgctgcacttgaaaaggaggtcgcagccctg gagaaa

The second DART-B polypeptide chain will contain, from the N-terminal to the C-terminal N terminal, the VL domain of a monoclonal antibody capable of binding to CD123 (VL_CD123), an intermediate linker peptide (linker 1) and the VH domain of a monoclonal antibody capable of binding to CD3 (VH_Cd3), intermediate linker 2, K-helical domain and C-terminus.

VL Domain_Cd₁₂3 the second DART-B polypeptide chain is characterized by the following sequence (SEQ ID NO: 27):

- 17 034142

DFVMTQSPDSLAVSLGERVTMSCKSSQSLLNSGNQKNYLTWYQQKPGQPPKLLIYWA STRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQNDYSYPYTFGQGTKLEIK

VH Domain_Cd3 the second DART-B polypeptide chain is characterized by the following sequence (SEQ ID NO: 24):

DIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGY TNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTT LTVSS

Thus, DART-B chain 2 consists of the following: 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 DART-B polypeptide chain is as follows (SEQ ID NO: 7):

DFVMTQSPDSLAVSLGERVTMSCKSSQSLLNSGNQKNYLTWYQQKPGQPPKKLIYWA

STRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQNDYSYPYTFGQGTKLEIKG

GGSGGGGDIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGY INPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLD YWGQGTKKKVKKALKALKALKALKALKALKALKALKALKALKALVALKAL

The DART-B polynucleotide coding chain 2 is SEQ ID NO: 8:

Gacttcgtgatgacacagtctcctgatagtctggccgtgagtctgggggagcgggtg actatgtcttgcaagagctcccagtcactgctgaacagcggaaatcagaaaaactat ctgacctggtaccagcagaagccaggccagccccctaaactgctgatctattgggct tccaccagggaatctggcgtgcccgacagattcagcggcagcggcagcggcacagat tttaccctgacaatttctagtctgcaggccgaggacgtggctgtgtactattgtcag aatgattacagctatccctacactttcggccaggggaccaagctggaaattaaagga ggcggatccggcggcggaggcgatatcaaactgcagcagtcaggggctgaactggca agacctggggcctcagtgaagatgtcctgcaagacttctggctacacctttactagg tacacgatgcactgggtaaaacagaggcctggacagggtctggaatggattggatac attaatcctagccgtggttatactaattacaatcagaagttcaaggacaaggccaca ttgactacagacaaatcctccagcacagcctacatgcaactgagcagcctgacatct gaggactctgcagtctattactgtgcaagatattatgatgatcattactgccttgac tactggggccaaggcaccactctcacagtctcctccggaggatgtggcggtggaaaa gtggccgcactgaaggagaaagttgctgctttgaaagagaaggtcgccgcacttaag gaaaaggtcgcagccctgaaagag

III. Modified variants of the characterized by the optimized sequence of CD123xCD3 bispecific diatel (DART-A)

A. Characterized by the optimized sequence of CD123xCD3 bispecific diatel containing albumin binding domain (DART-A with ABD w / ABD)

According to a second embodiment of the present invention, a CD123xCD3 sequence optimized bispecific diatel (DART-A) will contain one or more albumin binding domains (ABD) (DART-A with ABD w / ABD) on one or both of the diabetic polypeptide chains.

As disclosed in International Patent Publication No. WO 2012/018687, in order to improve the in vivo pharmacokinetic properties of diabody diabodies, they can be modified to contain a polypeptide portion of a protein binding protein to serum proteins at one or more ends of the diabody. Such a polypeptide portion of a protein that binds to whey proteins will most preferably be inserted at the C-terminus of the diatel molecule. A particularly preferred polypeptide portion of the whey protein binding protein for this purpose is the albumin binding domain (ABD) of Streptococcus G protein. The albumin binding domain 3 (ABD3) of protein G of strain Streptococcus G148 is particularly preferred.

The albumin binding domain 3 (ABD3) of protein G of Streptococcus G148 strain consists of 46 amino acid residues forming a stable three-helix structure and is characterized by broad albumin binding specificity (Johansson, MU et al. (2002) Structure, Specificity, And Mode Of Interaction For Bacterial Albumin-Binding Modules J. Biol. Chem. 277 (10): 8114-8120). Albumin is the most common protein in plasma and is characterized by a half-life of 19 days in humans. Albumin contains several low molecular weight binding sites that allow it to non-covalently bind to other proteins and, thereby, prolong their serum half-lives.

Thus, the first polypeptide chain of such an bispecific diabetic diabetic containing an albumin binding domain characterized by a CD123xCD3 sequence contains a third linker (linker 3) that separates the E-helix (or K-helix) of such a polypeptide chain from the albumin binding domain. A preferred sequence of such linker 3 is SEQ ID NO: 31: GGGS. A preferred albumin binding domain (ABD) is characterized by the following sequence (SEQ ID NO: 36):

LAEAKVLANRELDKYGVSDYYKNLIDNAKSAEGVKALIDEILAALP.

Thus, a preferred first strand of a CD123xCD3 sequence optimized bispecific diabetic diabetic containing an albumin binding domain is characterized by

- 18 034142 is terized by the following sequence (SEQ ID NO: 9):

QAVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRA PWTPARFSGSLLGGKAALTITGAQAEDEADYYCALWYSNLWVFGGGTKLTVLGGGGS GGGGEVQLVQSGAELKKPGASVKVSCKASGYTFTDYYMKWVRQAPGQGLEWIGDIIP SNGATFYNQKFKGRVTITVDKSTSTAYMELSSLRSEDTAVYYCARSHLLRASWFAYW GQGTLVTVSSGGCGGGEVAALEKEVAALEKEVAALEKEVAALEKGGGSLAEAKVLAN RE L DKYGVS DYYKNLIDNAKSAE GVKALIDEILAAL P

An albumin-binding domain characterized by an optimized CD123xCD3 sequence consists 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 polynucleotide encoding such a characterized by an optimized CD123xCD3 sequence diatel containing an albumin binding domain derivative is SEQ ID NO: 10:

caggctgtggtgactcaggagccttcactgaccgtgtccccaggcggaactgtgacc ctgacatgcagatccagcacaggcgcagtgaccacatctaactacgccaattgggtg cagcagaagccaggacaggcaccaaggggcctgatcgggggtacaaacaaaagggct ccctggacccctgcacggttttctggaagtctgctgggcggaaaggccgctctgact attaccggggcacaggccgaggacgaagccgattactattgtgctctgtggtatagc aatctgtgggtgttcgggggtggcacaaaactgactgtgctgggagggggtggatcc ggcggcggaggcgaggtgcagctggtgcagtccggggctgagctgaagaaacccgga gcttccgtgaaggtgtcttgcaaagccagtggctacaccttcacagactactatatg aagtgggtcaggcaggctccaggacagggactggaatggatcggcgatatcattcct tccaacggggccactttctacaatcagaagtttaaaggcagggtgactattaccgtg gacaaatcaacaagcactgcttatatggagctgagctccctgcgctctgaagataca gccgtgtactattgtgctcggtcacacctgctgagagccagctggtttgcttattgg ggacagggcaccctggtgacagtgtcttccggaggatgtggcggtggagaagtggcc gcactggagaaagaggttgctgctttggagaaggaggtcgctgcacttgaaaaggag gtcgcagccctggagaaaggcggcgggtctctggccgaagcaaaagtgctggccaac cgcgaactggataaatatggcgtgagcgattattataagaacctgattgacaacgca aaatccgcggaaggcgtgaaagcactgattgatgaaattctggccgccctgcct

The second polypeptide chain of such an albumin binding domain-characterized CD123xCD3 sequence-optimized diatel is characterized by the sequence described above (SEQ ID NO: 3) and encoded by a polynucleotide characterized by the sequence of SEQ ID NO: 4.

B. Characterized by the optimized sequence of CD123xCD3 bispecific diabodies containing the Fc IgG domain (DART-A with Fc w / Fc)

According to a third embodiment of the present invention, there is provided a bispecific diatel characterized by a sequence optimized CD123xCD3 consisting of three polypeptide chains and containing an IgG Fc domain (version 1 and version 2 of DART-A with Fc w / Fc) (Fig. 3A-3B).

To form such an IgG Fc domain, the first and third polypeptide chain of the diabody contain from the N-terminal to the C-terminal a cysteine-containing peptide (most preferably peptide 1, characterized by the amino acid sequence (SEQ ID NO: 55): DKTHTCPPCP), some or all CH2 domain and / or some or all of the CH3 domain of the complete immunoglobulin Fc domain and C-terminus. Some or all of the CH2 domain and / or some or all of the CH3 domain is associated with the formation of the immunoglobulin Fc domain of the bispecific monovalent Fc domain diabodies of the present invention. The first and second polypeptide chains of the bispecific monovalent Fc-dies of the present invention are covalently linked to each other, for example, by disulfide linking 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 do not have to be identical and are preferably modified so as to facilitate complex formation between the two polypeptides. For example, an amino acid substitution (preferably, substitution with an amino acid containing a bulky side group forming a ridge, for example tryptophan) can be introduced into the CH2 or CH3 domain so that the steric effect prevents interaction with a similarly mutated domain and forces the mutated domain to pair with the domain in which the complementary or locational mutation was constructed, i.e. 'cavity' (e.g. glycine replacement). Such mutation sets can be constructed in any pair of polypeptides containing a bispecific monovalent Fc-diatel molecule, and, in addition, constructed in any part of the polypeptide chains of the specified pair. Methods of protein engineering to promote heterodimerization instead of homodimerization are well known in the art, in particular with respect to the engineering of immunoglobulin-like molecules, and are provided herein (see, for example, Ridgway et al. (1996) Knobs-Into-Holes' Engineering Of Antibody CH3 Domains For Heavy Chain Heterodimerization, Protein Engr. 9: 617-621, Atwell et al. (1997) Stable Heterodimers From Remodeling The Domain Interface Of A Homodimer Using A Phage Display Library, J. Mol. Biol. 270: 26-35, and Xie et al. (2005) A New Format Of Bispecific Antibody: Highly Efficient Heterodimerization, Expression And Tumor Cell Lysis, J. Immunol. Methods 296: 95-101; each of which is fully incorporated into this

- 19,034,142 document by reference). Preferably, a “protrusion” is constructed in the CH2-CH3 domains of the first polypeptide chain, and a “cavity” is constructed in the CH2-CH3 domains of the third polypeptide chain. Thus, the ridge will assist in preventing homodimerization of the first polypeptide chain through its CH2 and / or CH3 domains. Since the third polypeptide chain preferably contains a substitution that provides the formation of a “depression”, it will be heterodimerized with the first polypeptide chain, as well as homodimerized with itself. A preferred protrusion is created by modifying the Fc domain of the native IgG Fc domain so that it contains the T366W modification. A preferred depression is created by modifying the Fc domain of the native IgG Fc domain to contain the modifications of T366S, L368A and Y407V. To facilitate the purification of the homodimer of the third polypeptide chain from the final bispecific monovalent Fc-diatel containing the first, second and third polypeptide chains, the protein A binding site of the CH2 and CH3 domains of the third polypeptide chain is preferably mutated using the amino acid substitution at position 435 (H435R) . Thus, the homodimer of the third polypeptide chain will not bind to protein A, while the bispecific monovalent Fc - diatel will retain its ability to bind protein A through the binding site to protein A on the first polypeptide chain.

A preferred sequence for the CH2 and CH3 domains of the Fc domain of an antibody present in the first polypeptide chain is (SEQ ID NO: 56):

APEAAGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNA KTKPREEQYNS TYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPR EPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD GSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

A preferred sequence for the CH2 and CH3 domains of the Fc domain of an antibody present in the third polypeptide chain is (SEQ ID NO: 11):

APEAAGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNA KTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPR EPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD GSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNRYTQKSLSLSPGK

C. Construct version 1 of DART-A w / Fc

To illustrate such Fc diabodies, a DART-A w / Fc version 1 construct is provided according to the present invention. The first DART-A w / Fc construct version 1 polypeptide contains, from the N-terminus to the C-terminus, the N-terminus, the VL domain of a monoclonal antibody capable of binding to CD123 (VL_CD123), an intermediate linker peptide (linker 1), the VH domain of a monoclonal antibody capable of binding to CD3 (VH_Cd3), linker 2, E-helical domain, linker 5, peptide 1, a polypeptide that contains the Fc domain CH2 and CH3 domains, and the C-terminus. A preferred linker 5 is characterized by the sequence (SEQ ID NO: 32): GGG. A preferred polypeptide that contains the Fc domain CH2 and CH3 domains is characterized by the following sequence (SEQ ID NO: 37):

APEAAGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNA KTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPR EPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD GSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Thus, the first polypeptide of such a construct of version 1 of DART-A w / Fc consists of the following: 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.

The preferred sequence of the first polypeptide of this construct version 1 of DART-A w / Fc is characterized by the following sequence (SEQ ID NO: 13):

DFVMTQSPDSLAVSLGERVTMSCKSSQSLLNSGNQKNYLTWYQQKPGQPPKLLIYWA STRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQNDYSYPYTFGQGTKLEIKG

GGSGGGGEVQLVESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVGR IRSKYNNYATYYADSVKDRFTISRDDSKNSLYLQMNSLKTEDTAVYYCVRHGNFGNS YVSWFAYWGQGTLVTVSSGGCGGGEVAALEKEVAALEKEVAALEKEVAALEKGGGDK THTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTI SKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKT TPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

A preferred polynucleotide encoding such a polypeptide is (SEQ ID NO: 14):

- 20 034142 gacttcgtgatgacacagtctcctgatagtctggccgtgagtctgggggagcgggtg actatgtcttgcaagagctcccagtcactgctgaacagcggaaatcagaaaaactat ctgacctggtaccagcagaagccaggccagccccctaaactgctgatctattgggct tccaccagggaatctggcgtgcccgacagattcagcggcagcggcagcggcacagat tttaccctgacaatttctagtctgcaggccgaggacgtggctgtgtactattgtcag aatgattacagctatccctacactttcggccaggggaccaagctggaaattaaagga ggcggatccggcggcggaggcgaggtgcagctggtggagtctgggggaggcttggtc cagcctggagggtccctgagactctcctgtgcagcctctggattcaccttcagcaca tacgctatgaattgggtccgccaggctccagggaaggggctggagtgggttggaagg atcaggtccaagtacaacaattatgcaacctactatgccgactctgtgaaggataga ttcaccatctcaagagatgattcaaagaactcactgtatctgcaaatgaacagcctg aaaaccgaggacacggccgtgtattactgtgtgagacacggtaacttcggcaattct tacgtgtcttggtttgcttattggggacaggggacactggtgactgtgtcttccgga ggatgtggcggtggagaagtggccgcactggagaaagaggttgctgctttggagaag gaggtcgctgcacttgaaaaggaggtcgcagccctggagaaaggcggcggggacaaa actcacacatgcccaccgtgcccagcacctgaagccgcggggggaccgtcagtcttc ctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcaca tgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtg gacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagc acgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaag gagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatc tccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgg gaggagatgaccaagaaccaggtcagcctgtggtgcctggtcaaaggcttctatccc agcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagacc acgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtg gacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggct ctgcacaaccactacacggaga

The second chain of such a DART-A w / Fc version 1 construct will contain, from the N-terminus to the C-terminus, the N-terminus, the VL domain of a monoclonal antibody capable of binding to CD3 (VLCD3), an intermediate linker peptide (linker 1), VH domain of a monoclonal antibody capable of binding to CD123 (VHCD123), linker 2, K-helical domain and C-terminus. Thus, a second polypeptide of such a construct of version 1 of DART-A w / Fc consists of the following: SEQ ID NO: 21 - SEQ ID NO: 29 - SEQ ID NO: 26 - SEQ ID NO: 30 - SEQ ID NO: 35. Such a polypeptide is characterized by the following sequence (SEQ ID NO: 15):

QAWTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRA PWTPARFSGSLLGGKAALTITGAQAEDEADYYCALWYSNLWVFGGGTKLTVLGGGGS GGGGEVQLVQS GAELKKPGASVKVS CKAS GYT FT DYYMKWVRQAPGQGLEWIGD11P SNGATFYNQKFKGRVTITVDKSTSTAYMELSSLRSEDTAVYYCARSHLLRASWFAYW GQGTLVTVSSGGCGGGKVAALKEKVAALKEKVAALKEKVAALKE

A preferred polynucleotide encoding such a polypeptide is characterized by the following sequence (SEQ ID NO: 16):

caggctgtggtgactcaggagccttcactgaccgtgtccccaggcggaactgtgacc ctgacatgcagatccagcacaggcgcagtgaccacatctaactacgccaattgggtg cagcagaagccaggacaggcaccaaggggcctgatcgggggtacaaacaaaagggct ccctggacccctgcacggttttctggaagtctgctgggcggaaaggccgctctgact attaccggggcacaggccgaggacgaagccgattactattgtgctctgtggtatagc aatctgtgggtgttcgggggtggcacaaaactgactgtgctgggagggggtggatcc ggcggcggaggcgaggtgcagctggtgcagtccggggctgagctgaagaaacccgga gcttccgtgaaggtgtcttgcaaagccagtggctacaccttcacagactactatatg aagtgggtcaggcaggctccaggacagggactggaatggatcggcgatatcattcct tccaacggggccactttctacaatcagaagtttaaaggcagggtgactattaccgtg gacaaatcaacaagcactgcttatatggagctgagctccctgcgctctgaagataca gccgtgtactattgtgctcggtcacacctgctgagagccagctggtttgcttattgg ggacagggcaccctggtgacagtgtcttccggaggatgtggcggtggaaaagtggcc gcactgaaggagaaagttgctgctttgaaagagaaggtcgccgcacttaaggaaaag gtcgcagccctgaaagag

A third polypeptide chain of this version 1 of DART-A w / Fc will contain the CH2 and CH3 domains of the IgG Fc domain. A preferred polypeptide consists of peptide 1 (SEQ ID NO: 55) and the Fc domain CH2 and CH3 domains (SEQ ID NO: 11) and is characterized by the sequence of SEQ ID NO: 54:

DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCWVDVSHEDPEVKFNW

YVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPSCLVKLSKDSKVFDSLVKLSKFVLSKFSKFVLSKFSKFVLSFKFVFWFK

A preferred polynucleotide that encodes such a polypeptide is characterized by the following sequence (SEQ ID NO: 12):

- 21 034142 gacaaaactcacacatgcccaccgtgcccagcacctgaagccgcggggggaccgtca gtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgag gtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactgg tacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtac aacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaat ggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaa accatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgccccca tcccgggaggagatgaccaagaaccaggtcagcctgagttgcgcagtcaaaggcttc tatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaagaaactac aagaccacgcctcccgtgctggactccgacggctccttcttcctcgtcagcaagctc accgtggacaagagcaggtgcgcgctgctgctgctgctgctgctgcgctgctgctgctgctgctgctgctgctgctgctgctgctgctgctgctgctgctgctgctgctgctgctgctgctgctgctgctgctgctgctgctgctgctgctgctgctgctgctgctgctgctgctgctgctgctgctgctgctgctgctgctgctgctgctgctgctgctgctcctccctcbcbcbbbbc

D. Construct version 2 of DART-A w / Fc

As a second example of such a DART-A w / Fc diabody, the present invention provides a three-chain diabody, version 2 of the DART-A w / Fc diabody (Fig. 3B).

The first polypeptide of this construct, version 2 of DART-A w / Fc, contains, from the N-terminus to the C-terminus, the N-terminus, a peptide linker (peptide 1), a polypeptide that contains the CH2 and CH3 domains of the Fc domain, connected (by linker 4) with the VL domain of a monoclonal antibody capable of binding to CD123 (VLCD123), an intermediate linker peptide (linker 1), the VH domain of a monoclonal antibody capable of binding to CD3 (VHCD3), linker 2, the K-helical domain and the C-terminus.

A preferred polypeptide that contains the Fc domain CH2 and CH3 domains is characterized by the following sequence (SEQ ID NO: 37):

APEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNA

KTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPR EPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD GSFFLYSKLGNKKHTVCHVKHTFKVTF

Linker 4 will preferably contain the amino acid sequence (SEQ ID NO: 57): APSSS. A preferred 4-linker is characterized by the sequence (SEQ ID NO: 33): APSSSPME. Thus, the first polypeptide of this construct version 2DART-A w / Fc consists of the following: 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 characterized by such a sequence is (SEQ ID NO: 17):

DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNW

YVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK

TISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNY KTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKA PSSSPMEDFVMTQSPDSLAVSLGERVTMSCKSSQSLLNSGNQKNYLTWYQQKPGQPP KLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQNDYSYPYTFGQG TKLEIKGGGSGGGGEVQLVESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGK GLEWVGRIRSKYNNYATYYADSVKDRFTISRDDSKNSLYLQMNSLKTEDTAVYYCVR HGNFGNSYVSWFAYWGQGTLVTVSSGGCGGGKVAALKEKVAALKEKVAALKEKVAAL KE

A preferred polynucleotide encoding such a polypeptide is characterized by the following sequence (SEQ ID NO: 18):

- 22 034142 gacaaaactcacacatgcccaccgtgcccagcacctgaagccgcggggggaccgtca gtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgag gtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactgg tacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtac aacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaat ggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaa accatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgccccca tcccgggaggagatgaccaagaaccaggtcagcctgtggtgcctggtcaaaggcttc tatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactac aagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctc accgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcat gaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaagcc ccttccagctcccctatggaagacttcgtgatgacacagtctcctgatagtctggcc gtgagtctgggggagcgggtgactatgtcttgcaagagctcccagtcactgctgaac agcggaaatcagaaaaactatctgacctggtaccagcagaagccaggccagccccct aaactgctgatctattgggcttccaccagggaatctggcgtgcccgacagattcagc ggcagcggcagcggcacagattttaccctgacaatttctagtctgcaggccgaggac gtggctgtgtactattgtcagaatgattacagctatccctacactttcggccagggg accaagctggaaattaaaggaggcggatccggcggcggaggcgaggtgcagctggtg gagtctgggggaggcttggtccagcctggagggtccctgagactctcctgtgcagcc tctggattcaccttcagcacatacgctatgaattgggtccgccaggctccagggaag gggctggagtgggttggaaggatcaggtccaagtacaacaattatgcaacctactat gccgactctgtgaaggatagattcaccatctcaagagatgattcaaagaactcactg tatctgcaaatgaacagcctgaaaaccgaggacacggccgtgtattactgtgtgaga cacggtaacttcggcaattcttacgtgtcttggtttgcttattggggacaggggaca ctggtgactgtgtcttccggaggatgtggcggtggaaaagtggccgcactgaaggag aaagttgctgctttgaaagagaaggtcgccgcacttaaggaaaaggtcgcagccctg aaagag

The second polypeptide chain of this construct, version 2 of DART-A w / Fc, contains, from the N-terminal to the C-terminal, the VL domain of a monoclonal antibody capable of binding to CD3 (VLCD3), an intermediate linker peptide (linker 1) and the VH domain of monoclonal antibodies capable of binding to CD123 (VHCD123) - This portion of the molecule is connected (via linker 2) to the helical domain. Thus, the third polypeptide of such a construct of version 2 of DART-A w / Fc consists of the following: SEQ ID NO: 21 - SEQ ID NO: 29 - SEQ ID NO: 26 - SEQ ID NO: 30 -SEQ ID NO: 34. A polypeptide characterized by such a sequence is (SEQ ID NO: 1), and is preferably encoded by a polynucleotide characterized by the sequence of SEQ ID NO: 2.

The third polypeptide chain will contain the CH2 and CH3 domains of the IgG Fc domain. A preferred polype of birds consists of peptide 1 (SEQ ID NO: 55) and domains CH2 and CH3 of the Fc domain (SEQ ID NO: 11) and is characterized by the sequence of SEQ ID NO: 54.

To evaluate the activity of the aforementioned CD123xCD3 bispecific diabodies (DART-A, DARTA w / ABD, DART-A w / Fc, DART-B), a control bispecific diabody (control DART) was obtained. The control DART is capable of simultaneously binding to FITC and CD3. Its two polypeptide chains are characterized by the following corresponding sequences:

Chain 1 control DART (SEQ ID NO: 19):

DWMTQTPFSLPVSLGDQASISCRSSQSLVHSNGNTYLRWYLQKPGQSPKVLIYKVS NRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHVPWTFGGGTKLEIKGG GSGGGGEVQLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGLEWVARI RSKYNNYATYYADSVKDRFTISRDDSKNSLYLQMNSLKTEDTAVYYCVRHGNFGNSY VSWFAYWGQGTLVTVSSGGCGGGEVAALEKEVAALEKEVAALEKEVAALEK

Chain 2 of the control DART (SEQ ID NO: 20):

QAWTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRA PWTPARFSGSLLGGKAALTITGAQAEDEADYYCALWYSNLWVFGGGTKLTVLGGGGS GGGGEVKLDETGGGLVQPGRPMKLSCVASGFTFSDYWMNWVRQSPEKGLEWVAQIRN KPYNYETYYSDSVKGRFTISRDDSKSSVYLQMNNLRVEDMGIYYCTGSYYGMDYWGQ GTSVTVSSGGCGGGKVAALKEKVAALKEKVAALKEKVAALKE

IV. Pharmaceutical Compositions

Compositions of the present invention include bulk drug compositions useful in the manufacture of pharmaceutical compositions (e.g., crude or non-sterile compositions) and pharmaceutical compositions (i.e., compositions that are suitable for administration to a subject or patient) that can be used in the preparation of standard dosage forms. Such compositions comprise a prophylactically or therapeutically effective amount of a CD123xCD3 sequence optimized bispecific diabetic according to the present invention or a combination of such agents and a pharmaceutically acceptable carrier. The compositions of the present invention preferably comprise a prophylactically or therapeutically effective amount of a sequence-optimized CD123xCD3 bispecific diabetic according to the present invention and a pharmaceutically acceptable carrier.

- 23 034142

The present invention also provides pharmaceutical compositions comprising a CD123xCD3 sequence optimized bispecific diabodies of the present invention and a second therapeutic antibody (e.g., a tumor-specific monoclonal antibody) that is specific for a particular cancer antigen, and a pharmaceutically acceptable carrier.

In a particular embodiment, the term “pharmaceutically acceptable” means approved by a regulatory agency of the federal or state government or listed in the United States Pharmacopeia or other generally recognized pharmacopeia for use in animals and more specifically in humans. The term “carrier” refers to a diluent, adjuvant (for example, Freund’s adjuvant (complete and incomplete), excipient or inert carrier with which the therapeutic agent is administered. Such pharmaceutical carriers may be sterile liquids, such as water and oils, including oils of mineral, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred carrier if the pharmaceutical composition is administered intravenously. Saline solutions and aqueous solutions of dextrose and glycerol solutions can also be used as liquid carriers, in particular for injection solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, skimmed milk powder, glycerin, propylene, glycol, water, ethanol and the like. The composition, if necessary, may also contain minor amounts of wetting agents or emulsifiers, or pH buffering agents. These compositions may take the form of solutions, suspensions, emulsions, tablets, dragees, capsules, powders, sustained release formulations and the like.

Typically, the ingredients of the compositions of the present invention are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or anhydrous concentrate in a hermetically sealed container such as an ampoule or sachet indicating the amount of active agent. If the composition is intended to be administered by infusion, it may be sold with an infusion vial containing sterile water or a pharmaceutical grade saline solution. If the composition is intended for administration by injection, an ampoule with sterile water for injection or saline may be provided so that the ingredients can be mixed before administration.

The compositions of the present invention can be formulated in neutral or salt forms. Pharmaceutically acceptable salts include, without limitation, salts formed by anions such as anions derived from hydrochloric, orthophosphoric, acetic, oxalic, tartaric acids, etc., and salts formed by cations, such as cations derived from sodium, potassium , ammonium, calcium, iron hydroxides, isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine, etc.

The present invention also provides a pharmaceutical bag or kit containing one or more containers filled with the CD123xCD3 characterizing sequence optimized bispecific diabodies of the present invention alone or with such a pharmaceutically acceptable carrier. In addition, one or more other prophylactic or therapeutic agents suitable for treating a disease may also be included in a pharmaceutical package or kit. The present invention also provides a pharmaceutical bag or kit containing one or more containers filled with one or more ingredients of the pharmaceutical compositions of the present invention. Optionally, such container container (s) may be accompanied by a notice in the form prescribed by the government body that regulates the production, use or sale of pharmaceuticals or biological products, and this notice reflects the permission of the relevant body of production, use or sale for administration to people.

According to the present invention, kits are provided that can be used in the methods described above. The kit may contain bispecific diabodies according to the present invention characterized by the optimized sequence of CD123xCD3. The kit may further comprise one or more other prophylactic and / or therapeutic agents suitable for treating a malignant tumor in one or more containers; and / or the kit may further comprise one or more cytotoxic antibodies that bind to one or more cancer antigens associated with the cancer. In certain embodiments, the other prophylactic or therapeutic agent is a chemotherapeutic agent. In other embodiments, the prophylactic or therapeutic agent is a biological or hormonal therapeutic agent.

- 24 034142

V. Methods of administration

The compositions of the present invention can be provided for treating, preventing and reducing the intensity of one or more symptoms associated with a disease, disorder or infection by administering to the subject an effective amount of a fusion protein or conjugated molecule of the present invention or a pharmaceutical composition comprising a fusion protein or conjugated molecule according to the present invention. According to a preferred aspect, such compositions are substantially purified (i.e., substantially free of substances that limit its effect or produce undesirable side effects). In a particular embodiment, the subject is an animal, preferably a mammal, such as a non-primate mammal (e.g., cow, horse, cat, dog, rodent, etc.) or a primate (e.g., a monkey, such as cynomolgus monkey, person, etc.). According to a preferred embodiment, the subject is a human.

Various delivery systems are known and can be used to administer the compositions of the present invention, for example, liposome encapsulation, microparticles, microcapsules, recombinant cells capable of expressing an antibody or fusion protein mediated by endocytosis receptor (see, for example, Wu et at. (1987) ) Receptor-Mediated In Vitro Gene Transformation By A Soluble DNA Carrier System, J. Biol. Chem. 262: 4429-4432), a nucleic acid construct as part of a retroviral or other vector, etc.

Methods for administering a molecule of the present invention include, without limitation, parenteral administration (e.g., intradermal, intramuscular, intraperitoneal, intravenous and subcutaneous), epidural and mucosal (e.g., intranasal and oral routes). According to a specific embodiment, the bispecific diabodies of the present invention characterized by the optimized sequence of CD123xCD3 are administered intramuscularly, intravenously or subcutaneously. The compositions can be administered in any convenient way, for example, by infusion or a bolus injection, by absorption through epithelial or mucosal membranes (for example, the mucous membrane of the oral cavity, the mucosa of the rectum and intestines, etc.) and can be administered together with other biologically active agents . The introduction may be systemic or local. In addition, pulmonary administration can also be used, for example, by using an inhaler or nebulizer and an aerosol formulation. See, for example, US Patent Nos. 6019968; 5,985,320; 5,985,309; 5,934,272; 5,874,064; 5,855,913; 5290540 and 4880078 and international patent publications according to PCT No. 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.

According to the present invention, it is also provided that the bispecific diabodies characterized by the optimized CD123xCD3 sequence according to the present invention are packaged in a hermetically sealed container, such as an ampoule or sachet indicating the amount of the molecule. In one embodiment, the CD123xCD3 sequence optimized bispecific diabodies of the present invention are supplied as a dry sterilized lyophilized powder or anhydrous concentrate in a hermetically sealed container and can be diluted, for example, with water or saline to an appropriate concentration for administration to a subject. The CD123xCD3 sequence-optimized bispecific diabodies of the present invention are preferably delivered as a dry sterile lyophilized powder in a hermetically sealed container in a unit dosage of at least 5 μg, more preferably at least 10 μg, at least 15 μg, at least 25 μg μg, at least 50 μg, at least 100 μg, or at least 200 μg.

Lyophilized, characterized by the optimized sequence of CD123xCD3 bispecific diabodies according to the present invention, must be stored at a temperature of 2 to 8 ° C in their original container and the molecules must be administered no later than 12 hours, preferably no later than 6 hours, no later than 5 h, no later than 3 hours or no later than 1 hour after their dilution. According to an alternative embodiment, the CD123xCD3 sequence optimized bispecific diabodies of the present invention are supplied in liquid form in a hermetically sealed container indicating the amount and concentration of the molecule, fusion protein or conjugated molecule. The liquid form of the CD123xCD3 sequence optimized bispecific diabodies of the present invention is preferably supplied in a hermetically sealed container in which the molecules are in a concentration of at least 1 μg / ml, more preferably at least 2.5 μg / ml, at least 5 μg / ml, at least 10 μg / ml, at least 50 μg / ml, or at least 100 μg / ml.

The amount of the composition according to the present invention, which will be effective in the treatment, prevention or reduction of the intensity of one or more associated with the violation

- 25,034,142 symptoms, can be determined using standard clinical techniques. The exact dose used in the composition will also depend on the route of administration and the severity of the condition, and should be determined in accordance with the decision of the attending physician and all the relevant circumstances of the patient. Effective doses can be extrapolated from dose-response curves from in vitro test systems or test systems — animal models.

For the bispecific diabodies characterized by the optimized CD123xCD3 sequence provided by the present invention, the dosage administered to the patient is preferably determined based on the body weight (kg) of the recipient subject. The dosage administered is typically from at least about 0.3 to about 0.9 ng / kg per day, from at least about 1 to about 3 ng / kg per day, from at least about 3 to about 9 ng / kg per day, from at least about 10 to about 30 ng / kg per day, from at least about 30 to about 90 ng / kg per day, from at least about 100 to about 300 ng / kg per day , from at least about 200 to about 600 ng / kg per day; from at least about 300 to about 900 ng / kg per day; from at least about 400 to about 800 ng / kg per day; from at least about 500 to about 1000 ng / kg per day, from at least about 600 to about 1000 ng / kg per day, from at least about 700 to about 1000 ng / kg per day, from at least about 800 to about 1000 ng / kg per day; from at least about 900 to about 1000 ng / kg per day; or at least about 1000 ng / kg per day.

According to another embodiment, a patient is given a treatment regimen comprising one or more doses of such a prophylactically or therapeutically effective amount of an optimized CD123xCD3 sequence of bispecific diabodies provided by the present invention, the treatment regimen being administered within 2 days, 3 days, 4 days, 5 days, 6 days or 7 days. In certain embodiments, a treatment regimen provides for periodically administering doses of a prophylactically or therapeutically effective amount of an optimized CD123xCD3 sequence of bispecific diabodies provided by the present invention (e.g., administering a dose on day 1, day 2, 3 day and day 4 of this week and the lack of dosing of a prophylactically or therapeutically effective amount of a CD123xCD3 bispecific diabetic characterized by the optimized sequence provided for in accordance with the present invention on day 5, day 6 and day 7 of the same week). Typically, 1, 2, 3, 4, 5 or more courses of treatment are given. Each course can take the same pattern or in different patterns.

According to another embodiment, the administered dose is increased over the first quarter, first half or first two-thirds or three quarters of the regimen (s) (for example, for the first, second or third treatment regimen of 4 courses) until the daily prophylactically or therapeutically effective amount is reached characterized by an optimized sequence of CD123xCD3 bispecific diabodies.

In the table. 1 presents 5 examples of various dosage regimens described above for a typical course of treatment. ___________________________________________________________________

Table 1

<td>Scheme</td><td>Day</td><td colspan="5">Dosage of diatel (ng of diatel per kg of body weight of the subject per day)</td>

<td rowspan="2"> 1</td><td> 1,2,3,4</td><td> 100</td><td> 100</td><td> 100</td><td> 100</td><td> 100</td>

<td> 5,6,7</td><td>not</td><td>not</td><td>not</td><td>not</td><td>not</td>

<td rowspan="2"> 2</td><td> 1,2,3,4</td><td> 300</td><td> 500</td><td> 700</td><td> 900</td><td> 1,000</td>

<td> 5,6,7</td><td>not</td><td>not</td><td>not</td><td>not</td><td>not</td>

<td rowspan="2"> 3</td><td> 1,2,3,4</td><td> 300</td><td> 500</td><td> 700</td><td> 900</td><td> 1,000</td>

<td> 5,6,7</td><td>not</td><td>not</td><td>not</td><td>not</td><td>not</td>

<td rowspan="2"> 4</td><td> 1,2,3,4</td><td> 300</td><td> 500</td><td> 700</td><td> 900</td><td> 1,000</td>

<td> 5,6,7</td><td>not</td><td>not</td><td>not</td><td>not</td><td>not</td>

The dosage and frequency of administration of the characterized by the optimized sequence of CD123xCD3 bispecific diabodies according to the present invention can be reduced or changed by enhancing the absorption and penetration of the characterized by the optimized sequence of CD123xCD3 bispecific diabodies into tissues using modifications such as, for example, lipidization.

The dosage administered to a patient characterized by the optimized CD123xCD3 sequence of bispecific diabodies of the present invention can be calculated for use as monotherapy. Alternatively, the bispecific diabodies of the present invention characterized by the optimized CD123xCD3 sequence are used in combination with other therapeutic compositions, and the dosage administered to the patient is less than when using these molecules as monotherapy.

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The pharmaceutical compositions of the present invention can be administered topically to a region in need of treatment; this can be achieved, for example, without limitation by local infusion, injection or by means of an implant, said implant being made of a porous, non-porous or gelatinous material, including membranes such as Sialastic membranes or fibers. When introducing a molecule according to the present invention, it is preferable to pay attention to the use of materials that do not absorb the molecule.

The compositions of the present invention can be delivered in a vesicle, in particular a liposome (see Langer (1990) New Methods Of Drug Delivery, Science 249: 1527-1533); Treat et al., In Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 353- 365 (1989); Lopez-Berestein, ibid., Pp. 3 17-327; see mainly in the same place).

The compositions of the present invention can be delivered in a controlled or sustained release system. Any technique known to one skilled in the art can be used to produce sustained release formulations containing one or more of the optimized CD123xCD3 sequence of bispecific diabodies of the present invention. See, for example, US patent No. 4526938; international patent publications according to PCT No. WO 91/05548; WO 96/20698; Ning et al. (1996) Intratumoral Radioimmunotheraphy Of A Human Colon Cancer Xenograft Using A SustainedRelease Gel, Radiotherapy &amp; Oncology 39: 179-189, Song et al. (1995) Antibody Mediated Lung Targeting Of Long-Circulating Emulsions, PDA Journal of Pharmaceutical Science &amp; 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 Of Recombinant 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, the pump may be used in a controlled release system (See Langer, previously; 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 Thrombosis, Surgery 88: 507-516; and Saudek et al. (1989) A Preliminary Trial Of The Programmable Implantable Medication System For Insulin Delivery, N. Engl. J. Med. 321: 574-579). In another embodiment, polymeric materials can be used to achieve controlled release of molecules (see, for example, Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Florida (1974); Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, New York (1984); Levy et al. (1985) Inhibition Of Calcification Of Bioprosthetic Heart Valves By Local Controlled-Release Diphosphonate, Science 228: 190-192; During et al. (1989) Controlled Release Of Dopamine From A Polymeric Brain Implant: In Vivo Characterization, Ann. Neural. 25: 351-356; Howard et al. (1989) Intracerebral Drug Delivery In Rats With Lesion-Induced Memory Deficits, J. Neurosurg. 7 (1): 105-112); U.S. Patent Nos. 5,679,377; 5,916,597; 5,912,015; 5,989,463; 5128326; international patent publications according to PCT No. WO 99/15154 and WO 99/20253). Examples of polymers used in sustained release formulations include, but are not limited to, 2-hydroxyethyl methacrylate polymer, methyl methacrylate polymer, acrylic acid polymer, ethylene vinyl acetate copolymer, methacrylic acid polymer, polyglycolides (PLG), polyanhydrides, N-vinyl pyrrolidone polymer, vinyl alcohol polymer , polyacrylamide, polyethylene glycol, polylactides (PLA), a copolymer of lactides and glycolides (PLGA) and polyorthoesters. a controlled release system can be placed close to a therapeutic target (e.g., lungs), so only a fraction of the systemic dose will be needed (see, for example, Goodson, Medical Applications of Controlled Release, previously vol. 2, pp. 115-138 (1984)). Compositions suitable as controlled release implants can be used according to Dunn et al. (see US patent No. 5945155). The specified specific method is based on the therapeutic effect of in situ controlled release of bioactive material from the polymer system. Implantation usually occurs anywhere in the body of a patient in need of therapeutic treatment. A non-polymer delayed delivery system can be used by which a non-polymer implant in the subject's body is used as a drug delivery system. When implanted into the body, the organic solvent of the implant will disperse, disperse, or leach from the composition into the surrounding tissue fluid, and the non-polymer material will gradually coagulate or precipitate to form a solid, microporous matrix (see US Patent No. 5888533).

Controlled release systems are discussed in a review by Langer (1990, New Methods Of Drug Delivery, Science 249: 1527-1533). Any technique known to a person skilled in the art can be used to produce sustained release formulations containing one or more therapeutic agents of the present invention. See, for example, US patent No. 4526938; international patent publications No. WO 91/05548 and WO 96/20698; Ning et al. (1996) Intratumoral Radioimmunotheraphy Of A Human Colon Cancer Xenograft Using A Sustained-Release Gel, Radiotherapy &amp; Oncology 39: 179-189, Song et at. (1995) Antibody Mediated Lung Targeting Of Long

- 27 034142

Circulating Emulsions, PDA Journal of Pharmaceutical Science &amp; 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 Of Recombinant 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.

If the composition according to the present invention is a nucleic acid encoding a bispecific diabetic according to the present invention characterized by the optimized sequence CD123xCD3, a nucleic acid can be introduced in vivo to stimulate the expression of a bispecific diatel encoded by it characterized by an optimized sequence of CD123xCD3 by constructing it as part of the corresponding nucleic acid expression vector and introducing it so that it becomes intracellular, for example, by using a retroviral vector (see US patent No. 4980286), or by direct injection, or by using microparticle bombardment (for example, a gene gun; Biolistic, Dupont), or by coating with lipids, or cell surface receptors, or by means of transfection, or by introducing it connected to a homeobox-like a peptide that is known to penetrate the nucleus (see, for example, Joliot et al. (1991) Antennapedia Homeobox Peptide Regulates Neural Morphogenesis, Proc. Natl. Acad. Sci. (USA) 88: 1864-1868), etc. d. Alternatively, the nucleic acid can be introduced intracellularly and inserted into the DNA of the host cell for expression by homologous recombination.

Treating a subject with a therapeutically or prophylactically effective amount of an optimized CD123xCD3 sequence of bispecific diabodies according to the present invention may comprise a single treatment or preferably may include a series of treatments. According to a preferred example, the subject is treated with the optimized sequence of CD123xCD3 bispecific diabodies of the present invention once a week for about 1-10 weeks, preferably 2-8 weeks, more preferably about 3-7 weeks and even more preferably for about 4, 5 or 6 weeks. The pharmaceutical compositions of the present invention can be administered once a day, twice a day, or three times a day. Alternatively, the pharmaceutical compositions may be administered once a week, twice a week, once every two weeks, once a month, every six weeks, once every two months, twice a year or once a year. In addition, it should be understood that the effective dosage of the molecules used for treatment may increase or decrease during the course of a particular treatment.

VI. The use of the compositions according to the present invention

Characterized by the optimized sequence of CD123xCD3 bispecific diabodies according to the present invention are characterized by the ability to provide treatment for any disease or condition associated with the expression of CD123 or characterized by the expression of CD123. Thus, without limitation, such molecules can be used in the diagnosis or treatment of acute myeloid leukemia (AML), chronic myeloid leukemia (CML), including the CML blast crisis and the Abelson oncogene associated with CML (VSG-ABL translocation), myelodysplastic syndrome ( MDS), acute B-lymphoblastic leukemia (B-ALL), chronic lymphocytic leukemia (CLL), including CLL with Richter syndrome or Richter transformation, hairy cell leukemia (ON), neoplasms from blast plasmacytoid dendritic cells (OBPDK), non-Hodkin lymphomas (NHL), including mantle cell lymphoma (MKL) and small cell lymphocytic lymphoma (MLL), Hodgkin lymphoma, systemic mastocytosis and Burkitt's lymphoma (see example 2); autoimmune lupus erythematosus (SLE), allergies, bronchial asthma and rheumatoid arthritis. In addition, the bispecific diabodies of the present invention can be used in the manufacture of drugs for the treatment of the conditions described above.

By presenting the description of the present invention in general terms, it will become clearer with reference to the following examples, which are presented for illustration and are not intended to limit the present invention unless otherwise indicated.

Example 1

Construction of CD123xCD3 bispecific diabodies and control protein Table 2 provides a list of bispecific diabodies that were expressed and purified. A bispecific diatel (DART-A) characterized by an optimized CD123xCD3 sequence and a bispecific diatel (DART-B) characterized by a non-optimized CD123xCD3 sequence are capable of simultaneously binding to CD123 and CD3. The control bispecific diatelo (control DART) is able to simultaneously bind to FITC and CD3. Bispecific diabodies are heterodimers or heterotrimers with the listed amino acid sequences. Methods for the formation of bispecific diabodies are presented in international patent publications No. WO 2006/113665, WO 2008/157379, WO 2010/080538, WO 2012/018687, WO 2012/162068 and WO 2012/162067.

- 28 034142

<td colspan="3">table 2</td>

<td>Bispecific diabodies</td><td>Amino Acid Sequences of the Polypeptide Chain</td><td>Nucleic Acid Coding Sequences</td>

<td>Characterized by the optimized sequence of CD123 x CD3 bispecific diatel (DART-A) (binds to CD3 on epitope 1)</td><td>SEQ ID N0: 1 SEQ ID N0: 3</td><td>SEQ ID N0: 2 SEQ ID N0: 4</td>

<td>Non-optimized CD123 x CD3 sequence bispecific diatel (DART-B) (binds to CD3 on epitope 2)</td><td>SEQ ID N0: 5 SEQ ID N0: 7</td><td>SEQ ID N0: 6 SEQ ID N0: 8</td>

<td>Characterized by the optimized sequence of CD123 x CD3 bispecific diatel containing albumin binding domain (DART-A w / ABD) (binds to CD3 on epitope 1), contains an albumin binding domain (ABD) to increase the in vivo half-life</td><td>SEQ ID N0: 9 SEQ ID N0: 3</td><td>SEQ ID N0: 10 SEQ ID N0: 4</td>

<td>Version 1 of the CD123 x CD3 sequence optimized bispecific diabetic containing the IgG Fc domain (DART-A w / Fc version 1) (binds to CD3 on epitope 1) contains the Fc domain to increase the in vivo half-life</td><td>SEQ ID N0: 54 SEQ ID N0: 13 SEQ ID N0: 15</td><td>SEQ ID N0: 12 SEQ ID N0: 14 SEQ ID N0: 16</td>

<td colspan="3">table 2</td>

<td>Bispecific diabodies</td><td>Amino Acid Sequences of the Polypeptide Chain</td><td>Nucleic Acid Coding Sequences</td>

<td>Version 2 of the CD123 x CD3 sequence optimized bispecific diabetic containing an IgG Fc domain (DART-A w / Fc version 2) (binds to CD3 on epitope 1) contains an Fc domain to increase the in vivo half-life</td><td>SEQ ID N0: 54 SEQ ID N0: 17 SEQ ID N0: 1</td><td>SEQ ID N0: 12 SEQ ID N0: 18 SEQ ID N0: 2</td>

<td>Control bispecific diatelo (control DART) (binds to CD3 on epitope 1) (binds to an irrelevant target - FFTC)</td><td>SEQ ID N0: 19 SEQ ID N0: 20</td><td></td>

Example 2. Antibody Labeling of Target Cells for Quantitative FACS (QFACS)

In total, 10 target cells were harvested from culture, resuspended in 10% human AB serum in FACS buffer (PBS + 1% BSA + 0.1% NaAzide) and incubated for 5 min to block Fc receptors. Antibody-labeled microspheres with different antibody binding capabilities (Quantum ™ Simply Cellular® (QSC), Bangs Laboratories, Inc., Fishers, IN) and target cells were labeled with PE anti-CD123 antibody (BD Biosciences) according to the manufacturer's instructions. Briefly, one drop of each QSC microsphere was added to a 5 ml polypropylene tube, and PE-labeled anti-CD123 antibody was added at a concentration of 1 μg / ml to both target cells and microspheres. The tubes were incubated in the dark for 30 min at 4 ° C. Cells and microspheres were washed by adding 2 ml FACS buffer and centrifuging at 2500xG for 5 minutes. One drop of a population of empty microspheres was added after washing. The microspheres were first analyzed on a flow cytometer to establish device-specific settings for analysis (PMT voltage and compensation). Using the same instrument settings, the geometric mean of the fluorescence values ​​of microspheres and target cells was recorded. A standard curve of antibody binding sites in populations of microspheres was obtained from the geometric mean fluorescence of populations of microspheres.

- 29 034142

The antibody binding sites on the target cells were calculated based on the geometric mean fluorescence of the target cells using the standard curve obtained for the microspheres in the QuickCal spreadsheet (Bangs Laboratories).

To determine suitable cell lines - targets for evaluating CD123xCD3 bispecific diabodies, levels of surface expression of CD123 on target lines Kasumi-3 (AML), Molml3 (AML), THP-1 (AML), TF-1 (erythroleukemia) and RS4-11 (ALL) was evaluated using quantitative analysis of FACS (QFACS). The absolute numbers of CD123 antibody binding sites on the cell surface were calculated using the QFACS kit. As shown in Table 3, the absolute number of CD123 antibody binding sites per cell was Kasumi-3 (high)&gt; Molml3 (medium)&gt; THP-1 (medium)&gt; TF-1 (moderately low)&gt; RS4-11 (low) . The three cell lines with the highest levels of expression were AML cell lines: Kasumi-3, MOLM13 and THP-1. Non-AML cell lines: TF-1 and RS4-11, were characterized by moderately low / low expression of CD123, respectively ._______________

<td colspan="2">Table 3</td>

<td>Cell line target</td><td>Surface expression of CD123 (antibody binding sites)</td>

<td>Kasumi-3</td><td> 118620</td>

<td>Molml3</td><td> 27311</td>

<td>THR-1</td><td> 58316</td>

<td>TF-1</td><td> 14163</td>

<td>RS4-11</td><td> 957</td>

<td>A498</td><td>Negative</td>

<td>NT29</td><td>Negative</td>

Example 3. CTL Cytotoxicity Assay (LDH Release Assay)

The attached tumor target cells were detached with 0.25% trypsin EDTA and collected by centrifugation at 1000 rpm for 5 minutes. A suspension of target cell lines was collected from the culture, washed with an analysis medium. Cell concentration and viability were measured by trypan blue displacement using a Beckman Coulter Vi-Cell counter. Target cells were bred to 4x10⁵ cells / ml in assay medium. 50 μl of the diluted cell suspension was added to a 96-well U-bottom cell culture-treated plate (Cat. No. BD Falcon 353077).

Three sets of controls were established to measure maximum target release (MR) independent of antibody T cell cytotoxicity (AICC) and spontaneous release from target cell (SR) as follows:

1) MR: 200 μl of assay medium without CD123xCD3 bispecific diabodies and 50 μl of target cells; detergent was added at the end of the experiment to determine the maximum LDH release.

2) AICC: 50 μl of assay medium without CD123xCD3 bispecific diabodies, 50 μl of target cells and 100 μl of T cells.

3) SR: 150 μl of medium without CD123xCD3 bispecific diabodies and 50 μl of target cells.

CD123xCD3 bispecific diabodies (DART-A, DART-A w / ABD and DART-B) and controls were initially diluted to a concentration of 4 μg / ml, and then serial dilutions to a final concentration of 0.00004 ng / ml (t e. 40 fmg / ml). 50 μl of the dilutions were added to the plate containing 50 μl of target cells / well.

The purified T cells were washed once with assay medium and resuspended in assay medium with a T cell density of 2x10⁶ cells / ml 2x10⁵ T cells in 100 μl were added to each well to a final ratio of effector to cell target (E: T) of 10: 1. The plates were incubated for approximately 18 hours at 37 ° C in 5% CO₂. After incubation, 25 μl of a 10x lysing solution (Promega, Cat. No. G182A) or 1 mg / ml of digitonin was added to the control wells of maximum release, mixed with a pipette 3 times and the plates were incubated for 10 min until the target cells were completely lysed. The plates were centrifuged at 1200 rpm for 5 minutes and 50 μl of the supernatant was transferred from each well of the assay plate to a flat-bottom ELISA plate and 50 μl of LDH substrate solution (Promega, Cat. G1780) was added to each well. The plates were incubated for 10-20 minutes at room temperature (RT) in the dark, then 50 μl of stop solution was added. Optical density (OD) was measured at 490 nm for 1 h on a Victor2 Multilabel plate reader (Perkin Elmer, Cat. No. 420-014).

Cytotoxicity in% was calculated as described below, and dose response curves were obtained using GraphPad PRISM5® software.

Specific cell lysis was calculated from OD data using the following formula:

Cytotoxicity (%) = 100x (OD sample - OD AICC) / (OD MR - OD SR)

Redirected cytolysis of target cell lines using different levels of surface CD123

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CD123xCD3 bispecific diabodies showed a pronounced ability to redirect cytolysis with concentrations that should reach 50% of maximum activity (EC50s) in the sub-ng / ml range, regardless of the specificity of binding to the CD3 epitope (DART-A compared to DART-B) in cell lines - targets with a high level of expression of CD123, Kasumi-3 (EC50 = 0.01 ng / ml) (Fig. 4, panel D), average expression level of CD123, Molml3 (EC50 = 0, H, 8 ng / ml) and THP-1 (EC50 = 0.24 ng / ml) (FIG. 4, panel C and E, respectively) and moderately low or low expression levels of CD123, TF-1 (EC50 = 0.46 ng / ml) and RS4-11 (EC50 = 0.5 ng / ml) (Fig. 4, panel B and A, respectively). Similarly, CD123xCD3 mediated bispecific molecules redirected cytolysis was also observed with numerous cell lines targeting T cells from various donors, and a lack of redirected cytolysis activity was observed in cell lines that do not express CD123. The results are summarized in table. 4.

<td colspan="4">Table 4</td>

<td>Cell line target</td><td>Surface expression of CD123 (antibody binding sites)</td><td>EC50 characterized by an optimized sequence of CD123 x CD3 bispecific diabodies (ng / ml) E: T = 10: 1</td><td>Max. % cytolysis</td>

<td>Kasumi-3</td><td> 118620</td><td> 0,01</td><td> 94</td>

<td>Molml3</td><td> 27311</td><td> 0,18</td><td> 43</td>

<td>THR-1</td><td> 58316</td><td> 0,24</td><td> 40</td>

<td>TF-1</td><td> 14163</td><td> 0,46</td><td> 46</td>

<td>RS4-11</td><td> 957</td><td> 0,5</td><td> 60</td>

<td>A498</td><td>Negative</td><td>No activity</td><td>No activity</td>

<td>NT29</td><td>Negative</td><td>No activity</td><td>No activity</td>

If it is necessary to reproduce the indicated example, it should be understood that a person skilled in the art will be able, within the limits of practical and acceptable limits, modify the protocol described above in an appropriate way to reproduce the described results. Thus, it is not intended that the illustrated protocol be followed in a precisely defined manner.

Example 4. T-cell activation during redirected cytolysis using bispecific bispecific diabodies characterized by the optimized CD123xCD3 sequence (DART-A, DART-A w / ABD and DART-A w / Fc)

The bispecific diabodies characterized by the optimized CD123xCD3 sequence showed a pronounced ability for redirected cytolysis, regardless of the presence or absence of technology for increasing the half-life (DART-A compared to DART-A w / ABD compared to DART-A w / Fc) in cell lines targeting high expression of CD123, Kasumi-3, and average expression of CD123, THP-1 (Fig. 5, panels A and B, respectively). In order to obtain T cell activation characteristics during a process mediated by a sequence-optimized CD123xCD3 sequence-specific bispecific redirect cytolysis diabetic, Cells from redirected cytolysis assays were stained for CD25 T-cell activation marker and analyzed by FACS. As shown in FIG. 5, panel D, CD25 was upregulated in CD8 T cells in a dose-dependent manner, indicating that characterized by optimized CD123xCD3 sequence bispecific diabodies induced cell activation during redirected cytolysis. In contrast, in the absence of target cells, activation of CD8 T cells was not observed (FIG. 5, panel C), which indicates that bispecific diabodies characterized by the optimized CD123xCD3 sequence do not activate T cells in the absence of target cells. Similarly, CD8 T cells did not activate upon incubation with target cells and a control bispecific diabel (control DART) (Fig. 5, panel D), indicating the need for cross-linking of T cells and target cells with characterized by an optimized sequence of CD123xCD3 bispecific diabodies.

Example 5. Intracellular staining for granzyme B and perforin

To determine the intracellular contents of granzyme B and perforin in T cells, CTL analysis was adjusted as described above. After approximately 18 hours, cells from the assay plate were stained with anti-CD4 and anti-CD8 antibodies by incubation for 30 minutes at 4 ° C. After surface staining, cells were incubated in 100 μl of fixation and permeabilization buffer (BD BioSciences) for 20 min at 4 ° C. The cells were washed with permeabilization / washing buffer (BD BioSciences) and incubated in 50 μl of a mixture of antibodies to granzyme B and perforin (obtained in IX permeabilization / washing buffer) at 4 ° C for 30 min. The cells were then washed with 250 μl permeabilization / washing buffer and resuspended in permeabilization / washing buffer to obtain

- 31,034,142 given by the FACS.

Positive regulation of granzyme B and perforin characterized by an optimized sequence of CD123xCD3 bispecific diatel (DART-A) in T cells during redirected cytolysis

In order to investigate the possible mechanism of cytotoxicity of T-cells mediated by the characterized, optimized sequence of CD123xCD3 bispecific diabetic (DART-A), the intracellular contents of granzyme B and perforin were measured in T-cells after redirected cytolysis. Dose-dependent up-regulation of granzyme B and perforin in both CD8 and CD4 T cells was observed after incubation of T cells and Kasumi-3 cells with DART-A (Fig. 6, panel A). Interestingly, upregulation was almost two times higher in CD8 T cells compared to CD4 T cells (Fig. 6, panel A). If the analysis was performed in the presence of granzyme B and perforin inhibitors, cell lysis was not observed. Positive regulation of granzyme B or perforin was absent in CD8 or CD4 T cells if T cells were incubated with Kasumi-3 target cells and a control bispecific diabetic (control DART) (Fig. 5, panel B). These data indicate that DART-A-mediated lysis of target cells can be mediated through mechanisms involving granzyme B and perforin.

Example 6. In vivo antitumor activity characterized by an optimized sequence of CD123xCD3 bispecific diatel (DART-A)

Isolation of PBMCs and T cells from human whole blood

RVMS from healthy donors - people were isolated from whole blood using Ficoll gradient centrifugation. Briefly, whole blood was diluted 1: 1 with sterile PBS. 35 ml of diluted blood was applied to 15 ml of Ficoll-Paque ™ Plus in 50 ml tubes and the tubes were centrifuged at 1400 rpm for 20 minutes with disinhibition. A white blood cell film between the two phases was collected in a 50 ml tube with a 45 ml PBS by centrifuging the tubes at 600xg (1620 rpm) for 5 minutes. The supernatant was removed and the cell pellet was washed once with PBS and the number of viable cells was determined by displacing the trypan blue dye. PBMC resuspended to a final concentration of 2.5 x 10⁶ cells / ml in complete medium (RPMI 1640, 10% FBS, 2 mM glutamine, 10 mM HEPES, 100 μM / 100 μM / ml penicillin / streptomycin (P / S).

T cell isolation

Unexposed T cells were isolated by negative selection from PBMC from whole human blood using the Dynabeads Untouched human T cell isolation kit (Life Technologies) according to the manufacturer's instructions. After isolation, T cells were cultured overnight in RPMI medium with 10% FBS, 1% penicillin / streptomycin.

Tumor model

Human T cells and tumor cells (Molml3 or RS4-11) were combined in a ratio of 1: 5 (1x10⁶ and 5x10⁶ respectively) and suspended in 200 μl of sterile saline and administered by subcutaneous injection (SC) on day 0 of the study (SD0). A bispecific diatel (DART-A) characterized by a sequence optimized CD123xCD3 or a control bispecific diatel (control DART) was administered intravenously (IV) by injection of 100 μl into the tail vein, as shown in Table 1. 5 (MOLM13) and tab. 6 (RS4-11).

<td colspan="4">Table 5 Study design for model MOLM13</td>

<td>Treatment group</td><td>Dose (mg / kg)</td><td>Scheme</td><td>Number of animals</td>

<td>Control - inert carrier (MOLM-13 cells individually implanted or + T cells)</td><td> -</td><td>SD0, 1, 2, 3</td><td> 8</td>

<td>DART-A</td><td> 0,5</td><td>SD0, 1, 2, 3</td><td> 8</td>

<td>DART-A</td><td> 0,2</td><td>SD0, 1, 2, 3</td><td> 8</td>

<td>DART-A</td><td> 0,1</td><td>SD0, 1, 2, 3</td><td> 8</td>

<td>DART-A</td><td> 0,02</td><td>SD0, 1, 2, 3</td><td> 8</td>

<td>DART-A</td><td> 0,004</td><td>SD0, 1, 2, 3</td><td> 8</td>

<td>DART-A</td><td> 0,0008</td><td>SD0, 1, 2, 3</td><td> 8</td>

<td>DART-A</td><td> 0,00016</td><td>SD0, 1, 2, 3</td><td> 8</td>

- 32 034142

<td colspan="4">Table 6 Study design for model RS4-11</td>

<td>Treatment group</td><td>Dose (mg / kg)</td><td>Scheme</td><td>Number of animals</td>

<td>Control - inert carrier (RS4-11 cells separately implanted)</td><td> -</td><td>SDO, 1, 2, 3</td><td> 8</td>

<td>Control - inert carrier (RS4-11 + implanted T cells)</td><td> -</td><td>SDO, 1, 2, 3</td><td> 8</td>

<td>Test DART</td><td> 0,2</td><td>SDO, 1, 2, 3</td><td> 8</td>

<td>DART-A</td><td> 0,5</td><td>SDO, 1, 2, 3</td><td> 8</td>

<td>DART-A</td><td> 0,2</td><td>SDO, 1, 2, 3</td><td> 8</td>

<td>DART-A</td><td>ο, ι</td><td>SDO, 1, 2, 3</td><td> 8</td>

<td>DART-A</td><td> 0,02</td><td>SDO, 1, 2, 3</td><td> 8</td>

<td>DART-A</td><td> 0,004</td><td>SDO, 1, 2, 3</td><td> 8</td>

Data collection and statistical analysis:

Animal weights - Weights of individual animals were recorded twice a week until the end of the study, starting from the moment of injection of tumor cells.

State of Agony / Mortality - Animals were monitored twice a week for general agony and daily for mortality. Fatalities in animals were assessed as drug-related or technical based on factors including macroscopic observations and weight loss; animal deaths were recorded daily.

Tumor volume - The volumes of individual tumors were recorded twice a week, starting no later than one week after implantation of the tumor and continuing until the completion of the study.

length (mm) x width²Tumor volume (mm³) = ---------------- Animals that died due to technical or drug-related death were excluded from the data calculations.

Tumor Growth Inhibition — Tumor growth inhibition (TGI) values ​​were calculated for each group containing treated animals using the following formula:

Average end tumor volume (subjected to treatment) - average beginning tumor volume (subjected to treatment) Ί -: --------------------------------------- ----------: ----------- XIUU

Average end tumor volume (control) - average beginning tumor volume (control)

Animals experiencing partial or complete remission, or animals dying due to technical or drug-related deaths, were excluded from TGI calculations. The criteria of the National Cancer Institute for the activity of the compound is TGI&gt; 58% (Corbett et al. (2004) Anticancer Drug Development Guide; Totowa, NJ: Humana 99-123).

Partial / complete tumor response - Individual mice characterized by the presence of tumors smaller than 1 mm³ on day 1, was classified as characterized by partial remission (PR) and the value of tumor regression in% (% TR) was determined using the following formula:

I Final tumor volume (mm³) _------- ------------» _h . _{χ 1000 / o}

The initial tumor volume (mm)

Individual mice that lacked palpable tumors were classified as undergoing complete remission (CR).

Tumor volume statistics - A statistical analysis was performed between the treated and control groups by comparing tumor volumes. For these analyzes, two-way analysis of variance was used, followed by the Bonferroni posterior test. All analyzes were performed using GraphPad PRISM® software (version 5.02). Data on mass and tumor from individual animals that died due to technical or drug-related death were excluded from the analysis.

However, data for animal tumors that showed partial or complete responses were included in these calculations.

MOLM13 Results

The AML cell line, MOLM13, was pre-mixed with activated T cells and implanted with SC NOD / SCID gamma (NSG) knockout mice (N = 8 / group) on day SD0, as described in detail below. MOLM13 tumors in the inert-vehicle-treated group (MOLM13 cells alone or together with T cells) showed a relatively in vivo growth profile (Fig. 7, panels A and B). On day SD8e, the average tumor volume of the group treated with an inert carrier was 129.8 ± 29.5 mm, and by day SD15 the tumors reached an average volume of 786.4 ± 156.7 mm. By the end of the experiment, on day SD18, the tumors reached medium

- 33,034,142 th volume of 1398.8 ± 236.9 mm.

DART-A treatment was started on the same day as the mixture of tumor cells and T cells [(SD0)] was implanted and continued further with daily injections for an additional 7 days to achieve a total of 8 daily injections. 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 μg / kg). The results are shown in FIG. 7, panels A (0.5, 0.2, 0.1, 0.02 and 0.004 mg / kg) and FIG. 7, Panel B (20, 4, 0.8, and 0.16 μg / kg). On study day 11, tumor growth of MOLM13 was significantly inhibited at dose levels of 0.16, 0.5, 0.2, 0.1, 0.02, and 0.004 mg / kg (p &lt;0.001). Moreover, treatment of MOLM13 tumor mice with dose levels of 20 and 4 μg / kg dose resulted in 8/8 and 7/8 CR, respectively. By the end of the experiment, on day SD18, the average volume of tumors treated with DART-A at a dose of 0.8 - 20 μg / kg ranged from 713.6.0 ± 267.4 to 0 mm³moreover, all tumors were significantly smaller than the tumors in the control group treated with an inert carrier. TGI values ​​were 100, 94, and 49% for groups receiving doses of 20, 4, and 0.8 μg / kg, respectively. Compared to the inert carrier-treated group with MOLM13 tumor cells, the groups that received DART-A at a dose level of 20 and 4 μg / kg achieved a statistical significance level by day SD15, whereas the group treated with 0.8 mcg / kg, reached a statistical significance level by day SD18.

Results RS4-11

The ALL cell line, RS4-11, was pre-mixed with activated T cells and implanted with SC NOD / SCID gamma (NSG) knockout mice (N = 8 / group) on day SD0, as described in detail below. RS4-11 tumors in the inert-vehicle-treated group (RS4-11 cells alone or together with T cells) showed a profile of relatively aggressive growth in vivo (Fig. 8).

DART-A treatment was started on the same day as the mixture of tumor cells and T cells [(SD0)] was implanted and continued further with daily injections for an additional 3 days to achieve a total of 4 daily injections. Animals were treated with DART-A at 5 dose levels (0.5, 0.2, 0.1, 0.02 and 0.004 mg / kg). The results are shown in FIG. 8.

The bispecific diatel (DART-A) characterized by the sequence optimized CD123xCD3 effectively inhibited the growth of both MOLM13 AML and RS4-11ALL tumors implanted with SC NOD / SCID mice in the context of the Winn model if doses were started on the day of implantation and continued for 3 or more consecutive days. Based on criteria established by the National Cancer Institute, DART-A at a dose level of 0.1 mg / kg or higher (TGI&gt; 58) is considered active in the RS4-11 model and a DART-A dose of 0.004 mg / kg and above, was active in the MOLM13 model. The lower doses of DART-A associated with inhibition of tumor growth in the MOLM13 model compared to the RS4-11 model are consistent with in vitro data demonstrating that MOLM13 cells are characterized by higher levels of CD123 expression than RS4-11 cells, which correlates with increased sensitivity to in vitro DART-A mediated cytotoxicity in MOLM13 cells.

If it is necessary to reproduce the indicated example, it should be understood that a person skilled in the art will be able, within the limits of practical and acceptable limits, modify the protocol described above in an appropriate way to reproduce the described results. Thus, it is not intended that the illustrated protocol be followed in a precisely defined manner.

Example 7. Surface expression of CD123 on leukemic power cells and stem cells in a primary tissue sample from patient 1 with AML

To determine the expression pattern of CD123 in primary samples from a 1 s patient, cryopreserved primary bone marrow samples and PBMCs of a patient with AML were evaluated for surface expression of CD123 on leukemic blast cells.

AML Bone Marrow Sample - Clinical Report

Age: 42

Gender Female

Subtype AML: M2

Percentage of malignant cells based on morphology:

67,5%

Bone marrow immunophenotyping:

CD15 = 19%, CD33 = 98.5%, CD38 = 28.8%, CD45 = 81.8%, CD64 = 39.7%,

CD117 = 42.9%, HLA-DR = 17%, CD2 = 1.8%, CD5 = 0.53%, CD7 = 0.2%, CD10 = 0.41%, CD19 = 1.1%, CD20 = l, 4%, CD22 = 0.71% CD34 = 0.82%

Expression of CD123 in leukemic blast cells in bone marrow mononuclear cells (BM MNC)

In general, 0.5x10⁶ bone marrow mononuclear cells (BM MNC) and peripheral cro mononuclear cells

- 34 034142 Wi (PBMC)) from patient 1 with AML was evaluated in relation to the expression of CD123. The Kasumi3 cell line was included as a control. Leukemic blast cells were identified using the myeloid marker CD33. As shown in FIG. 9, Panel A, 87% of cells from bone marrow AML from patient 1 expressed CD123 and CD33. CD123 expression levels were slightly lower than that of expressing CD123 at a high level of the AML cell line, Kasumi-3 (Fig. 9, panel B).

Example 8. Autologous CTL cytolysis analysis using primary samples from a patient with AML

The cryopreserved primary AML sample (bone marrow mononuclear cells (BMNC) and peripheral blood mononuclear cells (PBMC)) from patient 1 with AML was thawed in RPMI 1640 with 10% FBS and allowed to recover overnight at 37 ° C in 5% CO2. The cells were washed medium for analysis (RPMI 1640 + 10% FBS) and the number of viable cells was determined by the displacement of trypan blue. 150,000 cells / well in 150 μl of assay medium was added to a 96-well U-bottom plate (BD Biosciences). The bispecific diatel (DART-A) characterized by the optimized CD123xCD3 sequence was diluted to 0.1 and 0.01 ng / ml and 50 μl of each dilution was added to each well (final volume = 200 μl). A control bispecific diatel (control DART) was diluted to 0.1 ng / ml and 50 μl of each dilution was added to each well (final volume = 200 μl). A separate assay plate was set for each time point (48, 72, 120, and 144 hours) and the plates were incubated at 37 ° C in an incubator with 5% CO₂. At each time point, cells were stained with antibodies to CD4, CD8, CD25, CD45, CD33 and CD123. Labeled cells were analyzed on a FACS Calibur flow cytometer equipped with CellQuest Pro data acquisition software, version 5.2.1 (BD Biosciences). Data analysis was performed using Flowjo v9.3.3 software (Treestar, Inc). T cell expansion was measured by gating on populations of CD4 + and CD8 + and activation was determined by measuring the average value of the fluorescence intensity of CD25 (MFI) in gated against CD4 + and CD8 + populations. The leukemic blast cell population was identified by gating CD45 + CD33 +.

Autologous deletion of tumor cells, T-cell expansion and activation using bispecific diatel (DART-A) characterized by the optimized CD123xCD3 sequence in primary samples from patient 1 with AML

In order to determine the activity mediated by the characterized optimized sequence of CD123xCD3 bispecific diabodyel (DART-A) in patient 1 with AML, samples from the patient were incubated with 0.1 or 0.01 ng / ml DART-A and the percentage ratios of leukemic blast cells and T cells were measured in various temporary points after treatment. Leukemic blast cells were identified by CD45 + / CD33 + gating. Incubation of primary bone marrow samples in AML with DART-A led to depletion of the leukemia cell population over time (Fig. 10, panel A), which was accompanied by a concomitant expansion of residual T cells (Fig. 10, panel B) and the induction of T- markers cell activation (Fig. 10, panel C). In DART-A-treated samples, T cells expanded from about 7% to about 80% by 120 hours. T-cell activation, as measured by expression of CD25 on CD4 and CD8 cells, peaked after 72 hours and decreased to a time point of 120 hours.

If it is necessary to reproduce the indicated example, it should be understood that a person skilled in the art will be able, within the limits of practical and acceptable limits, modify the protocol described above in an appropriate way to reproduce the described results. Thus, it is not intended that the illustrated protocol be followed in a precisely defined manner.

Example 9. Surface expression of CD123 on leukemic power cells and stem cells in a primary tissue sample from a patient with ALL

To determine the expression pattern of CD123 in primary samples from a patient with ALL, a cryopreserved primary sample of PBMC from a patient with ALL was evaluated for surface expression of CD123 on leukemic blast cells.

Expression of CD123 in leukemic blast cells in peripheral blood mononuclear cells (PBMC)

In general, 0.5x10⁶ peripheral blood mononuclear cells (PBMCs) from a healthy donor and a patient with ALL were evaluated for CD123 expression. As shown in FIG. 11, panels E-H, the vast majority of bone marrow ALL cells expressed CD123. In contrast, in a normal donor, B cells were negative for CD123 and pDC, and monocytes were positive for CD123 (Fig. 11, panel D).

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

- 35 034142

Example 10

CTL autologous cytolysis analysis using primary samples from a patient with ALL

The cryopreserved primary ALL sample (peripheral blood mononuclear cells (PBMC)) was thawed in RPM 11640 with 10% FBS and allowed to recover overnight at 37 ° C in 5% CO₂. Cells were washed with assay medium (RPMI 1640 + 10% FBS) and the number of viable cells was determined by the displacement of trypan blue. 150,000 cells / well in 150 μl of assay medium was added to a 96-well U-bottom plate (BD Biosciences). The bispecific diatel (DART-A) characterized by the optimized CD123xCD3 sequence was diluted to 10, 1 ng / ml and 50 μl of each dilution was added to each well (final volume = 200 μl). A separate assay plate was set for each time point (48, 72, 120, and 144 hours) and the plates were incubated at 37 ° C in an incubator with 5% CO₂. At each time point, cells were stained with antibodies to CD4, CD8, CD25, CD45, CD33 and CD123. Labeled cells were analyzed on a FACS Calibur flow cytometer equipped with CellQuest Pro data acquisition software, version 5.2.1 (BD Biosciences). Data analysis was performed using Flowjo v9.3.3 software (Treestar, Inc). Cellular expansion was measured by gating on populations of CD4 + and CD8 + and activation was determined by measuring MFI of CD25 in gated against CD4 + and CD8 + populations. The leukemic blast cell population was identified by gating CD45 + CD33 +.

Autologous deletion of tumor cells, T-cell expansion and activation using a bispecific diatel (DART-A) characterized by a sequence-optimized CD123xCD3 in primary samples from patients with ALL

To determine the mediated, characterized by the optimized sequence of CD123xCD3 bispecific diatel (DART-A) activity in primary samples from a patient with ALL, patient samples were incubated with 1 ng / ml DART-A and the percentage ratios of leukemic blast cells and T cells were measured at different time points after treatment . Leukemic blast cells were identified by CD45 + / CD33 + gating. Incubation of primary bone marrow samples in ALL with DART-A led to depletion of the leukemia cell population over time compared to the untreated control or control DART (Fig. 13, panel H compared to panels F and G). If T cells were counted (staining for CD8 and CD4) and activation (staining for CD25) was analyzed, T cells were expanded and activated in a DART-A sample (FIG. 14, panels I and L, respectively) compared to untreated samples or treated with control DART samples (FIG. 14, panels H, G, K, and J, respectively).

Example 11. Surface expression of CD123 on leukemic power cells and stem cells in a primary tissue sample from patient 2 with AML

To determine the expression pattern of CD123 in primary samples of patient 2 with AML, cryopreserved primary samples of bone marrow and PBMC of a patient with AML were evaluated for surface expression of CD123 on leukemic blast cells.

Expression of CD123 in leukemic blast cells in bone marrow mononuclear cells (BMNC)

Overall 0.5x10⁶ bone marrow mononuclear cells (BM MNC) and peripheral blood mononuclear cells (PBMC) from patient 2 with AML were evaluated to identify leukemic blast cells. Leukemic blast cells were identified using myeloid markers CD33 and CD45. As shown in FIG. 15, Panel B, 94% of the cells from bone marrow with AML were leukemic blast cells. The T cell population was identified by expression of CD3. As shown in FIG. 15, Panel C, approximately 15% of the cells from the bone marrow sample of AML and PBMC were T cells.

Example 12. Autologous CTL cytolysis analysis using primary samples from patient 2 with AML

The cryopreserved primary sample of AML (bone marrow mononuclear cells (BM MNC) and peripheral blood mononuclear cells (PBMC)) from patient 2 with AML was thawed in RPM 11640 with 10% FBS and allowed to recover overnight at 37 ° C in 5% CO₂. Cells were washed with assay medium (RPMI 1640 + 10% FBS) and the number of viable cells was determined by the displacement of trypan blue. 150,000 cells / well in 150 μl of assay medium was added to a 96-well U-bottom plate (BD Biosciences). The bispecific diatel (DART-A) characterized by the optimized sequence CD123xCD3 and the control bispecific diatel (control DART) were diluted to 0.1 and 0.01 ng / ml and 50 μl of each dilution was added to each well (final volume = 200 μl). A separate assay plate was set for each time point (48, 72, 120, and 144 hours) and the plates were incubated at 37 ° C in an incubator with 5% CO2. At each time point, cells were stained with antibodies to CD4, CD8, CD25, CD45, CD33 and CD123. Labeled cells were analyzed on a FACS Calibur flow cytometer equipped with CellQuest Pro data acquisition software, version 5.2.1 (BD Biosciences). Data analysis was performed using Flowjo v9.3.3 software (Treestar, Inc). T cell expansion was measured by gating on populations of CD4 + and CD8 + and activation was determined by measuring MFI of CD25 in gated against CD4 + and CD8 + populations. Gay blast cell population identified by gay

- 36 034142 CD45 + CD33 +.

Autologous deletion of tumor cells, T-cell expansion and activation in primary samples from patient 2 with AML

In order to determine the activity mediated by the characterized optimized sequence CD123xCD3 bispecific diatel (DART-A) in primary samples from patient 2 with AML, patient samples were incubated with 0.1 or 0.01 ng / ml DART-A and percentage ratios of leukemic blast cells and T cells measured at various time points after treatment. Incubation of primary bone marrow samples in AML with DART-A led to depletion of the leukemia cell population over time (FIG. 16, panel A), which was accompanied by a concomitant expansion of residual T cells (both CD4 and CD8) (FIG. 16, panel B and FIG. 16, panel C, respectively). To determine whether T cells were activated, cells were stained for CD25 or Ki-67, both markers of T cell activation. As shown in Figure 17, Panels A and B, incubation of primary bone marrow samples in AML with DART-A resulted in T-cell activation. The indicated data represented a time point of 144 hours.

Intracellular staining for granzyme B and perforin

To determine the intracellular contents of granzyme B and perforin in T cells, CTL analysis was performed. After approximately 18 hours, cells from the assay plate were stained with anti-CD4 and anti-CD8 antibodies by incubation for 30 minutes at 4 ° C. After surface staining, cells were incubated in 100 μl of fixation and permeabilization buffer for 20 min at 4 ° C. The cells were washed with permeabilization buffer / and incubated in 50 μl of a mixture of antibodies to granzyme B and perforin obtained in IX permeabilization / washing buffer at 4 ° C for 30 min. The cells were then washed with 250 μl permeabilization / washing buffer and resuspended in permeabilization / washing buffer to obtain this FACS

Positive regulation of granzyme B and perforin characterized by an optimized sequence of CD123xCD3 bispecific diatel (DART-A) in T cells during redirected cytolysis

In order to investigate the possible mechanism of cytotoxicity of T-cells mediated by the characterized, optimized sequence of CD123xCD3 bispecific diabetic (DART-A), the intracellular contents of granzyme B and perforin were measured in T-cells after redirected cytolysis. If T cells were incubated with a control bispecific diabel (control DART), upregulation of granzyme B and perforin was not observed. A positive regulation of the contents of granzyme B and perforin in both CD8 and CD4 T cells was observed under the action of the bispecific diabetic character (DARTA) characterized by the optimized CD123xCD3 sequence (Fig. 17, panels C and D). Interestingly, upregulation was almost two times higher in CD8 T cells compared to CD4 T cells (Fig. 17, panel C and Fig. 17, panel D). These data indicate that DART-A-mediated lysis of target cells is mediated via a pathway involving granzyme B and perforin.

Example 13

The bispecific diatel characterized by the optimized sequence of CD123xCD3 cross-reacts with non-human primate CD123 and CD3 proteins

BIACORE ™ assays were performed to quantify the degree of binding between the bispecific diabetic specific antibody (DART-A) characterized by the optimized sequence of CD123xCD3 and CD3. BIACORE ™ assays measure the dissociation rate constant, kd. The binding affinity (KD) between an antibody and its target is a function of the kinetic constants for association (association rate constant, k_a) and dissociation (dissociation rate constant, kd) according to the formula: KD = [kd] / [ka]. BIACORE ™ analysis uses surface plasmon resonance to measure directly specified kinetic parameters. Recombinant human or Javanese macaque CD3 was directly immobilized on a support. Purified human or Javanese macaque CD123 was captured and immobilized on a substrate. The dissociation time was measured and bivalent data approximation was performed. Binding and affinity constants were obtained using a 1: 1 approximation of binding. The results of the BIACORE assays, which compared the binding to human CD123 and CD3 proteins compared to cynomolgus macaque CD123 and CD3 proteins, are shown in FIG. 18. The binding affinities of cynomolgus macaque CD123 proteins (FIG. 18D) and cynomolgus macaque CD3 (FIG. 18B) are comparable with binding affinities for human CD123 proteins (FIG. 18C) and human CD3 (FIG. 18A).

Example 14. Autologous depletion of monocytes in vitro with PBMC human and cynomolgus monkey

PBMCs from whole blood samples of human or cynomolgus monkey were added to U-bottom plates with a T-cell density of 200,000 cells / well in 150 μl of assay medium. Dilutions of bispecific diabodies characterized by the optimized CD123xCD3 sequence (DART-A or DART-A w / ABD) were obtained in assay medium. 50 μl of each dilution of DART-A or DART-A w / ABD was added to the plate containing PBMC in wells in two parallel. Tablets

- 37 034142 were incubated for -18-24 hours at 37 ° C. Supernatants were used to determine cytotoxicity as described above. As shown in FIG. 19 (panels A and B), pDC cell depletion was observed in both human PBMCs (FIG. 19, panel A) and cynomolgus PBMCs (FIG. 19, panel B). These results indicate that pDC circulating in the bloodstream can be used as a pharmacodynamic marker for preclinical toxicological tests in cynomolgus monkeys.

If it is necessary to reproduce the indicated example, it should be understood that a person skilled in the art will be able, within the limits of practical and acceptable limits, modify the protocol described above in an appropriate way to reproduce the described results. Thus, it is not intended that the illustrated protocol be followed in a precisely defined manner.

Example 15

Plasma cytoid dendritic cell depletion in cynomolgus monkeys treated with an optimized CD123xCD3 sequence of bispecific diabetic (DART-A)

As part of a toxicological study to search for a dose range, cynomolgus monkeys were injected with a CD123xCD3 optimized sequence characterized by a bispecific diatel (DART-A) in the form of 4-day infusions in doses of 0.1, 1, 10, 30, 100, 300 or 1000 ng / kg . Control DART was administered at a dose of 100 ng / kg. To identify pDC and populations of RVMS of cynomolgus macaque monocytes, cells were labeled with anti-CD14-FITC antibody. Monocytes identified as CD14 + population and pDC identified as CD14CD123¹ population. As shown in FIG. 20, panels K and L, pDC were depleted no later than 4 days after infusion at a dose of not more than 10 ng / kg DART-A. The depletion of pDC was not observed in monkeys treated with a control bispecific diabetic (control DART) or treated with an inert carrier with a monkey filler at a time point of 4 days (Fig. 20, panels G, H, C and D, respectively) . The cytokine contents: interferon-gamma, TNF-alpha, IL6, IL5, IL4 and IL2, were determined 4 hours after infusion. A slight increase or no increase in cytokine levels was observed in animals treated with DART-A compared to those treated with control DART or treated with an inert carrier.

In FIG. Figures 21 and 22 show the results of FACS analysis for B-cells (CD20 +) (Fig. 21, panel A), monocytes (CD14 +) (Fig. 21, panel B), NK cells (CD159 + CD16 +) (Fig. 21, panel C) pDC (CD123^Hi, CD14-) (FIG. 21, panel D) and T cells (generally CD4 + and CD8 +) (FIG. 22, panel A, FIG. 22, panel B and FIG. 22, panel D, respectively).

Treatment of monkeys with control DART did not lead to noticeable effects on T or B lymphocytes, NK cells, monocytes and pDC. Treatment of monkeys with DART-A at doses of 10 ng / kg / day or higher resulted in the elimination of pDC (Fig. 21, panel D). PDC depletion was complete and prolonged, returning to pre-treatment levels a few weeks after completion of dosing. The content of T-lymphocytes circulating in the bloodstream decreased with the introduction of DART-A, but returned to levels before treatment at the end of each weekly cycle, which indicates changes in migration, and not real depletion. Both CD4 and CD8 T lymphocytes were characterized by the same pattern. The T-lymphocyte activation marker, CD69 (Fig. 22, panel C), was only slightly positive among the circulating cells and did not change following the administration of a dose of DART-A. The contents of B-lymphocytes, monocytes and NK-cells fluctuated during the course of the administration of doses of DART-A with significant variability observed among monkeys. A tendency towards increased contents of circulating B-lymphocytes and monocytes was observed in both monkeys at the highest doses.

So, the above results demonstrate the therapeutic efficacy of a bispecific diabatel (DART-A) characterized by the optimized CD123xCD3 sequence. The bispecific diatel (DART-A) characterized by the optimized CD123xCD3 sequence can be used as a therapeutic agent for the treatment of numerous diseases and conditions, including the following: AML, ABL (ALL), CLL, MDS, pDCL, mantle cell lymphoma, hairy cell leukemia, CLL with Richter transformation, blast crisis in CML, BLL (subclass is CD123 +) (see example 2); systemic lupus erythematosus (SLE), allergies (basophils are CD123 +), bronchial asthma, etc.

Example 16. Comparative properties characterized by an optimized sequence of CD123xCD3 bispecific diabetic (DART-A) and characterized by a non-optimized sequence of CD123xCD3 bispecific diabetic (DART-B)

Unexpected advantage and properties of the optimized sequence of CD123xCD3 bispecific diabodies

As discussed above, DART-A and DART-B were constructed in a similar fashion, and the first polypeptide of both constructs contains, from the N-terminus to the C-terminus, the N-terminus, the VL domain of a monoclonal antibody capable of binding to CD3 (VL_Cd3), an intermediate linker peptide (linker 1), the VH domain of a monoclonal antibody capable of binding to CD123 (VHCD123), linker 2, E

- 38 034142 helical domain and C-terminus. Similarly, the second polypeptide of both constructs contains, from the N-terminus to the C-terminus, the N-terminus, the VL domain of a monoclonal antibody capable of binding to CD123 (VLCD123), an intermediate linker peptide (linker 1), the VH domain of a monoclonal antibody capable of bind to CD3 (VHCD3), linker 2, K-helical domain and C-terminus.

As shown in Example 1, it was found that both CD123xCD3 bispecific diabodies are capable of simultaneously binding to CD3 and CD123. In addition, as disclosed in example 3 and in FIG. 4, panels C and D, two CD123xCD3 bispecific diabodies showed a pronounced ability to redirect cytolysis with concentrations that should reach 50% of the maximum activity (EC50) in the sub-ng / ml range, regardless of the specificity of binding to the CD3 epitope (DART-A compared with DART-B) in cell lines - targets with high expression of CD123. Thus, small variations in specific CD123xCD3 sequences of bispecific diabodies do not completely negate biological activity.

However, in all studied cell lines, it was found that DART-A was more active and more effective in redirected cytolysis than DART-B (see, for example, Figure 4, panels A, C and D). Thus, DART-A has shown an unexpected advantage over similar DART-B.

Example 17. Pharmacology DART-A in non-human primate for the treatment of hematological malignancies

Interleukin 3 receptor alpha chain (IL-3), CD123, is overexpressed on malignant cells in a wide range of hematologic 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 an unfavorable prognosis (Vergez F. et al. (2011) High Levels OfCD34 + CD38low / -CD123 + Blasts Are Predictive Of An 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 leukemic stem cells (LSC) (Jordan, ST. Et al. (2000) The Interleukin-3 Receptor Alpha Chain Is A Unique Marker For Human Acute Myelogenous Leukemia Stem Cells Leukemia 14: 1777-1784; Jin L. et al. (2009) Monoclonal Antibody-Mediated Targeting Of CD123, IL-3 Receptor Alpha Chain, Eliminates Human Acute Myeloid Leukemic Stem Cells Cell Stem Cell 5: 31-42), which is a promising feature that allows you to target the underlying cause of such diseases. According to this conclusion, CD123 also takes part in the autocrine loop of IL-3, which supports the onset and development of leukemia, as shown by the ability of a CD123 blocking monoclonal antibody to reduce the engraftment of leukemic stem cells and improve survival in the mouse model of acute myeloid leukemia (AML) ( Jin, L. et al. (2009) Monoclonal Antibody-Mediated Targeting Of CD123, IL-3 Receptor Alpha Chain, Eliminates Human Acute Myeloid Leukemic Stem Cells Cell Stem Cell 5: 31-42). However, in a phase 1 study in patients at high risk for AML, the monoclonal antibody did not show anti-leukemia activity (Roberts, AW et al. (2010) A Phase I Study Of Anti-CD123 Monoclonal Antibody (mAb) CSL360 Targeting Leukemia Stem Cells (LSC ) In AML J. Clin. Oncol. 28 (Suppl): el3012). Therefore, alternative approaches for targeting CD123 are needed, including depletion strategies. Although CD123 is expressed by a subclass of normal hematopoietic progenitor cells (LDCs), hematopoietic stem cells (HSC) express little or no CD123 (Jordan, CT et al. (2000) The Interleukin-3 Receptor Alpha Chain Is A Unique Marker For Human Acute Myelogenous Leukemia Stem Cells Leukemia 14: 1777-1784; Jin, W. et al. (2009) Regulation OfThU Cell Differentiation And EAE Induction By MAP3K NIK Blood 113: 6603-6610), indicating that strategies based on cell depletion from CD123 provide recovery through normal hematopoiesis.

Targeting the patient's own T-lymphocytes to target leukemia cells is a promising immunotherapeutic strategy for the treatment of hematological malignancies. An attempt to apply the therapeutic potential of this approach was carried out using blinatumomab (BiTE based on a bispecific antibody characterized by the ability to bind to CD3 and B19 CD19 antigen) in patients with B-cell lymphomas and pre-B-cell acute lymphoblastic leukemia ((Klinger M. et al . (2012) Immunopharmacologic Response Of Patients With B-Lineage Acute Lymphoblastic Leukemia To Continuous Infusion Of T Cell-Engaging CD19 / CD3-Bispecific BiTE Antibody Blinatumomab Blood 119: 6226-6233; Topp, MS et al. (2012) LongTerm Follow-Up Of Hematologic Relapse-Free Survival In A Phase 2 Study Of Blinatumomab In Patients With MRD In B-Lineage ALL Blood 120: 5185-5187; Topp, MS et al. (2011) Targeted Therapy With The T-Cell Engaging Antibody Blinatumomab Of Chemotherapy-Refractory Minimal Residual Disease In B-Lineage Acute Lymphoblastic Leukemia Patients Results In High Response Rate And Prolonged Leukemia-Free Survival J. Clin. Oncol. 29: 2493-2498).

Molecules CD123xCD3 bespecifically diabetic according to the present invention, such as

- 39 034142

DART-A contains an alternative bispecific, antibody-based modality that provides improved stability and more robust properties for manufacturing feasibility (Johnson S. et al. (2010) Effector Cell Recruitment With Novel Fv-Based Dual-Affinity Re-Targeting Protein Leads To Potent Tumor Cytolysis And In Vivo B-Cell Depletion J. Mol. Biol. 399: 436-449; Moore PA et al. (2011) Application Of Dual Affinity Retargeting Molecules To Achieve Optimal Redirected T-Cell Killing Of B-Cell Lymphoma Blood 117: 4542-4551).

To demonstrate the superiority and efficacy of the CD123xCD3 molecules of the bispecific diabetic according to the present invention, the biological activity of the above-described DART-A in vitro and preclinical models of leukemia, as well as its pharmacokinetics, pharmacodynamics and safe pharmacology on cynomolgus monkeys (Masas fascicularis) were evaluated in relation to either the above control DART (bispecific for CD3 and fluorescein), or control DART-2, which is bispecific for CD123 and fluorescein).

The amino acid sequence of the first polypeptide chain of the control DART-2 (CD123VL - linker - 4-4420VH - linker - E-helix; linkers are underlined) (SEQ ID NO: 58):

MSCKSSQSLL

SGSGTDFTLT GEVKLDETGG QIRNKPYNYE GSYYGMDYWG LEK

LAVSLGERVT

ESGVPDRFSG

EIKGGGSGGG

SPEKGLEWVA

VEDMGIYYCT

VAALEKEVAA

NSGNQKNYLT ISSLQAEDVA GLVQPGRPMK TYYSDSVKGR QGTSVTVSSG

DFVMTQSPDS KLLIYWASTR PYTFGQGTKL SDYWMNWVRQ SVYLQMNNLR EKEVAALEKE

WYQQKPGQPP VYYCQNDYSY LSCVASGFTF FTISRDDSKS

GCGGGEVAAL

Amino acid sequence of the second polypeptide chain of the control DART-2 (4420VL - linker - CD123VH - linker - K-helix) (SEQ ID NO: 59):

DWMTQTPFS LPVSLGDQAS ISCRSSQSLV HSNGNTYLRW YLQKPGQSPK

VLIYKVSNRF SGVPDRFSGS GSGTDFTLKI SRVEAEDLGV YFCSQSTHVP

WTFGGGTKLE IKGGGSGGGG EVQLVQSGAE LKKPGASVKV SCKASGYTFT

DYYMKWVRQA PGQGLEWIGD IIPSNGATFY NQKFKGRVTI TVDKSTSTAY

MELSSLRSED TAVYYCARSH LLRASWFAYW GQGTLVTVSS GGCGGGKVAA

LKEKVAALKE KVAALKEKVA ALKE

Bifunctional ELISA

The MaxiSorp ELISA plate (Nunc), onto which soluble human or cynomolgus monkey IL3R alpha (0.5 μg / ml) was applied overnight 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 sequential addition of the human CD3ε5-BIOTIN conjugate and streptavidin-HRP (Jackson ImmunoResearch). HRP activity was detected by the conversion of tetramethylbenzidine (BioFX) as a substrate for 5 minutes; the reaction was interrupted with 40 μl / well of 1% H2SO4 and the absorbance was read at 450 nm.

Surface plasmon resonance analysis

The ability of DART-A to bind to human and cynomolgus macaque CD3 or CD123 proteins was analyzed using a BIAcore 3000 biosensor (GE, Healthcare) as described by Johnson, S. et al. (2010) (Effector Cell Recruitment With Novel Fv-Based Dual-Affinity Re-Targeting Protein Leads To Potent Tumor Cytolysis And In Vivo B-Cell Depletion J. Mol. Biol. 399: 436-449) and Moore, PA et al. (2011) (Application Of Dual Affinity Retargeting Molecules To Achieve Optimal Redirected T-Cell Killing Of B-Cell Lymphoma Blood 117: 4542-4551). Briefly, carboxyl groups on the CM5 sensor chip were activated by injection of 0.2 M Noethyl-L- (3-diethylaminopropyl) carbodiimide and 0.05 M N-hydroxysuccinimide. Soluble CD3 or CD123 (1 μg / ml) was injected onto the surface of activated CM5 in 10 mM sodium acetate, pH 5.0, at a volumetric flow rate of 5 μl / min, after which 1 M ethanolamine was added for deactivation. Binding experiments were performed in 10 mM HEPES, pH 7.4, 150 mM NaCl, 3 mM EDTA and 0.005% P20 surfactant. The immobilized receptor surfaces were regenerated by pulsed injection of 10 mM glycine, pH 1.5. KD values ​​were determined by global approximation of the binding curves to the 1: 1 Langmuir binding model (BIAevaluation v4.1 software).

Cytolysis assay

Cell lines used for cell lysis assays were obtained from the American Type Culture Collection (ATCC) (Manassas, VA). PBMCs were isolated from the blood of healthy donors using the Ficoll-Paque Plus kit (GE Healthcare); T cells were purified using a negative selection kit (Life Technologies). Density of CD123 on the cell surface was determined using Quantum Simply Cellular granules (Bangs Laboratories, Inc., Fishers, IN). Cytotoxicity assays were performed as described by Moore, PA et al. (2011) (Application Of Dual Affinity Retargeting Molecules To Achieve Optimal Redirected T-Cell Killing Of B-Cell Lymphoma Blood 117: 4542-4551). Briefly, target cell lines (10⁵ cells / ml) were treated with serial dilutions of proteins such as DART-A or control DART in the presence of T cells in the indicated ratios of effector cells to target cells and incubated at 37 ° C overnight. Cell lysis was determined by the release of lactate dehydrogenase (LDH, Promega) in the culture supernatant. For flow cytolysis analysis

- 40 034142 cytometry of the target cell was labeled with CMTMR (Life Technologies) and cytolysis was monitored using a FACSCalibur flow cytometer. Data were analyzed using PRISM® 5 software (GraphPad) and presented as cytotoxicity in%.

Pharmacology for cynomolgus macaque

Non-human primate experiments were performed at Charles River Laboratories (Reno, NV), according to the guidelines of the Local Institutional Animal Care and Use Committee (IACUC). Chinese-born Macaque macaques (Masasa fascicularis) of Chinese origin (age range 2.5–9 years, mass range 2.7–5 kg) were specially bred, previously not subject to experiments, by an inert carrier or DART-A via intravenous infusion through port systems into femoral and jugular vein 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 analyzes were performed using a Fortessa LSR analyzer (BD Biosciences) equipped with lasers with wavelengths of 488, 640 and 405 nm, and using the following antibodies: CD4-V450, CD8-V450, CD123-PE-Cy7, CD45 -PerCP, CD4-APC-H7, CD8-FITC, CD25PE-Cy7, CD69-PerCP, PD-l-PE, TIM3-APC, CD3-Pacific Blue, CD95-APC, CD28-BV421, CD16-FITC, CD3Alexa488, CD38-PE, CD123-PE-Cy7, CD117-PerCP-Cy5,5, CD34-APC, CD90-BV421, CD45RA-APC-H7 and CD33-APC (BD Biosciences). The absolute number of cells was determined using TruCOUNT (BD Biosciences). Serum contents of IL-2, IL-4, IL-5, IL-6, TNF-α, and IFN-γ cytokines were measured using a kit of cytometric granules with monoclonal antibodies to the Th1 / Th2 cytokine of non-human primate (BD Bioscience). The concentration of DART-A in monkey serum samples was measured using sandwich immunoassay with electroluminescent detection (MesoScale Diagnostics, MSD, Rockville, MD). Briefly, recombinant human IL-3 Ra (R&amp;D System) was applied to an assay plate (MSD) and the plate was blocked with 5% BSA. Calibration standards or diluted test samples were applied, followed by the addition of a biotinylated monoclonal antibody exhibiting specific binding for the E-helical (SEQ ID NO: 34) and K-helical (SEQ ID NO: 35) domains of the molecule described above. SULFO-TAG ™ labeled streptavidin conjugate (MSD) was added and complex formation was analyzed in an MSD SECTOR® imaging device. DART-A concentrations were determined from standard curves created by approximating light emission intensity data in a five-parameter logistic model.

Determination of the physicochemical characteristics of purified DART-A showed a homogeneous heterodimer with a molecular weight of 58.9 kDa (Fig. 23; Figs. 24A-24B), which is stable at 2-8 ° C for up to 12 months in PBS . SPR analysis showed almost identical binding affinities for DART-A with the corresponding soluble antigens of human CD3 and CD123 and cynomolgus macaque (Fig. 25A-25D and table. 7). In addition, DART-A simultaneously bound to both antigens in ELISA format, which used human CD123 or monkeys to capture and human CD3 for detection (Fig. 26A-26B), and showed similar binding to human and monkey T-lymphocytes (Fig. .26C-26L). The data in the table. 7 are average values ​​from 3 independent experiments, each of which was carried out in two parallels.

<td colspan="4">Table 7 Equilibrium dissociation constants (K_D) for binding of DART-A to human CD3 and CD123 and cynomolgus monkey</td>

<td>Antigens</td><td>k_a(± SD) (mV)</td><td>k_d(± SD) (s'¹)</td><td>K_d(± SD) (nM)</td>

<td>CD3e / 5 people</td><td>5.7 (± 0.6) x 10⁵</td><td>5.0 (± 0.9) x 10³</td><td> 9,0 ±2,3</td>

<td>CD3e / 5 cynomolgus macaque</td><td>5.5 (± 0.5) x 10⁵</td><td>5.0 (± 0.9) x 10^_3</td><td> 9,2 ±2,3</td>

<td>CD 123-His Human</td><td>1.6 (± 0.4) x 10⁶</td><td>1.9 (± 0.4) x 10 '⁴</td><td> 0,13 ±0,01</td>

<td>CD 123-His Cynomolgus Macaque</td><td>1.5 (± 0.3) x 10⁶</td><td>4.0 (± 0.7) x 10 '⁴</td><td> 0,27 ± 0,02</td>

DART-A mediates redirected cytolysis by human or cynomolgus T-lymphocytes

DART-A mediated the redirected cytolysis of the target cells by human or monkey effector cells against Kasumi-3 CD123 + leukemia cell lines (Fig. 27A-27D), which was accompanied by the induction of activation markers. No activity was observed against CD123 negative targets (U937 cells) or using a control DART, indicating that T-cell activation is highly dependent on the involvement of target cells and that the monovalent involvement of CD3 with DART-A was insufficient to trigger T cell activation. Since CD123 is expressed in a subclass of normal circulating white blood cells, including pDC and monocytes (FIG. 27E), the effect of DART-A was further investigated in normal human and monkey PBMCs.

The effect of a step change was observed among RVMS of a person with a dose-dependent fast

- 41,034,142 deletion of CD14-CD123^hlgh cells (pDC and basophils) observed no later than 3 hours after the start of treatment, while the monocytes (CD14 + cells) were not exposed at this time point (Fig. 27F-27G). Depletion CD14-CD123^high cells increased over time at all concentrations of the DART-A molecule, while the number of monocytes slightly decreased by 6 hours and was depleted after 18 hours and at concentrations higher than 1 ng / ml. Incubation of the PBMC of the monkey with DART-A resulted in a comparable dose-dependent depletion of CD14-CD123^lg cells (Fig. 27H), further confirming the relevance of this species to the pharmacology of DART-A (CD14 + monkey cells express CD123 to a small extent or do not express at all, and they do not undergo depletion).

DART-A Pharmacokinetics in Cynomolgus Macaques

Javanese macaque was chosen as an acceptable pharmacological model for DARTA analysis based on the equivalent distribution of both target antigens in this form compared to humans based on immunohistochemistry with mAb precursors, according to published information (Munoz L. et al. (2001) Interleukin-3 Receptor Alpha Chain (CD123) Is Widely Expressed In Hematologic Malignancies, Haematologica 86: 1261-1269; Korpelainen, EI et al. (1996) IL-3 Receptor Expression, Regulation And Function In Cells Of The Vasculature Immunol. Cell Biol. 74: 1-7).

The study carried out according to the present invention included 6 treatment groups consisting of 8 cynomolgus monkeys per group (4 males, 4 females) (Table 8). All groups received an inert carrier - control for the first infusion; then an inert carrier or DART-A was administered intravenously for 4 weekly cycles. Group 1 animals received an inert carrier control over all 4 subsequent infusions, while groups 2-5 received weekly increasing doses of DART-A for 4 days a week for all subsequent infusions. Group 6 animals were treated with 7-day continuous weekly increasing doses of DART-A for all infusions. Schemes of 4 days of administration / 3 days without administration and 7 days of administration were developed in order to distinguish between long-term and temporary effects associated with the introduction of DART-A. Two males and 2 females for each group were killed at the end of the treatment phase (36 days), while the remaining monkeys were killed after 4 weeks of recovery (65 days). A subgroup of monkeys that have developed anti-drug antibodies (ADA) directed against humanized Fv of both CD3 and CD123, and data after the occurrence of ADA were excluded from the analysis of PK. All monkeys were exposed to DART-A during the study period ._______________________________________

Table 8

<td rowspan="3">Infusion number</td><td rowspan="3">Study days</td><td rowspan="2">Inert wear</td><td colspan="5">DART-A infusion</td>

<td colspan="4">(4 days of administration / 3 days without administration) ng / kg / day [ng / kg / 4 days]</td><td>(7 days of administration) ng / kg / day [ng / kg / 7days]</td>

<td>Group 1</td><td>Group 2</td><td>Group 3</td><td>Group 4</td><td>Group 5</td><td>Group 6</td>

<td> 1</td><td> 1</td><td>Inert carrier</td><td>Inert carrier</td><td>Inert carrier</td><td>Inert carrier</td><td>Inert carrier</td><td>Inert carrier</td>

<td> 2</td><td> 8</td><td>Inert carrier</td><td> 100 [400]</td><td> 100 [400]</td><td> 100 [400]</td><td> 100 [400]</td><td> 100 [700]</td>

<td> 3</td><td> 15</td><td>Inert carrier</td><td> 100 [400]</td><td> 300 [1200]</td><td> 300 [1200]</td><td> 300 [1200]</td><td> 300 [2100]</td>

<td> 4</td><td> 22</td><td>Inert carrier</td><td> 100 [400]</td><td> 300 [1200]</td><td> 600 [2400]</td><td> 600 [2400]</td><td> 600 [4200]</td>

<td> 5</td><td> 29</td><td>Inert carrier</td><td> 100 [400]</td><td> 300 [1200]</td><td> 600 [2400]</td><td> 1000 [4000]</td><td> 1000 [7000]</td>

<td>Recovery</td><td> 36-65</td><td></td><td></td><td></td><td></td><td></td><td></td>

The two-compartment model was used to evaluate the parameters of the RK (table. 9 and Fig. 28). Τ¹/ 2α was short (4-5 min), reflecting rapid binding to circulating targets; Τ¹/ 2β was also fast as expected for a molecule of a size that is excreted through the kidneys. Analysis of serum samples collected at the end of each infusion from group 6 monkeys showed a dose-dependent increase in C_max DART-A. In the table. 9, the inert carrier was PBS, pH 6.0, containing 0.1 mg / ml recombinant human albumin, 0.1 mg / ml PS-80 and 0.24% benzyl alcohol were used for all inert carrier infusions for the first 4 days each week of infusion, followed by the introduction of the same composition without benzyl alcohol for the remaining 3 days of each weekly infusion. DART-A was administered at the same time points as the continuous intravenous infusion of a PBS solution, pH 6.0, containing 0.1 mg / ml recombinant human albumin, 0.1 mg / ml PS80 and 0.24% benzyl alcohol in the required concentration.

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<td colspan="3">Table 9 Two-compartment analysis of the parameters of the Republic of Kazakhstan DART-A in cynomolgus monkeys</td>

<td>Indicator</td><td>300 ng / kg / day (mean ± SD)</td><td>600 ng / kg / day (mean ± SD)</td>

<td>^FROMtach &lt;^{PG / ML})</td><td> 77,4 ± 9,4</td><td> 113,8 ±33,5</td>

<td>AUC (h * pg / ml)</td><td> 7465 ±913</td><td> 11188 ±3282</td>

<td>U ^ Sl / kg)</td><td> 1,078 ±0,511</td><td> 2,098 ± 1,846</td>

<td>t_1/2alpha (h)</td><td> 0,07 ±0,018</td><td> 0,067 ± 0,023</td>

<td>tl / 2 beta (h)</td><td> 13,79 ±4,928</td><td> 21,828 ± 18,779</td>

<td>MRT (h)</td><td> 6,73 ± 3,327</td><td> 9,604 ±8,891</td>

Cytokine Release in Treated with DART-A-Javanese Macaques

Taking into account the properties of DART-A with respect to T-cell activation, an increase in circulating cytokines was concomitant with the infusion and, therefore, a low initial dose was used as a desensitization strategy based on previous data obtained with similar compounds (see, for example, Torr MS et al. (2011) Targeted Therapy With The TCell-Engaging Antibody Blinatumomab Of Chemotherapy-Refractory Minimal Residual Disease In B-Lineage Acute Lymphoblastic Leukemia Patients Results 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 Of AT Cell-Engaging Antibody Science 321: 974-977). Of the cytokines studied, IL-6 showed the most significant changes during infusion, albeit temporary in nature, of a minimum size and with large values ​​of variability between animals and between groups (Fig. 29A-29C). Small, temporary changes in IL-6 were also observed after infusions of an inert carrier (all infusions in group 1 and all infusions on day 1), which indicates the sensitivity of this cytokine to stress on manipulation. However, serum-dependent DART-A increases (&lt;80 pg / ml) of IL-6 in serum were observed in some monkeys after the first DART-A infusion (100 ng / kg / day), which returned to their original value by 72 hours. Interestingly, the release of IL-6 decreased with each subsequent infusion of DART-A, even if the dose level increased up to 1000 ng / kg / day. Minimum and transient serum TNF-α increases in DART-A were also observed (&lt;10 pg / ml); as with IL-6, the highest TNF-α release was observed after the first infusion. No DART-A-related changes in IL-5, IL-4, IL-2, or IFN-y levels were observed during the entire study compared to controls. It can be concluded that the release of cytokines in response to the treatment of monkeys with DART-A was minimal, temporary, and represented an effect on the first dose, controlled by increasing the dose within one subject.

DART-A-mediated deletion of circulating CD14- / CD123 + leukocytes in vivo

The absolute contents of circulating CD14- / CD123 + cells were measured during the study as a pharmacodynamic end result. Along with the fact that the number of CD123 + cells in control group 1 remained stable over time, treatment with DART-A was associated with an extensive depletion of circulating CD14- / CD123 + cells (94-100% of the initial value before the study), observed starting with the first measured time point (72 h) after the start of the first DART-A infusion (100 ng / kg / day) in all animals in all active treatment groups (Fig. 30A-30C). Depletion was prolonged, as it continued for a 3-day weekly break in the dose in group 2-5, returning to baseline levels only during a prolonged recovery period. To eliminate the possibility of masking DART-A or modulating CD123 (an unlikely scenario giving low levels of circulating DART-A), the amount of pDC was counted using an orthogonal marker, CD303. According to data on CD123, CD303 + pDC were similarly depleted in monkeys treated with DART-A (Fig. 30D-30F).

Contents of circulating T-lymphocytes, activation and analysis of subclasses

In contrast to the stable depletion of circulating CD123 + cells, DART-A, administered according to the 4-day / 3-day regimen without administration (groups 2-5), was associated with weekly fluctuations in the contents of circulating T-cells, with the introduction of continuous 7 -day infusions led to a similar decrease in the content of circulating T cells after the first injection, which slowly recovered without fluctuation even during the period of administration of doses (Figs. 31A-31C). The difference between the two dose strategies indicates that the effect of DART-A on T lymphocytes is consistent with the migration and / or accumulation of cells along the edge of the site of inflammation, and not with depletion. After the cessation of dose administration, the T-cell content unexpectedly rose to levels approximately 2 times higher than the initial values ​​during the recovery period. DART-A infusion was associated with an effect-dependent, progressive, increased frequency of occurrence of T cells expressing the late activation marker, PD-1, especially on CD4 + cells, with dosage group 6 showing the highest total contents (Figs. 31D-31I and Figs. 32A-32F and Fig. 33A

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33F). Tim-3, a marker associated with T cell depletion, was not found on CD4 + T cells and only with a low frequency among CD8 + cells (5.5–9.7%) and was found in 20.5–35.5% CD8 + / PD-1 + double-positive cells. There was no consistent change in the marker for early T cell activation, CD69, and only small changes in the expression of CD25 among circulating cells.

To exclude depletion after in vivo exposure, the ex vivo cytotoxic potential of effector cells isolated from cynomolgus monkeys receiving multiple DART-A infusions was compared with this parameter in previously non-experimental monkeys. As shown in FIG. 34, PBMCs isolated from DART-A treated monkeys showed cytotoxicity comparable to that of cells isolated from previously non-experimental monkeys, indicating that in vivo DART-A did not adversely affect effects on the ability of T cells to destroy target cells.

Exposure to DART-A increased the relative incidence of central CD4 memory cells and effector CD8 + memory cells due to the corresponding previously unaffected T cell population (Figs. 35A-35F and Figs. 32A-32F and Figs. 33A-33F), indicating that exposure to DART-A stimulated the expansion and / or mobilization of these cells.

Effects on hematopoiesis and bone marrow precursors

DART-A was well tolerated by monkeys at all doses studied; however, reversible decreases in red blood cell parameters were observed at the highest doses (Figs. 36A-36C). Rapid blood sampling could make a potential contribution to this, since animals treated with an inert carrier showed a slight decrease in red blood cell count. A reticulocyte response was observed in all animals; at the highest level of exposure (group 6), however, the response was slightly less pronounced for a similar decrease in red blood cell count (Fig. 36D-36F). Morphological analysis of bone marrow smears during the study was unremarkable. Flow cytometry, however, revealed that the incidence of CD123 + cells within the negative for immature cell lines (Lin-) of bone marrow populations decreased in animals treated with DART-A at the end of the dose period, returning to the original values ​​in the end of the recovery period (Fig. 37A-37B). HSC (defined as Lin- / CD34 + / CD38- / CD45RA- / CD90 + cells (Pang, WW et al. (2011) Human Bone Marrow Hematopoietic Stem Cells Are Increased In Frequency And Myeloid-Biased With Age Proc. Natl. Acad Sci. (USA) 108: 20012-20017)) showed greater variability between groups; group 4-6 monkeys treated with DART-A showed some visible decrease compared to the corresponding levels before dosing, however, no decrease was observed in all treated groups compared to animals treated with an inert carrier. These data indicate that HSCs were less sensitive to targeted effects using DART-A and are consistent with the observed reversibility of the negative effects of DART-A treatment on hematopoiesis.

As demonstrated above, for infusions over 4 weeks, according to the weekly schedule of 4 days of administration / 3 days without administration or according to the scheme of 7 days of administration weekly with initial doses of 100 ng / kg / day, which increase in stages every week to 300, 600 and 1000 ng / kg / day, administration of DART-A to cynomolgus monkeys was well tolerated. The deletion of circulating CD123 + cells, including pDC, was observed after the start of the first administration and continued throughout the study at all doses and regimens. A reversible decrease in bone marrow precursor CD123 + was also observed. Cytokine release, as a significant safety risk for CD3-targeted therapies, was correctable and consistent with the effect of the first dose. Slight reversible anemia was observed at the highest doses, but no other adverse effects (associated with the purpose of exposure or not associated with the purpose of exposure) were noted.

Javanese macaque is an acceptable animal model for the pharmacological evaluation of DART-A, given the high degree of homology between orthologs and the ability of DART-A to bind with similar affinity for antigens and to mediate redirected T-cell cytolysis in both species. In addition, both antigens are consistently expressed in monkeys and humans, including similar expression by hematopoietic precursors and in the endothelial cytoplasm of numerous tissues. Minor exceptions are expression in Leydig cells in humans, not monkeys, and low or no CD123 in monkey monocytes compared to humans.

The primary concern associated with therapeutic strategies in which cell activation is involved includes the release of cytokines and non-targeted cytotoxic effects. A recent study with CD3xCD123 bispecific immune construct for fusion of scFv with bivalent recognition of CD3 demonstrated anti-leukemia activity in vitro, but caused nonspecific T-cell activation and IFN-y secretion (Kuo SR et al. (2012) Engineering A CD123xCD3 Bispecific scFv Immunofusion For The Treatment Of Leukemia And Elimination Of Leukemia Stem Cells Protein Eng. Des. Sel. 25: 561-569). The monovalent nature of each of the binding arms and the highly homogeneous form of DART-A ensures that T-cell activation depends solely on the involvement of target cells: T-cell activation is not observed

- 44 034142 was performed in the absence of target cells or when using a control DART molecule, which included only the shoulder of targeted action on CD3. In addition, high doses (up to 100 μg / kg / day) of the control DART molecule did not trigger the release of cytokines in cynomolgus monkeys.

The initial dose of the DART-A molecule, 100 ng / kg / day, was well tolerated, with minimal cytokine release. However, the cytokine storm did begin at a high initial dose (5 mcg / kg / day); however, such a dose could be achieved safely through phased weekly dose increases, indicating that DART-A mediated release of cytokines is likely to be mainly the effect of the first dose. The deletion of CD123 + target cells, thereby eliminating the source of ligation with CD3, may explain the effect of the first dose: an almost complete depletion of CD123 + cells was observed in doses not exceeding 3-10 ng / kg / day, indicating that in vivo release of cytokines there is a biased dose-dependent effect compared with cytotoxicity. Dose-related profiles for cytotoxicity and cytokine release by human T cells are also consistent with the presented observation.

T-cell desensitization, in which DART-A-mediated upregulation of PD1 may play a role, also appears to contribute to limiting the release of cytokines after the first DART-A infusion. Recent studies show that increased PD-1 expression after antigen-induced T-cell retention in areas of inflammation contributes, through interactions with PD-L1, to stop signal termination, thereby releasing and desensitizing cells (Honda, T. et al. (2014) Tuning Of Antigen Sensitivity By T Cell Receptor-Dependent Negative Feedback Controls T Cell Effector Function In Inflamed Tissues, Immunity 40: 235-247; Wei F. et al. (2013) Strength Of PD-1 Signaling Differentially Affects T-Cell Effector Functions, Proc. Natl. Acad Sci. (USA) 110: E2480-E2489). The counteraction of PD-1 TCR signal strength is not informative: while the proliferation and production of cytokines are most sensitive to PD-1 inhibition, the effect on cytotoxicity is minimal (Wei F. et al. (2013) Strength Of PD-1 Signaling Differentially Affects T-Cell Effector Functions, Proc. Natl. Acad. Sci. (USA) 110: E2480E2489). Naturally, the ex vivo cytotoxic potential of T cells from monkeys exposed to multiple DART-A infusions was comparable to that of T cells from monkeys not previously exposed to experiments, despite the elevated PD-1 content in the former. In addition, upregulation of PD-1 was not accompanied by the expression of TIM3, a hallmark of T cell depletion, as shown for T cells subjected to prolonged stimulation with CD3 antibodies or chronic infections (Gebel HM et al. (1989) D Cells From Patients Successfully Treated With OKT3 Do Not React With The T-Cell Receptor Antibody, Hum. Immunol. 26: 123-129; Wherry EJ (2011) T Cell Exhaustion, Nat. Immunol. 12: 492-499).

The deletion of circulating CD123 + cells in monkeys treated with DART-A was quick and lasted for a weekly break in the 4-day / 3-day dose-free schedule, which is consistent with the elimination of the target cells. In contrast, temporary fluctuations in the number of circulating T cells were probably the result of migration from / to tissues and lymphoid organs depending on DART-A. Exposure to DART-A stimulates the expansion and / or mobilization of antigen-exposed T-lymphocytes, cells that are preferably located in tissues and exhibit faster cytotoxic effector function (Mirenda V. et al. (2007) Physiologic And Aberrant Regulation Of Memory T-Cell Trafficking By The Costimulatory Molecule CD28 Blood 109: 2968-2977; Marelli-Berg, FM et al. (2010) Memory T-Cell Trafficking: New Directions For Busy Commuters, Immunology 130: 158-165).

The depletion of CD123 + normal cells may involve potential risks. pDC and basophils express high CD123 contents compared to lower monocyte and eosinophil contents (Lopez AF et al. (1989) Reciprocal Inhibition Of Binding Between Interleukin 3 And Granulocyte-Macrophage Colony-Stimulating Factor To Human Eosinophils Proc. Natl. Acad. Sci. Sci. . (USA) 86: 7022-7026; Munoz, L. et al. (2001) Interleukin-3 Receptor Alpha Chain (CD123) Is Widely Expressed In Hematologic Malignancies Haematologica 86: 1261-1269; Masten BJ et al. (2006) Characterization Of Myeloid And Plasmacytoid Dendritic Cells In Human Lung J. Immunol. 177: 7784-7793; Korpelainen EI 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). PDC has been shown to play a role in the control of certain viruses in infection models in mice or monkeys, although they have not been critical for controlling the immune response in influenza (Colonna M. et al. (1997) Specificity And Function Of Immunoglobulin Superfamily NK Cell Inhibitory And Stimulatory Receptors Immunol. Rev. 155: 127-133; Smit JJ et al. (2006) Plasmacytoid Dendritic Cells Inhibit Pulmonary Immunopathology And Promote Clearance Of Respiratory Syncytial Virus J. Exp. Med. 203: 1153-1159). PDC tumor models can stimulate tumor growth and metastasis, while pDC depletion led to tumor inhibition (Sawant, A. et al. (2012) Depletion Of Plasmacytoid Dendritic Cells Inhibits Tumor Growth And Prevents Bone Metastasis Of

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Breast Cancer Cells J. Immunol. 189: 4258-4265). Temporary, minor, dose-dependent facial edema was observed in some monkeys treated with DART-A; however, these monkeys did not observe elevated histamine levels or when human basophils were lysed by DART-A-mediated T cell lysis. Monocyte depletion may include potential infection risks; the consequence of pDC, the depletion of basophils or eosinophils in humans, therefore, should be monitored.

Commited hematopoietic precursors that express CD123, such as common myeloid precursor (CMP) (Jordan St. et al. (2000) The Interleukin-3 Receptor Alpha Chain Is A Unique Marker For Human Acute Myelogenous Leukemia Stem Cells, Leukemia 14: 1777- 1784; Rieger MA et al. (2012) Hematopoiesis Cold Spring Harb. Perspect. Biol. 4: a008250) can be targeted by DART-A, which is a possible explanation for the slight anemia observed after administration of DART-A at the highest dose. The erythropoietic reticulocytic response is likely to be effective at all dose levels of DART-A; however, in relation to adequate reductions in red blood cell parameters, animals exposed to the highest doses of DART-A (group 6, 7-day infusion) showed a reduced reticulocyte response, indicating a possible cytotoxic activity on precursors (e.g., CMP). The effect was reversible after discontinuation of treatment with DART-A, which is consistent with repopulation from stored HSCs with low or no CD123.

Alternative approaches for the depletion of CD123 + cells include a CD123-specific second generation monoclonal antibody with an enhanced Fc region (Jin, L. et al. (2009) Monoclonal Antibody-Mediated Targeting Of CD123, IL-3 Receptor Alpha Chain, Eliminates Human Acute Myeloid Leukemic Stem Cells Cell Stem Cell 5: 31-42; Roberts, AW et al. (2010) A Phase I Study Of AntiCD123 Monoclonal Antibody (mAb) CSL360 Targeting Leukemia Stem Cells (LSC) In AML J. Clin. Oncol. 28 (Suppl): el3012) associated with IL-3 diphtheria toxin (Frankel, A. et al. (2008) Phase I Clinical Study Of Diphtheria Toxin-Interleukin 3 Fusion Protein In Patients With Acute Myeloid Leukemia And Myelodysplasia Leuk. Lymphoma 49 : 543-553), cytokine-induced (CIK) killer cells expressing CD123 specific chimeric antigen receptors (CAR) (Tettamanti, S. et al. (2013) Targeting Of Acute 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) Efficacy Against Human Acute Myeloid Leukemia And Myeloablation Of Normal Hematopoiesis In A Mouse Model Using Chimeric Antigen Receptor-Modified T Cells Blood 123 (15) : 2343-2354; Mardiros A. et al. (2013) T Cells Expressing CD123-Specific Chimeric Antigen Receptors Exhibit Specific Cytolytic Effector Functions And Antitumor Effects Against Human Acute Myeloid Leukemia Blood 122: 3138-3148). CAR Cells showed pronounced in vitro leukemic blast cell cytolysis and anti-leukemia activity in an xenogenic model of disseminated AML (Mardiros A. et al. (2013) T Cells Expressing CD123-Specific Chimeric Antigen Receptors Exhibit Specific Cytolytic Effector Functions And Antitumor Effects Against Human Acute Myeloid Leukemia Blood 122: 3138-3148). A recent study reported ablation of normal hematopoiesis in NSG mice with transplanted human CD34 + cells after transfer of CD123 CAR T cells (Gill S. et al. (2014) Efficacy Against Human Acute Myeloid Leukemia And Myeloablation Of Normal Hematopoiesis In A Mouse Mo del Using Chimeric Antigen Receptor-Modified T Cells Blood 123 (15): 2343-2354), although other authors have not observed similar effects in vitro or in vivo (Tettamanti S. et al. (2013) Targeting Of Acute Myeloid Leukaemia By Cytokine-Induced Killer Cells Redirected With A Novel CDI23-Specific Chimeric Antigen Receptor Br. J. Haematol. 161: 389-401; Pizzitola,

I. et al. (2014) Chimeric Antigen Receptors Against CD33 / CD123 Antigens Efficiently Target Primary Acute Myeloid Leukemia Cells in vivo Leukemia doi: 10.1038 / leu.2014.62). In the experiments discussed above, the deletion of CD123 + populations of bone marrow precursors was observed, but it was reversible during the recovery period; in addition, the depletion of this small population did not lead to changes in the saturation of bone marrow cells or in the ratio of erythroid cells to myeloid (E: M) for all studied dose levels of DART-A. These differences underline the potential advantages of DART-A over cellular therapies, as it provides a titratable system that is based on autologous T cells as opposed to ultra-efficient ex vivo transduced cells that are more difficult to control. CD123 is overexpressed in some hematologic malignancies, including AML, hairy cell leukemia, plasmacytoid dendritic cell bladder neoplasms (OBPDK), pre-B-cell acute lymphoblastic leukemia (B-ALL) and chronic lymphocytic leukemia, Hodgkin's disease with Reed-Sternberg, as well as myelodysplastic syndrome and systemic mastocytosis (Kharfan-Dabaja, MA et al. (2013) Diagnostic and Therapeutic Advances in Elastic Plasmacytoid Dendritic Cell Neoplasm: A Focus On Hematopoietic Cell Transplantation Biol. Blood Marrow Transplant. 19: 1006-1012; Florian S. et al. (2006) Detection Of Molecular Targets On The Surface Of CD34 + / CD38-Stem Cells In Various My eloid 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 Analysis Of CD123 Is Useful For Immunophenotyping

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Classical Hodgkin Lymphoma Cytometry In Clin. Cytom. 80: 91-99). The predicted pharmacodynamic activity and controlled safety profile observed in nonhuman primates additionally confirms the clinical applicability and effectiveness of DART-A as immunotherapy for these disorders.

In conclusion, it should be noted that DART-A is an antibody-based molecule involving the CD3e TCR subunit to redirect T lymphocytes against cells expressing CD123, an antigen upregulated in some hematologic malignancies. DART-A binds both antigens of both human and cynomolgus macaques with similar affinities and redirects T cells of both species to kill CD123 + cells. Monkeys that were infused 4 or 7 days a week with weekly increasing doses of DARTA showed a depletion of circulating CD123 + cells 72 hours after the start of treatment, which lasted for 4 weeks of treatment, regardless of the dosage regimen. A decrease in circulating T cells also occurred, but was restored to its initial value until the next infusion in monkeys receiving treatment according to the scheme, providing for 4 days of dose administration, which is consistent with DART-A mediated mobilization. Administration of DART-A increased circulating PD1 +, but not TIM-3 +, T cells; in addition, ex vivo analysis of T cells from treated monkeys revealed unchanged redirected lysis of the target cells, indicating no depletion. Toxicity was limited by the minimal temporary release of cytokines after the first DART-A infusion, but not after the next administrations, even if the dose was increased, and by a minimal decrease in the number of red blood cells with a concomitant decrease in CD123 + bone marrow precursors. A clinical trial of DART-A for hematologic malignancies is warranted.

All publications and patents mentioned in the present description of the invention are included in this document to the same extent as if it were provided that each individual publication or patent application is expressly and individually incorporated by reference in its entirety. Along with the fact that the present invention has been described using specific embodiments thereof, it should be understood that further modifications are possible, and it is contemplated that the present application covers any changes, applications, or adaptations of the present invention, following generally the principles of the present invention and including such deviations from the present disclosure that are consistent with known or generally accepted practice in the art, to which the present invention belongs, and which can be applied to the main features set forth hereinabove.