EA040028B1 - BISPECIFIC MONOVALENT DIABODIES WHICH ARE ABLE TO BIND CD19 AND CD3 AND THEIR APPLICATIONS - Google Patents

Description

Link to related application

This patent application claims priority under U.S. Provisional Application Serial No. 62/055695 (filed September 26, 2014, pending), which application is hereby incorporated by reference in its entirety. completeness.

Link to sequence listing

This application includes one or more sequence listings in accordance with 37 C.F.R. 1.821 et seq., which are disclosed on machine-readable media (file named 1301_0116PCT_Sequence_Listing_ST25.txt, created on September 18, 2015 and having a size of 47329 bytes), the file of which is incorporated herein by reference in its entirety.

The field of technology to which the present invention relates

The present invention relates to bispecific monovalent diabodies that contain two polypeptide chains and which have one binding site specific for the CD19 epitope and one binding site specific for the CD3 epitope (i.e., a bispecific monovalent diabody for CD19 x CD3 ). Most preferably, such CD19 x CD3 bispecific monovalent diabodies consist of three polypeptide chains and have one binding site specific for the CD19 epitope and one binding site specific for the CD3 epitope, and additionally contain an immunoglobulin Fc domain ( i.e. bispecific monovalent Fc-diabody to CD19 x CD3). The bispecific monovalent diabodies and the bispecific monovalent Fc diabodies of the present invention are capable of simultaneously binding to CD19 and CD3. The present invention relates to pharmaceutical compositions which contain such bispecific monovalent diabodies or such bispecific monovalent Fc diabodies. The present invention further relates to methods of using such diabodies in the treatment of disease, in particular hematological malignancies.

Background of the Invention

A. CD19.

CD19 (B-lymphocyte surface antigen B4, Genbank accession number M28170) is a 95 kDa type I transmembrane glycoprotein of the immunoglobulin superfamily (Stamenkovic, I. et al. (1988) CD19, The Earliest Differentiation Antigen Of The B Cell Lineage, Bears Three Extracellular Immunoglobulin-Like Domains And An Epstein-Barr Virus-Related Cytoplasmic Tail, J. Exper. Med. 168(3):1205-1210; Tedder, T. F. et al. (1989) Isolation Of cDNAs Encoding The CD19 Antigen Of Human And Mouse B Lymphocytes A New Member Of The Immunoglobulin Superfamily, J Immunol 143(2):712-717 Zhou, L J et al (1991) Structure And Domain Organization Of The CD19 Antigen Of Human, Mouse, And Guinea Pig In Lymphocytes Conservation Of The Extensive Cytoplasmic Domain, J Immunol 147(4):14241432). CD19 is expressed on follicular dendritic cells and on all B cells from early B progenitor cells during heavy chain rearrangement up to the plasma cell stage when CD19 expression is downregulated. CD19 is not expressed on hematopoietic stem cells or on B cells prior to the B cell progenitor stage (Sato et al. (1995) The CD19 Signal Transduction Molecule Is A Response Regulator Of B-Lymphocyte Differentiation, Proc. Natl. Acad. Sci. (U.S.A.) 92:11558-62 Loken et al (1987) Flow Cytometric Analysis of Human Bone Marrow II Normal B Lymphocyte Development, Blood, 70:1316-1324 Wang et al (2012) CD19: A Biomarker For B Cell Development, Lymphoma Diagnosis And Therapy, Exp Hematol and Oncol 1:36).

CD19 is a component of the B cell receptor (BCR) complex and is also a positive regulator of B cell signaling that modulates the threshold for B cell activation and humoral immunity. CD19 interacts with CD21 (CR2, C3d fragment receptor) and CD81 via two C2-type extracellular Ig-like domains, forming the BCR complex with CD225. The intracellular domain of CD19 is involved in intracellular signal transduction cascades with regulation primarily, but not exclusively, of signals downstream of BCR and CD22 (Mei et al. (2012) Rationale of Anti-CD19 Immunotherapy: An Option To Target Autoreactive Plasma Cells In Autoimmunity , Arthritis Res and Ther. 14 (Suppl. 5): S1; Wang et al. (2012) CD19: A Biomarker For B Cell Development, Lymphoma Diagnosis And Therapy, Exp. Hematol. and Oncol. 1:36); Delnagro et al. (2005) CD19 Function in Central and Peripheral B-Cell Development, Immunologic Res. 31:119-131).

Several properties of CD19 support its possible potential as a target for immunotherapy. CD19 is one of the most commonly expressed antigens in the B-cell lineage and is expressed in >95% of B-cell-associated malignancies, including acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), and non-Hodgkin's lymphoma (NHL). ) (Wilson, K. et al. (2010) Flow Minimal Residual Disease Monitoring Of Candidate Leukemic Stem Cells Defined By The Immunophenotype, CD34+CD38lowCD19+ In BLineage Childhood Acute Lymphoblastic Leukemia, Haematologica 95(4):679-683; Maloney, D.G. et al (1997) IDEC-C2B8 (Rituximab) Anti-CD20 Monoclonal Antibody Therapy In Patients With Relapsed LowGrade Non-Hodgkin's Lymphoma, Blood 90(6):2188-2195; Vose, J.M. (1998) Current Approaches To The Management Of Non-Hodgkin's Lymphoma, Semin Oncol 25(4):483-491 Nagorsen, D. et al (2012) Bli-1 040028 natumomab: A Historical Perspective, Pharmacol Ther 136(3):334-342 Topp, M.S. et al (2011) Targeted Therapy With The T-Cell-Engagi ng 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. 2011 Jun 20; 29(18):2493-2498; Nadler, et al. (1983) B4, A Human B Lymphocyte-Associated Antigen Expressed On Normal, Mitogen-Activated, And Malignant B Lymphocytes. J. Immunol.; 131:244-250; Ginaldi et al. (1998) Levels Of Expression Of CD19 And CD20 In Chronic In Cell Leukaemias, J. Clin. Pathol. 51:364-369; Anderson et al. (1984) Expression of Human in Cell-Associated Antigens on Leukemias and Lymphomas: A Model of Human in Cell Differentiation, Blood 63:1424-1433). CD19 is expressed on few, if any, other cell types and is not expressed on terminally differentiated plasma cells, thus being useful in CD19-targeted therapies. CD19 does not enter the circulation and can be rapidly internalized (Ma et al. (2002) Radioimmunotherapy For Model B Cell Malignancies Using 90Y-Labeled Anti-CD19 And Anti-CD20 Monoclonal Antibodies, Leukemia 16:60-66; Raufi et al. (2013 ) Targeting CD19 in B-cell lymphoma: emerging role of SAR3419, Cancer Management Res 3:225-233). Notably, CD19 expression is maintained on cell lymphomas that become resistant to anti-CD20 therapy (Davis et al. (1999) Therapy of B-cell Lymphoma With Anti-CD20 Antibodies Can Result In The Loss Of Cd2o Antigen Expression. Clin Cancer Res 5:611-615, 1999). CD19 has also been proposed as a target for the treatment of autoimmune diseases (Tedder (2009) CD19: A Promising B Cell Target For Rheumatoid Arthritis, Nat. Rev. Rheumatol. 5:572-577).

B-cell malignancies represent a heterogeneous group of disorders with widely varying characteristics and clinical behavior. Historically, most patients with symptomatic disease have received a combination of non-cross-reacting genotoxic agents to achieve long-term remission and, in some cases, cure. Despite their effectiveness, many traditional regimens are also associated with significant acute and long-term toxicities (see, for example, Stock, W. et al. (2013) Dose Intensification Of Daunorubicin And Cytarabine During Treatment Of Adult Acute Lymphoblastic Leukemia: Results Of Cancer And Leukemia Group In Study 19802, Cancer 2013 Jan 1;119(1):90-98). The anti-CD20 monoclonal antibody rituximab is used to treat many B-cell disorders for which it can be used in combination with standard of care treatments or used alone (Fowler et al. (2013) Developing Novel Strategies to Target B-Cell Malignancies , Targeted Pathways, B-Cell Lymphoma in ASCO Educational Book, pp. 366-372 Bargou, R. et al (2008) Tumor Regression In Cancer Patients By Very Low Doses Of A T Cell-Engaging Antibody, Science 321(5891) Thomas, D. A. et al (2010) Chemoimmunotherapy With A Modified Hyper-CVAD And Rituximab Regimen Improves Outcome In De Novo Philadelphia Chromosome-Negative Precursor B-Lineage Acute Lymphoblastic Leukemia, J. Clin Oncol 28(24): 3880-3889). Despite encouraging clinical results, retreatment of patients with slow-growing lymphomas with the single agent rituximab was associated with a response rate of only 40%, indicating that resistance in malignant B cells may occur in response to long-term exposure to rituximab (Smith (2003). ) Rituximab (Monoclonal Anti-CD20 Antibody): Mechanisms Of Action And Resistance Oncogene 22:7359-68 Davis et al (1999) Therapy of B-cell Lymphoma With Anti-CD20 Antibodies Can Result In The Loss Of CD20 Antigen Expression, Clin Cancer Res, 5:611-615, 1999 Gabrilovich, D. et al (2003) Tumor Escape From Immune Response: Mechanisms And Targets Of Activity, Curr. Drug Targets 4(7):525-536). Rituximab-resistant cell lines developed in vitro have been reported to exhibit cross-resistance to many chemotherapeutic agents (Czuczman et al. (2008) Acquirement Of Rituximab Resistance In Lymphoma Cell Lines Is Associated With Both Global CD20 Gene And Protein Down-Regulation Regulated At The Pretranscriptional And Post-transcriptional Levels, Clin Cancer Res. 14:1561-1570; Olejniczak et al. (2008) Acquired Resistance To Rituximab Is Associated With Chemotherapy Resistance Resulting From Decreased Bax And Bak Expression, Clin Cancer Res. 14: 1550-1560).

Additional B-cell lymphoma surface antigens that are targets of therapeutic antibodies include CD19 (Hoelzer (2013) Targeted Therapy With Monoclonal Antibodies In Acute Lymphoblasitic Leukemia, Curr. Opin. Oncol. 25:701-706; Hammer (2012) CD19 As An Attractive Target For Antibody-Based Therapy, mAbs 4:571-577). However, despite high activity against lymphoma associated with complex internalization and intracellular release of free drug, various anti-CD19 antibodies, anti-CD19 binding moiety bispecific antibodies, or antibody-drug conjugates (ADCs) have been reported, with antitumor effects of such anti-CD19 or ADC antibodies were variable, indicating that the specific properties of the antibodies, such as epitope binding, ability to induce CD19 oligomerization, or differences in intracellular signaling, affect their effectiveness (Du et al. (2008) Differential Cellular Internalization Of Anti-CD19 And -CD22 Immunotoxins Results In Different Cytotoxic Activity, Cancer Res. 68:6300-6305; Press et al. (1994) Retention Of B-Cell-Specific Monoclonal Antibodies By Human Lymphoma Cells. Blood, 83:1390- 1397;Szatrowski et al.(2003) Lineage Specific Treatment Of Adult Patients With Acute Lymphobla stic Leukemia In First Re- 2 040028 mission With Anti-B4-Blocked Ricin Or High-Dose Cytarabine: Cancer And Leukemia Group In Study 9311,

Cancer 97:1471-1480; Frankel et al. (2013) Targeting T Cells To Tumor Cells Using Bispecific Antibodies,

Curr. Opin. Chem. Biol. 17:385-392).

Thus, despite improvements in survival for many, the majority of patients with B-cell malignancies continue to relapse after standard chemoimmunotherapy, and more than 15,000 patients in the United States still die each year from B-cell malignancies. Therefore, treatments with novel, non-cross-resistance compounds are needed to further improve patient survival.

B. CD3.

CD3 is a T-cell co-receptor consisting of four separate chains (Wucherpfennig, K.W. 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;Chetty, R. et al.(1994) CD3: Structure, Function, And Role Of Immunostaining In Clinical Practice, J. Pathol.173(4):303307;Guy , C. S. et al (2009) Organization Of Proximal Signal Initiation At The TCR:CD3 Complex, Immunol Rev. 232(1):7-21).

In mammals, the complex contains a CD3y chain, a CD3δ chain, and two CD3ε chains. These chains associate with a molecule known as the T cell receptor (TCR) to generate an activation signal in T lymphocytes (Smith-Garvin, J. E. et al. (2009) T Cell Activation, Annu. Rev. Immunol. 27:591- 619). In the absence of CD3, TCRs do not bind properly and are destroyed (Thomas, S. et al. (2010) Molecular Immunology Lessons From Therapeutic T-Cell Receptor Gene Transfer Immunology 129(2):170-177). It was found that CD3 binds to the membranes of all mature T cells and is virtually absent in another cell type (see Janeway, C.A. et al. (2005) In: Immunobiology: The Immune System In Health And Disease, 6th ed. Garland Science Publishing, NY, pp. 214-216 Sun, Z. J. et al.(2001) Mechanisms Contributing To T Cell Receptor Signaling And Assembly Revealed By The Solution Structure Of An Ectodomain Fragment Of The CD3ε:y Heterodimer Cell 105(7):913-923 ; Kuhns, M. S. et al. (2006) Deconstructing The Form And Function Of The TCR/CD3 Complex Immunity 2006 Feb; 24(2):133-139).

The invariant signal transduction component CD3ε of the T cell receptor (TCR) complex on T cells was used as a target to promote the formation of an immunological synapse between T cells and tumor cells. The cooperative interaction of CD3 and tumor antigen activates T cells that trigger the lysis of tumor cells expressing the tumor antigen (Baeuerle et al. (2011) Bispecific T Cell Engager For Cancer Therapy In: Bispecific Antibodies, Kontermann, RE. (Ed.) Springer-Verlag ; 2011:273-287). This approach provides a global interaction of bispecific antibodies with the T-cell compartment with high specificity for tumor cells and is widely applicable to a wide range of tumor cell surface antigens.

C. Antibodies and other binding molecules.

Antibodies are immunoglobulin molecules capable of specifically binding to a target, such as a carbohydrate, polynucleotide, lipid, polypeptide, etc., via at least one antigen recognition site located in the variable region of the immunoglobulin molecule. As used herein, the term includes not only intact polyclonal or monoclonal antibodies, but also their mutants, naturally occurring variants, fusion proteins containing an antibody portion with an antigen recognition site of the required specificity, humanized antibodies, chimeric antibodies, and any other modified configuration of an immunoglobulin molecule, which contains the antigen recognition site of the required specificity.

The ability of an intact, unmodified antibody (eg, IgG) to bind an antigen epitope depends on the presence of variable domains in the immunoglobulin light and heavy chains (ie, the VL and VH domains, respectively). The interaction of an antibody light chain and an antibody heavy chain, and in particular the interaction of its VL and VH domains, form one of the epitope binding sites in an antibody. In contrast, the scFv construct contains the antibody VL and VH domains on the same polypeptide chain, with the domains separated by a flexible linker of sufficient length to allow the two domains to self-assemble into a functional epitope binding site. If self-assembly of the VL and VH domains is not possible due to insufficient linker length (less than about 12 amino acid residues), two of the scFv constructs interact with each other to form a bivalent molecule in which the VL of one strand associates the VH of the other (as discussed in Marvin et al. (2005) Recombinant Approaches To IgG-Like Bispecific Antibodies Acta Pharmacol Sin 26:649-658).

In addition to their known diagnostic applications, antibodies have been shown to be useful as therapeutic agents. In the past few decades, interest in the therapeutic potential of antibodies has revived, and antibodies have become one of the leading classes of biotechnologically produced drugs (Chan, C.E. et al. (2009) The Use Of Antibodies In The Treatment Of Infectious Diseases Singapore Med. J 50(7):663-666). Nearly 200 antibody-based drugs have been approved or are in development.

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Natural antibodies are only able to bind to one kind of epitope (ie, monospecific), although they can bind multiple copies of that kind (ie, exhibit bivalence or multivalence). A wide range of recombinant bispecific antibody formats have been developed (see, for example, PCT Publication Nos. WO 2008/003116, WO 2009/132876, WO 2008/003103, WO 2007/146968), most of which use linker peptides either to merge the antibody against the nuclear antigen (IgA, IgD, IgE, IgG or IgM) with an additional binding protein (eg scFv), or to merge, for example, two Fab fragments or scFv. As a rule, such approaches involve trade-offs and alternatives. For example, PCT Publication Nos. WO 2013/174873, WO 2011/133886 and WO 2010/136172 disclose that the use of linkers can cause problems in therapeutic plans and describe a trispecific antibody in which the CL and CH1 domains are switched from their respective natural positions, and the VL and VH domains have been diversified (WO 2008/027236; WO 2010/108127) to allow them to bind to more than one antigen. Thus, the molecules disclosed in these documents change the binding specificity to the ability to bind an additional kind of antigen. PCT Publication Nos. WO 2013/163427 and WO 2013/119903 disclose a modification of the CH2 domain containing a fusion protein adduct comprising a binding domain. The document notes that the CH2 domain probably plays only a minimal role in mediating effector function. PCT Publication Nos. WO 2010/028797, WO 2010028796 and WO 2010/028795 disclose recombinant antibodies in which the Fc regions have been replaced with additional VL and VH domains to form trivalent binding molecules. PCT Publication Nos. WO 2003/025018 and WO 2003012069 disclose recombinant diabodies in which individual strands contain scFv domains. PCT Publication No. WO 2013/006544 discloses multivalent Fab molecules that are synthesized as a single polypeptide chain and then subjected to proteolysis to obtain heterodimeric structures. Thus, the molecules disclosed in these documents change all or some of the ability to mediate an effector function to the ability to bind an additional kind of antigen. PCT Publication Nos. WO 2014/022540, WO 2013/003652, WO 2012/162583, WO 2012/156430, WO 2011/086091, WO 2007/075270, WO 1998/002463, WO 1992/030349 1 and additional binding domains or functional groups in an antibody or antibody portion (e.g., adding a diabody to an antibody light chain, or adding additional VL and VH domains to an antibody light and heavy chain, or adding a heterologous fusion protein, or linking multiple Fab domains to each other ). Thus, the molecules disclosed in these documents change the native structure of an antibody to the ability to bind an additional kind of antigen.

In addition, the ability to produce diabodies that differ from such natural antibodies by the ability to bind two or more different kinds of epitope (i.e. exhibit bispecificity or multispecificity in addition to bivalence or multivalence) has been noted in the art (see, for example, Holliger et al (1993) Diabodies: Small Bivalent And Bispecific Antibody Fragments, Proc Natl Acad Sci (U.S.A.) 90:6444-6448 US 2004/0058400 (Hollinger et al) US 2004/0220388 (Mertens et al.) Lu, D. et al (2005) A Fully Human Recombinant IgG-Like Bispecific Antibody To Both The Epidermal Growth Factor Receptor And The InsulinLike Growth Factor Receptor For Enhanced Antitumor Activity, J Biol Chem 280(20):19665-19672, WO 02/02781 (Mertens et al.), Olafsen, T. et al (2004) CEA Diabody Allows SiteSpecific 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, P.A. et al. (2009) Bispecific T-Cell Engaging Antibodies For Cancer Therapy Cancer Res. 69(12):4941-4944).

The design of the diabody is based on single chain fragments of the variable region (scFv). Such molecules are obtained by linking the variable regions of the light and/or heavy chain using a short binding peptide. Bird et al. (1988) (Single-Chain Antigen-Binding Proteins Science 242:423-426) disclose an example of binding peptides that are an approximately 3.5 nm bridge between the carboxy terminus of one variable region and the amino terminus of another variable region. Other sequence linkers have been designed and used (Bird et al. (1988) Single-Chain Antigen-Binding Proteins, Science 242:423-426). Linkers, in turn, can be modified for additional functions such as drug attachment or attachment to solid supports. Single chain variants can be produced either recombinantly or synthetically. For synthetic production of scFv, an automatic synthesizer can be used. For recombinant scFv production, a suitable plasmid containing a polynucleotide that encodes an scFv can be introduced into a suitable host cell, either eukaryotic, such as yeast, plant, insect, or mammalian cells, or prokaryotic, such as E. coli. Polynucleotides encoding the scFv of interest,

- 4 040028 can be obtained by routine manipulations such as ligation of polynucleotides. The resulting scFv can be isolated using standard protein purification techniques known in the art.

United States Patent No. 7585952 and United States Patent Document Publication No. 2010-0173978 relate to scFv molecules that are immunospecific for ErbB2. Bispecific T cell activators (BiTE), a type of scFv molecule, have been described (WO 05/061547; Baeuerle, P et al. (2008) BiTE: A New Class Of Antibodies That Recruit T Cells Drugs of the Future 33:137-147; Bargou, et al 2008 Tumor Regression in Cancer Patients by Very Low Doses of a T Cell-Engaging Antibody, Science 321: 974-977. Such molecules consist of a single polypeptide chain molecule having two antigen-binding domains, one of which binds immunospecifically to a CD3 epitope and the second of which binds immunospecifically to an antigen present on the surface of the target cell.

Bispecific molecules have been described that target both CD19 and CD3. Blinatumomab, bispecific for scFv-CD19 x CD3 BiTE, is in clinical trials and is reported to have a CD3 affinity of ~100 nM and a CD19 affinity of ~1 nM. Blinatumomab demonstrates an EC ₅₀ for in vitro target cell lysis of ~10-100 pg/mL (Frankel et al. (2013) Targeting T Cells To Tumor Cells Using Bispecific Antibodies, Curr. Opin. Chem. Biol. 17:385-392) . Dual-affinity CD19 x CD3 bispecific retargeting antibodies (DART) have also been developed (Moore 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). Compared to the bispecific scFv-CD19 x CD3 BiTE, this molecule exhibited similar binding affinities but had a lower EC ₅₀ for in vitro target cell lysis of ~0.5-5 pg/mL and also exhibited consistently higher levels of maximal lysis AFM11 has also been described as bispecific for CD19 x CD3 Tandab. AFM11 has been reported to have a lower _EC50 for in vitro target cell lysis of ~0.5-5 pg/mL (Zhukovsky et al. (2013) A CD19/CD3 Bispecific Tandab, AFM11, Recruits T Cells To Potently and Safely Kill CD19+ Tumor Cells, J Clin Oncol 31, 2013 (suppl; abstr 3068)). None of these constructs includes an Fc domain.

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 in higher yield and with higher activity than non-optimized diabodies. The present invention thus addresses the problem of providing polypeptides that are specifically designed and optimized for the formation of heterodimeric diabodies. The present invention solves this problem by providing exemplary optimized anti-CD19 x CD3 diabodies.

Brief summary of the present invention

The present invention relates to bispecific monovalent diabodies that contain two polypeptide chains and which have one binding site specific for the CD19 epitope and one binding site specific for the CD3 epitope (i.e., a bispecific monovalent diabody for CD19 x CD3 ). Most preferably, such CD19 x CD3 bispecific monovalent diabodies consist of three polypeptide chains and have one binding site specific for the CD19 epitope and one binding site specific for the CD3 epitope, and additionally contain an immunoglobulin Fc domain ( i.e. bispecific monovalent Fc-diabody to CD19 x CD3). The bispecific monovalent diabodies and the bispecific monovalent Fc diabodies of the present invention are capable of simultaneously binding to CD19 and CD3. The present invention relates to pharmaceutical compositions which contain such bispecific monovalent diabodies or such bispecific monovalent Fc diabodies. The present invention further relates to methods of using such diabodies in the treatment of disease, in particular hematological malignancies.

The anti-CD19 x CD3 bispecific monovalent diabodies and the bispecific Fc monovalent diabodies of the present invention thus comprise at least two different polypeptide chains. The polypeptide chains of such diabodies associate with each other in a heterodimeric manner to form one binding site specific for the CD19 epitope and one binding site specific for the CD3 epitope. The anti-CD19 x CD3 bispecific monovalent diabody or Fc bispecific monovalent diabody according to the present invention is thus monovalent in that it is able to bind only one copy of the CD19 epitope and only one copy of the CD3 epitope, but also bispecific in that one diabody able to bind simultaneously to the CD19 epitope and to the CD3 epitope. Each of the diabody polypeptide chains is covalently linked to the other diabody polypeptide chain, for example, via a disulfide bond of cysteine residues located in such polypeptide chains, thereby forming a covalently linked complex. In specific embodiments, the diabodies of the present invention further have an immunoglobulin Fc domain and/or an albumin binding domain to extend the in vivo half-life.

In more detail, the present invention relates to a bispecific monovalent CD19 x CD3 Fc diabody capable of specific binding to CD19 and CD3, wherein the diabody contains a first, second and third polypeptide chain, wherein the polypeptide chains form a covalently linked complex, and wherein:

I) the first polypeptide chain contains in the direction from the N-terminus to the C-terminus:

A) an IA domain containing:

(1) a subdomain (IA1) that contains a VL domain capable of binding to either CD19 (VL _CD19 ) or CD3 (VLcd3); and (2) a subdomain (IA2) that contains a VH domain capable of binding to either CD19 (VH _CD19 ) or CD3 (VHcd3);

while the subdomains IA1 and IA2 are separated from each other by a polypeptide linker and are either (a) VLcdi9 and VHcd3; or ^(b) VLCD3 ^and VHCD19;

B) an IB domain containing a charged heterodimer-promoter domain, wherein the IB domain is separated from the IA domain by a polypeptide linker;

C) an IC domain containing the CH2-CH3 domain of the antibody; And

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

A) an IIA domain containing:

(1) a subdomain (IIA1) that contains a VL domain capable of binding to either CD19 (VL _CD19 ) or Cd3 (VL _cd3 ); and (2) a subdomain (IIA2) that contains a VH domain capable of binding to either CD19 (VH _CD19 ) or CD3 (VHcd3);

while the subdomains IIA1 and IIA2 are separated from each other by a polypeptide linker and are:

(a) VL _cd19 and VH _cd3 if the subdomains of IA1 and IA2 are VL _cd3 and VH _cd19 ; or (b) VL _cd3 and VH _CD19 if the subdomains of IA1 and IA2 are VL _CD19 and VH _cd3 ;

B) an IIB domain containing a charged heterodimer-promoter domain, wherein the IIB domain is separated from the IIA domain by a polypeptide linker, and wherein the charged heterodimer-promoter domain of the IB domain and the charged heterodimer-promoter domain of the IIB domain have opposite charges; And

iii) the third polypeptide chain contains in the direction from the N-terminus to the C-terminus the IIIC domain, which contains the CH2-CH3 domain of the antibody;

wherein the _CD19 VL and _CD19 VH domains form a CD19-binding domain, and the _cd3 VL and _cd3 VH domains form a CD3-binding domain; and the CH2-CH3 domains of the first and third polypeptide chains form an Fc domain capable of binding to the Fc receptor, thereby forming a CD19 x CD3 bispecific monovalent diabody.

The present invention further relates to an embodiment of the aforementioned anti-CD19 x CD3 bispecific monovalent Fc diabody, wherein:

(A) each of the IB and IIB domains contains a cysteine residue that covalently links the first polypeptide chain to the second polypeptide chain via a disulfide bond; and (B) each of the IC and IIIC domains contains a cysteine residue that covalently links the first polypeptide chain to the third polypeptide chain via a disulfide bond.

The present invention further relates to an embodiment of any of the aforementioned anti-CD19 x CD3 bispecific monovalent Fc diabodies, wherein _CD19 VL has the amino acid sequence of SEQ ID NO: 17 and _CD19 VH has the amino acid sequence of SEQ ID NO: 21.

The present invention further relates to an embodiment of any of the aforementioned bispecific anti-CD19 x CD3 monovalent Fc diabodies, wherein the _cd3 VL has the amino acid sequence of SEQ ID NO: 25 and the _cd3 VH has the amino acid sequence of SEQ ID NO: 29.

The present invention further relates to an embodiment of any of the aforementioned bispecific anti-CD19 x CD3 monovalent Fc diabodies wherein the CH2-CH3 domain of the IC domain has the amino acid sequence of SEQ ID NO: 15 and the CH2-CH3 domain of the IIIC domain has the amino acid sequence SEQ ID NO: 16.

The present invention further provides an embodiment of any of the aforementioned anti-CD19 x CD3 bispecific monovalent Fc diabodies wherein:

(A) the charged heterodimer-promoter domain of domain IB has the amino acid sequence of SEQ ID NO: 10, and the charged heterodimer-promoter domain of domain IIB has the amino acid sequence of SEQ ID NO: 11; or (B) the charged heterodimer-promoter domain of the IB domain has an amino acid sequence

- 6 040028 the identity of SEQ ID NO: 12, and the charged heterodimer-promoter domain of domain IIB has the amino acid sequence of SEQ ID NO: 13.

The present invention further provides an embodiment of any of the aforementioned anti-CD19 x CD3 bispecific monovalent Fc diabodies wherein:

(A) the first polypeptide chain has the amino acid sequence of SEQ ID NO: 35;

(B) the second polypeptide chain has the amino acid sequence of SEQ ID NO: 37; and (C) the third polypeptide chain has the amino acid sequence of SEQ ID NO: 39.

The present invention further relates to an embodiment of any of the aforementioned bispecific anti-CD19 x CD3 monovalent Fc diabodies that is capable of cross-reacting with both human and primate CD19 and CD3.

The present invention further relates to any of the anti-CD19 x CD3 bispecific monovalent Fc diabodies described above for use as a pharmaceutical agent.

The present invention further provides any of the anti-CD19 x CD3 bispecific monovalent Fc diabodies described above for use in the treatment of a disease or condition associated with or characterized by CD19 expression, or in a method of treating a disease or condition characterized by CD19 expression, in in particular, if the disease or condition associated with or characterized by CD19 expression is a cancer, and more specifically, if the cancer is selected from the group consisting of acute myeloid leukemia (AML), chronic myelogenous leukemia (CML), including CML blast crisis and CML-associated Abelson oncogene (Bcr-ABL translocation), myelodysplastic syndrome (MDS), acute B-cell lymphoblastic leukemia (B-ALL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, chronic lymphocytic leukemia ( CLL), including Richter's syndrome or Richter's transformation CLL, vol squamous cell leukemia (HCL), blast plasmacytoid dendritic cell neoplasms (BPDCN), non-Hodgkin's lymphomas (NHL), including mantle cell leukemia (MCL) and small cell lymphocytic lymphoma (SLL), Hodgkin's lymphoma, systemic mastocytosis, and Burkitt's lymphoma.

The present invention further relates to a covalently linked polypeptide complex, wherein the polypeptide complex comprises a first polypeptide chain and a second polypeptide chain, wherein:

(A) the first polypeptide chain contains a polypeptide with the amino acid sequence of SEQ ID NO: 2 associated with a heterodimer-promoter domain with the amino acid sequence of SEQ ID NO: 12; and (B) the second polypeptide chain comprises a polypeptide with the amino acid sequence of SEQ ID NO: 2 linked to a heterodimer promoter domain with the amino acid sequence of SEQ ID NO: 13;

wherein the heterodimer promoter domains of the first polypeptide chain and the heterodimer promoter domains of the second polypeptide chain are covalently linked to each other via a disulfide bond.

The present invention further provides a pharmaceutical composition comprising any of the anti-CD19 x CD3 bispecific monovalent Fc diabodies described above and a physiologically acceptable carrier. The present invention further relates to the use of such a pharmaceutical composition in the treatment of a disease or condition associated with or characterized by CD19 expression, in particular where the disease or condition associated with or characterized by CD19 expression is a malignant tumor, and more specifically, if the malignant tumor is selected from the group consisting of acute myeloid leukemia (AML), chronic myelogenous leukemia (CML), including CML blast crisis and CML-associated Abelson oncogene (Bcr-ABL translocation), myelodysplastic syndrome (MDS), acute B-cell lymphoblastic leukemia (B-ALL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, chronic lymphocytic leukemia (CLL), including Richter's syndrome or Richter's transformation CLL, hairy cell leukemia (HCL), blast neoplasms plasmacytoid dendritic cells (BPDCN), non-Hodgkin's lymphomas (NHL), in including mantle cell leukemia (MCL) and small cell lymphocytic lymphoma (SLL), Hodgkin's lymphoma, systemic mastocytosis and Burkitt's lymphoma.

The CD19 x CD3 bispecific monovalent diabodies and CD19 x CD3 bispecific monovalent Fc diabodies of the present invention are preferably capable of exhibiting a similar binding affinity for human or cynomolgus monkey soluble CD3 and a 10-fold difference in binding to CD19 as analyzed by surface plasmon resonance technology ( SPR) (BIAcore) or as analyzed by flow cytometry using a population of gated as CD4+ and CD8+ T cells and a population of gated as CD20+ B cells.

The CD19 x CD3 bispecific monovalent diabodies and CD19 x CD3 bispecific monovalent Fc diabodies of the present invention are preferably capable of mediating redirected killing of target tumor cells using human T cells in an assay using any of the 3 target lines B- cellular lymphoma, Raji/GF (Burkitt's lymphoma), HBL-2 (mantle cell lymphoma) or Jeko-1 (mantle cell lymphoma), as well as using purified human primary T cells as effector cells. In such an assay, the killing of target tumor cells is measured by the lactate dehydrogenase (LDH) release assay, in which the enzymatic activity of LDH released from cells as a result of cell death is quantified, or by the luciferase assay, in which the relative light unit (RLU) of luciferase is an indicator for determining the relative viability of Raji/GF target cells that have been engineered to express both green fluorescent protein (GFP) and luciferase genes. The observed _EC50 of such redirected killing is approximately 5 pM or less, approximately 3 pM or less, approximately 1 pM or less, approximately 0.5 pM or less, approximately 0.3 pM or less, approximately 0.2 pM or less, approximately 0.1 pM or less, about 0.05 pM or less, about 0.04 pM or less, about 0.03 pM or less, about 0.02 pM or less, or about 0.01 pM or less.

The CD19 x CD3 bispecific monovalent diabodies and CD19 x CD3 bispecific monovalent Fc diabodies of the present invention are preferably capable of mediating cytotoxicity against human and cynomolgus monkey peripheral blood mononuclear cells (PBMCs) to cause dose-dependent depletion of CD20+ B cells in both of these cell systems at ratios of effector cells to T cells of 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, or 9:1. The observed EC ₅₀ of human CD20+ B cell depletion is about 7 pM or less, about 5 pM or less, about 3 pM or less, about 1 pM or less, about 0.5 pM or less, about 0.3 pM or less, about 0.2 pM or less, about 0.1 pM or less, about 0.05 pM or less, about 0.04 pM or less, about 0.03 pM or less, about 0.02 pM or less, or about 0.01 pM or less. The observed EC ₅₀ of CD20+ B cell depletion in cynomolgus monkeys is approximately 1000 pM or less, approximately 900 pM or less, approximately 800 pM or less, approximately 500 pM or less, approximately 300 pM or less, approximately 100 pM or less, approximately 50 pM or less, about 20 pM or less, about 10 pM or less, about 5 pM or less, about 2 pM or less, or about 1 pM or less, so that the ratio of autologous human B cell depletion to cynomolgus B cell depletion is about 500:1 or less, about 300:1 or less, about 100:1 or less, about 50:1 or less, about 20:1 or less, or about 10:1 or less.

The CD19 x CD3 bispecific monovalent diabodies and CD19 x CD3 bispecific monovalent Fc diabodies of the present invention are preferably capable of mediating the release of cytokines (e.g., IFN-γ, TNF-α, IL-2, IL-4, IL-6 and IL- 10, and especially IFN-γ and TNF-α) from human and cynomolgus PBMCs. This release is about 5000 pg/mL or less, about 4000 pg/mL or less, about 3000 pg/mL or less, about 2000 pg/mL or less, about 1000 pg/mL or less, about 500 pg/mL or less , about 2000 pg/ml or less, or about 100 pg/ml or less. The average EC ₅₀ of such cytokine release from human PBMC is about 100 pM or less, about 90 pM or less, about 80 pM or less, about 50 pM or less, about 30 pM or less, about 20 pM or less, about 10 pM or less, or about 5 pM or less. The average EC50 of this cytokine release from cynomolgus monkey PBMC is about 3000 pM or less, about 2000 pM or less, about 1000 pM or less, about 500 pM or less, about 300 pM or less, about 200 pM or less, about 100 pM or less, or about 50 pM or less.

The CD19 x CD3 bispecific monovalent diabodies and CD19 x CD3 bispecific monovalent Fc diabodies of the present invention are preferably capable of mediating inhibition of human B-cell lymphoma tumor growth in a co-mixed xenograft in which such molecules are administered to NOD/SCID mice together with HBL tumor cells. -2 (lymphomas from human mantle zone cells) or Raji (Burkitt's lymphomas) and activated human T cells at a ratio of 1:5. Preferably, such diabodies are capable of inhibiting tumor growth when provided at a concentration greater than 100 μg/mL, at a concentration of approximately 100 μg/kg, at a concentration of approximately 80 μg/kg, at a concentration of approximately 50 μg/kg, at a concentration of approximately 20 μg/kg , at a concentration of approximately 10 mcg/kg, at a concentration of approximately 5 mcg/kg, at a concentration of approximately 2 mcg/kg, at a concentration of approximately 1 mcg/kg, at a concentration of approximately 0.5 mcg/kg, at a concentration of approximately 0.2 mcg /kg, at a concentration of approximately 0.1 μg/kg, at a concentration of approximately 0.05 μg/kg, at a concentration of approximately 0.02 μg/kg, at a concentration of approximately 0.01 μg/kg, or at a concentration approximately 0.005 mcg/kg, or at a concentration less than 0.005 mcg/kg.

The CD19 x CD3 bispecific monovalent diabodies and the CD19 x CD3 bispecific monovalent Fc diabodies of the present invention are preferably capable of exhibiting antitumor activity in an HBL-2 (human mantle cell lymphoma) xenograft model in female NSG B2m -/- mice with implanted tumor cells. HBL-2 cells intradermally (ID) on day 0 followed by intraperitoneal (IP) injection of PBMC on day 4 and with diabody administration on day 17 to cause a decrease in tumor volume of about 10%, about 20%, about 40%, about 60 %, about 80%, about 90% or more than 90%.

The CD19 x CD3 bispecific monovalent diabodies and CD19 x CD3 bispecific monovalent Fc diabodies of the present invention are preferably capable of exhibiting prolonged half-lives when administered to a human or non-human recipient. Such half-lives can be measured by injecting a diabody into a humFcRn transgenic mouse (United States Patent Nos. 6992234 and 7358416; Haraya, K. (2014) Application Of Human FcRn Transgenic Mice As A Pharmacokinetic Screening Tool Of Monoclonal Antibody, Xenobiotica 2014 Jul 17 :1-8; Proetzel, G. et al. (2014) Humanized FcRn mouse models for evaluating pharmacokinetics of human IgG antibodies, Methods 65(1):148-153; Stein, C. et al. (2012) Clinical Chemistry Of Roopenian, DC et al (2010) Human FcRn Transgenic Mice For Pharmacokinetic Evaluation Of Therapeutic Antibodies, Methods Molec Biol 602:93-104) . The measurement is performed using a B6.Cg-Fcgrt ^tmlDcr (CAGFCGRT)276Dcr/DcrJ mouse (accession number 004919) available from Jackson Laboratories, Bar Harbor, Maine, US.

For the avoidance of doubt, the diabodies of the present invention may exhibit one, two, three, more than three, or all of the functional features described herein. Thus, diabodies in accordance with the present invention may exhibit any combination of the functional features described herein.

The diabody according to the present invention can be used as a pharmaceutical agent. Preferably, diabodies are used in the treatment of a disease or condition associated with or characterized by CD19 expression. The present invention also relates to the use of a diabody according to the present invention in the manufacture of a pharmaceutical composition, preferably for the treatment of a disease or condition associated with or characterized by CD19 expression, as further defined herein.

The disease or condition associated with or characterized by CD19 expression may be B-cell malignancy (Campo, E. et al. (2011) The 2008 WHO Classification Of Lymphoid Neoplasms And Beyond: Evolving Concepts And Practical Applications, Blood 117(19 ):5019-5032)). For example, the cancer may be selected from the group consisting of acute myeloid leukemia (AML), chronic myelogenous leukemia (CML), including CML blast crisis and CML-associated Abelson oncogene (Bcr-ABL translocation), myelodysplastic syndrome (MDS). ), acute B-cell lymphoblastic leukemia (B-ALL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, chronic lymphocytic leukemia (CLL), including Richter's syndrome or Richter's transformation CLL, hairy cell leukemia (HCL), neoplasms from blast plasmacytoid dendritic cells (BPDCN), non-Hodgkin's lymphomas (NHL), including mantle cell leukemia (MCL) and small cell lymphocytic lymphoma (SLL), Hodgkin's lymphoma, systemic mastocytosis and Burkitt's lymphoma.

The present invention further relates to a pharmaceutical composition comprising any of the diabodies described above and a physiologically acceptable carrier. The present invention particularly relates to such a pharmaceutical composition for treating a disease or condition that is refractory to treatment with rituximab.

The present invention further relates to the use of a pharmaceutical composition as described above in the treatment of a disease or condition associated with or characterized by CD19 expression.

The present invention particularly relates to an embodiment of such use wherein the disease or condition associated with or characterized by CD19 expression is a B-cell malignancy (specifically, a malignancy selected from the group consisting of acute myeloid leukemia ( AML), chronic myelogenous leukemia (CML), including CML blast crisis and CML-associated Abelson oncogene (Bcr-ABL translocation), myelodysplastic syndrome (MDS), acute B-cell lymphoblastic leukemia (B-ALL), diffuse large cell B-cell lymphoma (DLBCL), follicular lymphoma, chronic lymphocytic leukemia (CLL), including Richter's syndrome or Richter's transformation CLL, hairy cell leukemia (HCL), neoplasms from blast plasmacytoid dendritic

- 9 040028 cells (BPDCN), non-Hodgkin's lymphomas (NHL), including mantle cell leukemia (MCL) and small cell lymphocytic lymphoma (SLL), Hodgkin's lymphoma, systemic mastocytosis and Burkitt's lymphoma).

Terms such as approximately should be understood to mean a 10% limit, more preferably a 5% limit of a certain value, unless the context dictates otherwise.

Brief description of graphic materials

In FIG. 1 illustrates the structure of a covalently linked bispecific monovalent diabody consisting of two polypeptide chains.

In FIG. 2A and 2B illustrate the structures of two versions of the first, second and third polypeptide chains of the three-chain bispecific anti-CD19 x CD3 monovalent Fc diabody according to the present invention (version 1, FIG. 2A; version 2, FIG. 2B).

In FIG. 3 shows the simultaneous interaction of DART-A with both CD19 and CD3 (•). Neither the control DART 1 () nor the control DART 2 (▲) are able to bind to both targets at the same time.

In FIG. 4A-4B show the binding of an anti-CD19 x CD3 bispecific molecule (DART-A) to human CD20+ B cells (FIG. 4A) and cynomolgus monkey (FIG. 4B).

In FIG. 5A-5B show the binding of an anti-CD19 x CD3 bispecific molecule (DART-A) to human CD4+ and CD8+ T cells (FIG. 5A) and cynomolgus monkey (FIG. 5B).

In FIG. 6A-6F show CD19 x CD3 bispecific molecule (DART-A) redirected killing of target cells compared to control DART 1. Dose-response curves of DART-A mediated cytotoxicity using primary human T cells as effector cells for target cells. lines: HBL-2 (Fig. 6A), Raji/GF (green fluorescent) (Fig. 6B), Jeko-1 (Fig. 6C), Molm-13 (Fig. 6D) and Colo205/Luc (Fig. 6F) with target cell killing as measured using LDH assay. In FIG. 6E shows Raji/GF cell killing determined using a luciferase assay (RLU) to measure relative cell viability. EC ₅₀ values for DART-A are shown in each graph for 1 typical experiment. DART-A: ▲; control DART 1: ▼.

In FIG. 7A-7D show CD19 x CD3 bispecific molecule (DART-A) mediated redirected killing of Raji/GF target cells at varying E:T cell ratios (10:1 (•), 5:1 () and 2.5:1 (▲)). Dose-response curves of DART-A mediated cytotoxicity (percentage cytotoxicity) with primary human T cells as effector cells for CD19+ target Raji/GF cells determined after 24 hours of incubation (Fig. 7A) and after 48 hours of incubation ( Fig. 7B). Dose-response curves of DART-A mediated cytotoxicity (percentage cytotoxicity) with primary human T cells as effector cells for CD19-negative JIMT-1 cells determined after 24 h incubation (Fig. 7C) and after 48 h incubation (Fig. .7D) were included as controls. Shown are 1 of 3 representative experiments, each using T cells from independent donors.

In FIG. 8A-8D show CD19 x CD3 bispecific molecule (DART-A) mediated redirected killing of target Raji/GF cells at lower E:T cell ratios of 2.5:1 (•) or 1:1 (). Dose-response curves of DART-A mediated cytotoxicity (percentage cytotoxicity) with primary human T cells as effector cells for CD19+ target Raji/GF cells determined after 72 hours of incubation (Fig. 8A) and after 96 hours of incubation ( Fig. 8B). Dose-response curves of DART-A mediated cytotoxicity (percentage cytotoxicity) with primary human T cells as effector cells for CD19-negative JIMT-1 cells determined after 72 hours of incubation (Fig. 8C) and after 96 hours of incubation (FIG. 8D) were included as controls. Shown are 1 of 3 representative experiments, each using T cells from independent donors.

In FIG. 9A-9B show CD19 x CD3 bispecific molecule (DART-A) mediated depletion of autologous B cells in human or cynomolgus PBMC. Dose-response curves are presented for DART-mediated (DART-A or control DART 1) depletion of autologous B cells. Fig. 9A: Percentage of human CD20+ cells after PBMC treatment with various doses of DART-A or control DART 1. FIG. 9B: Percentage of CD20+ cynomolgus monkey cells after PBMC treatment with various doses of DART-A or control DART 1. Each graph shows EC ₅₀ values for DART-A. The percentage of CD3+ cells was analyzed in parallel and used as an internal reference control, no change was observed at any tested concentration. DARTA: ▲; control DART 1: ▼.

In FIG. 10 shows CD19 x CD3 bispecific molecule (DART-A) mediated redirected killing of cynomolgus monkey PBMC target cells at E:T=30:1. DART-A: ▲; control DART 1: ▼.

In FIG. 11A-11C show CD19 x CD3 bispecific molecule (DART-A) mediated cytokine release from human T cells in the presence of target cells. Human T cells were treated with DART-A or control DART 1 for approximately 24 hours in the presence of

- 10 040028 Raji/GF target cells. Culture supernatants were collected and subjected to ELISA-based cytokine measurement. Shown is one typical experiment out of 2 using different donors.

Fig. 11A: release of γ-interferon (IFNy); fig. 11B: tumor necrosis factor-α (TNF-α) release; fig. 11C: release of interleukin-2 (IL-2). DART-A: ▲; control DART 1: ▼.

In FIG. 12A-12D show human T cell activation during redirected cell killing with an anti-CD19 x CD3 bispecific molecule (DART-A) compared to control DART 1. Dose-response curves of DART-A mediated induction of CD25 T-cell activation markers (Fig. 12A-12B) and CD69 (FIGS. 12C-12D) in CD8+ (FIGS. 12A and 12C) and CD4+ (FIGS. 12B and 12D) human T cells using Raji/GF target cells. Shown is one typical experiment out of 2, each using T cells from different donors. DART-A: ▲; control DART 1: ▼.

In FIG. 13A-13D show cynomolgus monkey T cell activation during redirected cell killing with an anti-CD19 x CD3 bispecific molecule (DART-A) compared to control DART 1. Dose-response curves of DART-A mediated induction of CD25 T-cell activation markers (FIG. 13A and 13B) and CD69 (FIGS. 13C and 13D) in CD8+ (FIGS. 13A and 13C) and CD4+ (FIGS. 13B and 13D) cynomolgus monkey T cells or PBMCs using Raji/GF target cells. One typical experiment using T cells is shown. DART-A: ▲; control DART 1: ▼.

In FIG. 14A-14D show that the CD19 x CD3 bispecific molecule (DART-A) and the control DART 1 are unable to mediate human T cell activation in the presence of CD19 negative JIMT-1 cells. Shown is a dose response curve of DART-A mediated induction of T cell activation markers CD25 (FIGS. 14A and 14B) and CD69 (FIGS. 14C and 14D) in CD8+ (FIGS. 14A and 14C) and CD4+ (FIGS. 14B and 14D). from the same donor as in Fig. 12A-12D. DART-A: ▲; control DART 1: ▼.

In FIG. 15A-15B show intracellular levels of granzyme B (FIG. 15A) and perforin (FIG. 15B) staining in CD4+ and CD8+ human T cells after incubation with Raji/GF target cells and DART-A or control DART 1 at varying concentrations for 24 hours at an E:T ratio of 10:1.

In FIG. 16A and 16B show the proliferation of CFSE-labeled human T cells by FACS assay after co-culture with HBL-2 target cells at an E:T ratio of 10:1 in the presence of DART-A or control DART 1 at 200 ng/mL for 24 h. (FIG. 16A) or 72 hours (FIG. 16B). Staining profiles are shown after co-cultivation with HBL-2 cells in the presence of DART-A (black lines) or in the presence of control DART 1 (gray shading).

In FIG. 17 shows inhibition of tumor growth by an anti-CD19 x CD3 bispecific molecule (DART-A) in mice implanted with HBL-2 tumor cells in the presence of activated human T cells. Non-obese diabetic/severe combined immunodeficient (NOD/SCID) female mice (n=8/group) were implanted subcutaneously (SC) with HBL-2 tumor cells + activated human T cells on day 0 followed by treatment with medium as a control, control DART 1 or DART-A on days 0-3, for a total of 4 IV doses. Human T cells and HBL-2 cells were incubated at a ratio of 1:5 (1x106 and 5x106 cells, respectively). Tumor volume is shown as group mean ± SEM.

In FIG. 18 shows inhibition of tumor growth by an anti-CD19 x CD3 bispecific molecule (DART-A) in mice implanted with Raji tumor cells in the presence of activated human T cells. Female NOD/SCID mice (n=8/group) were implanted with Raji SC tumor cells + activated human T cells on day 0 followed by treatment with vehicle control, control DART 1 or DART-A on days 0-3, in total 4 doses administered IV. Human T cells and Raji cells were incubated at a ratio of 1:5 (1x106 and 5x106 cells, respectively). Tumor volume is shown as group mean ± SEM.

In FIG. 19A-19B show the change in tumor volume over time in mice implanted with HBL-2 tumor cells. Fig. 19A: Female NSG B2m −/− mice (n=8 per group) were implanted intradermally (ID) with HBL-2 tumor cells (5x106) on day 0. On day 4, mice were implanted with PBMCs (5x107) intraperitoneally (IP). Mice were then treated intravenously (IV) with vehicle, control DART 1 or CD19 x CD3 bispecific DART-A as indicated (see Rx arrows). Tumor volume is shown as group mean ± SEM. Fig. 19B: NSG B2m -/- female mice (n=8 per group) were implanted intradermally (ID) with HBL-2 tumor cells (5x106) on day 0. On day 4, mice were implanted with PBMCs (5x107) intraperitoneally (IP). Mice were then treated intravenously (IV) with vehicle, control DART 1 or CD19 x CD3 bispecific DART-A as indicated (see Rx arrows). Tumor volume is shown as group mean ± SEM.

In FIG. 20A-20B show circulating B cell levels as a function of time in cynomolgus monkeys after IV administration of up to 4 cycles of weekly incremental doses of anti-CD19 x CD3 bispecific DART-A. Cynomolgus monkeys in group 1 (n=4) were treated with medium v 0.5 v 5 v 50 v 50 µg/kg DART-A administered IV on days 1, 8, 15, 22 and 29 (shown by vertical dotted lines) ( Fig. 20A). Cynomolgus monkeys in group 2 (n=4) were treated with medium v 2 v 10 v 100 v 100 µg/kg DART-A administered IV on days 1, 8, 15, 22 and 29 (shown by vertical dotted lines)

- 11 040028 (Fig. 20B). Data for each cynomolgus monkey is shown.

In FIG. 21A-21D show circulating B cell levels versus time in cynomolgus monkeys after IV administration of up to 3 cycles of weekly fixed doses or up to 5 cycles every 3rd day of fixed doses of DART-A. Cynomolgus monkeys in groups 3-5 (n=2 per group) were treated with CD19 x CD3 bispecific DART-A. Group 3 (FIG. 21A) received 5 ng/kg DART-A, group 4 (FIG. 21B) received 50 ng/kg DART-A, and group 5 (FIG. 21C) received 500 ng/kg DART-A. DART-A was administered IV on days 1, 8 and 15 (shown by vertical dotted lines). Cynomolgus monkeys in group 6 (n=2) (FIG. 21D) were treated with 500 ng/kg DART-A administered IV on days 1, 4, 8, 11 and 15 (shown by vertical dotted lines). Data for each cynomolgus monkey is shown.

In FIG. 22 shows a pharmacokinetic (PK) analysis of DART-A in humFcRn transgenic mice. mAb 1, mAb 2 and mAb 3 are irrelevant humanized IgG1 antibodies used as a control to allow determination of the half-life of a representative antibody in transgenic animals.

In FIG. 23 shows survival curves in the Raji-luc cell model of disseminated leukemia in human PBMC reconstituted mice after treatment with 500 µg/kg DART-A (), 500 µg/kg control DART 1 (▲) or medium as control (• ).

In FIG. 24 shows survival curves in the Raji-luc cell model of disseminated leukemia in mice reconstituted with human PBMC after treatment with 500 µg/kg (•), 100 µg/kg (), 20 µg/kg (o), 4 µg/kg (▲), 0.8 µg/kg (▼) or 0.16 µg/kg (□) DART-A, 500 mg/kg (Δ) DART control 1 or medium as control (♦).

In FIG. 25 shows survival curves in the Raji.luc cell model of lymphoma in human PBMC-reconstituted mice after treatment with 0.5mg/kg DART-A (▲), 0.1mg/kg DARTA (), or medium as control ( ·).

In FIG. 26 shows survival curves in the Raji.luc cell model of lymphoma in human PBMC-repaired mice after treatment with 500 µg/kg (▲), 20 µg/kg (▼), 4 µg/kg ( ♦), 0.8 µg/kg (□) or 0.16 µg/kg (Δ) DART-A, 0.1 mg/kg DART 1 control (o) or medium as control (•).

In FIG. 27 shows the serum concentration-time profile for DART-A in a cynomolgus monkey dosed with 10 μg/kg DART-A.

In FIG. 28 shows the mean±SEM number of CD45+/CD20+ cells/µl as determined by flow cytometry for study day and group. Vertical dotted lines on days 8, 15, 22 and 29 indicate each dose (D)

DART-A by 2-hour IV infusion for animals of groups 2-5, while animals of group 1 continued to receive medium as a control. The x-axis indicating the day of the study is divided into 3 segments covering days -7 - -1, days 0-63 and days 64-121.

In FIG. 29A-29I show a typical FACS analysis of malignant B cells (CD20+/CD5+) (FIGS. 29A-29C), T cells (CD4+ or CD8+) (FIGS. 29D-29E) and T cell activation (CD25 to CD4+ (FIGS. . 29F29G) or CD8+ (FIGS. 29H-29I) T cells) in CLL PBMCs (E:T=1:23) after no treatment (FIG. 29A), treatment with DART-A (FIGS. 29C, 29E, 29G and 29I) or control DART 1 (FIGS. 29B, 29D, 29F and 29H) for 6 days.

Detailed disclosure of the present invention

The present invention relates to bispecific monovalent diabodies that contain two polypeptide chains and which have one binding site specific for the CD19 epitope and one binding site specific for the CD3 epitope (i.e., a bispecific monovalent diabody for CD19 x CD3 ). Most preferably, such CD19 x CD3 bispecific monovalent diabodies consist of three polypeptide chains and have one binding site specific for the CD19 epitope and one binding site specific for the CD3 epitope, and additionally contain an immunoglobulin Fc domain ( i.e. bispecific monovalent Fc-diabody to CD19 x CD3). The bispecific monovalent diabodies and the bispecific monovalent Fc diabodies of the present invention are capable of simultaneously binding to CD19 and CD3. The present invention relates to pharmaceutical compositions which contain such bispecific monovalent diabodies or such bispecific monovalent Fc diabodies. The present invention further relates to methods of using such diabodies in the treatment of disease, in particular hematological malignancies.

A. Antibodies and other binding molecules.

1. Antibodies.

As used herein, the term antibodies includes not only intact polyclonal or monoclonal antibodies, but also their mutants, naturally occurring variants, fusion proteins containing an antibody portion with an antigen recognition site of the required specificity, humanized antibodies and chimeric antibodies, as well as any other modified configuration. an immunoglobulin molecule that contains an antigen recognition site of the required specificity. Throughout the present invention, light and heavy chain amino acid residues

- 12 040028 antibodies are numbered according to the EU index according to Kabat et al. (1992) Sequences of Proteins of Immunological Interest, National Institutes of Health Publication No. 91-3242. As used herein, the term antigen-binding fragment of an antibody refers to the portion of an antibody that has at least one antigen recognition site. As used herein, the term encompasses fragments (such as Fab, Fab', F(ab') ₂ Fv) and single strand (scFv).

The term monoclonal antibody refers to a homogeneous population of antibodies, wherein the monoclonal antibody consists of amino acids (naturally occurring and non-naturally occurring) that participate in the selective binding of an antigen. Monoclonal antibodies are highly specific, directed against a single antigenic site. The term monoclonal antibody covers not only intact monoclonal antibodies and full-length monoclonal antibodies, but also their fragments (such as Fab, Fab', F(ab')2 Fv), single chain (scFv), their mutants, fusion proteins containing part antibodies, humanized monoclonal antibodies, chimeric monoclonal antibodies, and any other modified configuration of an immunoglobulin molecule that contains an antigen recognition site of the required specificity and is capable of binding to an antigen. No limitation is intended as to the source of the antibody or the manner in which it is produced (eg, hybridoma, phage selection, recombinant expression, transgenic animals, etc.). The term includes whole immunoglobulins as well as fragments and the like as described above under the definition of the term antibody. Methods for producing monoclonal antibodies are known in the art. One method that may be used is that of Kohler, G. et al. (1975) Continuous Cultures Of Fused Cells Secreting Antibody Of Predefined Specificity, Nature 256:495-497, or a modification thereof. Typically, monoclonal antibodies are developed in mice, rats, or rabbits. Antibodies are obtained by immunizing an animal with an immunogenic amount of cells, cell extracts, or protein preparations that contain the desired epitope. The immunogen may be, without limitation, primary cells, cultured cell lines, cancer cells, proteins, peptides, nucleic acids, or tissue. The cells used for immunization may be cultured for a period of time (eg, at least 24 hours) prior to their use as an immunogen. The cells can be used as immunogens alone or in combination with a non-denaturing adjuvant such as Ribi. As a general rule, cells should be kept intact and preferably viable when used as immunogens. Intact cells may provide better antigen detection in immunized animals than destroyed cells. The use of denaturing or aggressive adjuvants, such as Freund's adjuvant, can destroy cells and is therefore not recommended. The immunogen may be administered several times at defined times, for example twice a week or once a week, or may be administered in a manner that maintains the viability of the animal (eg, in a tissue recombinant). Alternatively, existing monoclonal antibodies and any other equivalent antibodies that are immunospecific for the desired pathogenic epitope can be sequenced and recombinantly generated by any means known in the art. In one embodiment, such an antibody is sequenced and then the polynucleotide sequence is cloned into a vector for expression or propagation. The sequence encoding the antibody of interest can be stored in the vector in the host cell, and the host cell can then be expanded and frozen for future use. The polynucleotide sequence of such antibodies can be used for genetic manipulation to create bispecific molecules in accordance with the present invention, as well as a chimeric antibody, a humanized antibody, or a caninized antibody to improve the affinity or other characteristics of the antibody. The general principle of humanizing an antibody is to retain the core sequence of the antigen-binding portion of the antibody while replacing the non-human remainder of the antibody with human antibody sequences. There are four main steps for humanizing a monoclonal antibody. These are: (1) determination of the nucleotide and putative amino acid sequence of the light and heavy variable domains of the parent antibody, (2) construction of a humanized antibody or caninized antibody, i.e. deciding which antibody framework region to use during the humanization or caninization process, (3) actual humanization or caninization methods/techniques, and (4) transfection and expression of the humanized antibody. See, for example, US Pat. Nos. 4,816,567; 5,807,715;

2. Bispecific antibodies, multispecific diabodies and DART™ diabodies.

Providing non-monospecific diabodies offers a significant advantage over antibodies: the ability to co-ligate and co-localize cells that express different epitopes. Thus, bivalent diabodies are widely used, including in therapy and immunodiagnostics. Bivalence allows greater flexibility in the design and construction of the diabody in various applications, allowing increased avidity for multimeric antigens, cross-linking of various antigens, and targeted targeting of specific cell types, given the presence of both target antigens. Due to their increased valency, low dissociation rates

- 13 040028 and rapid clearance from the circulation (for small diabodies of ~50 kDa or less) diabody molecules known in the art have also found particular application 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 co-ligation of different cells, such as ligation of cytotoxic T cells to tumor cells (Staerz et al. (1985) Hybrid Antibodies Can Target Sites For Attack By T Cells, Nature 314:628-631, and Holliger et al. ( 1996) Specific Killing Of Lymphoma Cells By Cytotoxic T-Cells Mediated By A Bispecific Diabody, Protein Eng. 9:299-305).

Epitope-binding domains of the diabody can also be targeted to B-cell surface determinants such as CD19, CD20, CD22, CD30, CD37, CD40, and CD74 (Moore, P.A. 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; Cheson, B.D. et al. (2008) Monoclonal Antibody Therapy For B-Cell Non-Hodgkin's Lymphoma N. Engl. J. Med. 359 (6):613-626 Castillo, J. et al (2008) Newer monoclonal antibodies for hematological malignancies, Exp Hematol 36(7):755-768). Many studies have also found that binding of the diabody to effector cell determinants, such as Fcγ receptors (FcyR), activates the 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-CD3 x Anti-CEA Bispecific Diabodies And B7 x Anti-CEA Bispecific Fusion Proteins , Cancer Res. 59:2909-2916; WO 2006/113665; WO 2008/157379; WO 2010/080538; WO 2012/018687; WO 2012/162068). Typically, effector cell activation is triggered by binding of an antigen-bound antibody to the effector cell by Fc-FcyR interaction, thus diabody molecules can therefore exhibit Ig-like functionality whether or not they contain an Fc domain (e.g., as analyzed in any effector function assay known in the art or provided herein (e.g. ADCC assay)). By cross-linking tumor and effector cells, the diabody not only brings the effector cell into close proximity to the tumor cells, but also results in effective tumor killing (see, e.g., Cao et al. (2003) Bispecific Antibody Conjugates In Therapeutics, Adv. Drug. Deliv Rev. 55:171-197).

However, the aforementioned advantages come with significant costs. The formation of such non-monospecific diabodies requires the successful assembly of two or more separate and distinct polypeptides (ie, such formation requires that diabodies be formed by heterodimerization of different kinds of polypeptide chains). This fact is in contrast to monospecific diabodies, which are formed by homodimerization of identical polypeptide chains. Because at least two heterogeneous polypeptides (i.e., two kinds of polypeptide) must be provided to form a non-monospecific diabody, and because homodimerization of such polypeptides results in 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 preparation of such polypeptides must be done in such a way as to prevent covalent bonding between polypeptides of the same species (i.e., 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). Thus, non-covalent binding of such polypeptides has been studied 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; 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 is known in the art that bispecific diabodies composed 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).

Despite this problem, the art has succeeded in developing stable, covalently bonded, heterodimeric, non-monospecific diabodies called DART™ (see, for example, United States Patent Document Publications Nos. 2013-0295121; 2010-0174053 and 2009-0060910; EP 2714079; EP 2601216; EP 2376109; EP 2158221 and PCT Publication Nos. WO 2012/162068; WO 2012/018687; WO 2010/080538; and Moore, P.A. et al. (2011) Application Of Dual Affinity Retargeting Mole Redirected T-Cell Killing Of B-Cell Lymphoma, Blood 117(17):4542-4551 Veri, M.C. et al (2010) Therapeutic Control Of B Cell Activation Via Recruitment

- 14 040028

Of Fcgamma Receptor IIb (CD32B) Inhibitory Function With A Novel Bispecific Antibody Scaffold Arthritis Rheum. 62(7):1933-1943; Johnson, S. et al. (2010) Effector Cell Recruitment With Novel Fv-Based DualAffinity Re-Targeting Protein Leads To Potent Tumor Cytolysis And in vivo B-Cell Depletion, J. Mol. Biol. 399(3):436-449). Such diabodies contain two or more covalently linked polypeptides and involve the construction of one or more cysteine residues into each of the polypeptide species used. For example, the addition of a cysteine residue at the C-terminus of such constructs has been shown to provide a disulfide bond between polypeptide chains, stabilizing the resulting heterodimer without affecting the binding characteristics of the bivalent molecule.

Each of the two polypeptides of the simplest DART™ contains three domains (FIG. 1). The first polypeptide comprises (i) a domain that contains a first immunoglobulin light chain variable domain binding region (VL1), (ii) a second domain that contains a second immunoglobulin heavy chain variable domain binding region (VH2), and (iii) a third domain that serves to provide heterodimerization with the second polypeptide and to covalently link the first polypeptide to the second polypeptide of the diabody. The second polypeptide contains a complementary first domain (VL2 domain), a complementary second domain (VH1 domain), and a third domain that binds to the third domain of the first polypeptide chain to allow heterodimerization and covalent binding to the first polypeptide chain. Such molecules are stable, effective and have the ability to simultaneously bind two or more antigens. They are able to provide redirected T-cell mediated killing of cells expressing target antigens.

In one embodiment, every third domain of the first and second polypeptides contains a cysteine residue that serves to link the polypeptides together via a disulfide bond. The third domain of one or both of the polypeptides may further have a CH2-CH3 domain sequence such that the formation of the diabody polypeptide complex forms an Fc domain that is capable of binding to the Fc receptor of cells (such as B lymphocytes, dendritic cells, natural killer cells, macrophages, neutrophils). , eosinophils, basophils, and mast cells) (FIGS. 2A-2B). Many variations of such molecules have been described (see, for example, United States Patent Document Publications No. 2013-0295121; 2010-0174053 and 2009-0060910; European Patent Publication No. EP 2714079; EP 2601216; EP 2376109; EP 2158221 and PCT Publication No. WO 2012/162068; WO 2012/018687; WO 2010/080538). Such DART Fc carriers may contain three polypeptide chains. The first polypeptide of such a diabody contains three domains: (i) a VL1-containing domain, (ii) a VH2-containing domain, (iii) a domain that allows heterodimerization and covalent binding to the first polypeptide chain of the diabody, and (iv) a domain containing the sequence CH2CH3 . The second polypeptide of such a DART™ contains (i) a VL2-containing domain, (ii) a VH1-containing domain, and (iii) a domain that allows heterodimerization and covalent binding to the first polypeptide chain of the diabody. The third polypeptide of this DART™ contains the CH2-CH3 sequence. Thus, the first and second polypeptide chains of such a DART™ bind together to form a VL1/VH1 binding site that is capable of binding to an epitope as well as a VL2/VH2 binding site that is capable of binding to a second epitope. The first and second polypeptides are linked to each other via a disulfide bond using cysteine residues in their respective third domains. It should be noted that the first and third polypeptide chains complex with each other to form an Fc domain that is stabilized by a disulfide bond. Such diabodies are characterized by enhanced efficiency. Such DART™ Fc carriers can have either of two orientations (Table 1).

Table 1

First orientation ^3rd chain NH2-CH2-CH3-COOH 1st ^chain NH2-VLI -UN2-heterodimer-promoter domain- CH2-CH3-COOH 2nd ^chain NH2-VL2-VH1 - heterodimer-promoter domaincoon Second orientation ^3rd chain NH2-CH2-CH3-COOH 1st ^chain NH2-CH2-CH3 -VL 1 -UN2-heterodimer-promoter domain-COOH 2nd ^chain NH2-VL2-VH1 -heterodimer-promoter domain- UNSD

B. Preferred anti-CD19 x CD3 bispecific monovalent diabodies of the present invention.

The present invention relates in particular to CD19 x CD3 bispecific monovalent diabodies that are capable of simultaneously binding to CD19 and CD3, as well as uses for such

- 15 040028 molecules in the treatment of hematological malignant diseases. Although non-optimized anti-CD19 x CD3 bispecific monovalent 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 CD19 x CD3 bispecific monovalent diabodies by modifying or refining their sequences.

Preferred bispecific anti-CD19 x CD3 monovalent diabodies according to the present invention are bispecific anti-CD19 x CD3 monovalent Fc diabodies which consist of three polypeptide chains that bind to each other to form one binding site specific for the CD19 epitope and one a binding site specific for the CD3 epitope (see FIGS. 2A-2B) to allow simultaneous binding to CD19 and CD3. Thus, such diabodies bind to a first antigen, which can be either CD3 or CD19, and to a second antigen, which is CD19 if the first epitope is CD3, and which is CD3 if the first epitope is CD19.

Preferably, as shown in FIG. 2A, the first of such three polypeptide chains will contain, in the N-terminal to C-terminal direction, the N-terminus, the antigen-binding domain of the light chain variable domain (VL) of the first antigen (either CD3 or CD19), antigen-binding heavy chain variable domain (VH) domain of the second antigen (CD19 if the first antigen was CD3; CD3 if the first antigen was CD19), a heterodimer-promoter domain, and a C-terminus. 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. Preferably, the antigen-binding domain of the heavy chain variable domain is connected to the heterodimer-promoter domain by an intermediate linker peptide (linker 2). In the case of a bispecific monovalent Fc diabody to CD19 x CD3, the C-terminus of the heterodimer promoter domain is connected to the CH2-CH3 domains of the Fc region (Fc domain) by an intermediate linker peptide (linker 3) or an intermediate spacer-linker peptide (spacer-linker 3 ). Most preferably, the first of the three polypeptide chains will thus contain in the direction from the N-terminus to the C-terminus VL _ne p _{By antigen} linker 1 - VH _{second antigen} - linker 2 - heterodimer-promoter domain - spacer-linker 3 - Fc -domain.

Alternatively, as shown in FIG. 2B, the first of these three polypeptide chains will contain, N-terminus to C-terminus, the N-terminus, linker 3, Fc region CH2-CH3 domains, an intermediate spacer peptide (linker 4), having, for example, the amino acid sequence of APSSS (SEQ ID NO: 47) or the amino acid sequence of APSSSPME (SEQ ID NO: 48), the antigen-binding domain of the light chain variable domain (VL) of the first antigen (either CD3 or CD19), the antigen-binding heavy chain variable domain (VH) domain of the second antigen (CD19 if the first antigen was CD3; CD3 if the first antigen was CD19), a heterodimer-promoter domain, and a C-terminus. 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. Preferably, the antigen binding domain of the heavy chain variable domain is linked to the heterodimer promoter domain by an intermediate linker peptide (linker 2). Most preferably, the first of the three polypeptide chains will thus contain, in N-terminal to C-terminal direction, linker 3 - Fc domain - linker 4 - VL _{first antigen} - linker 1 VH _{second antigen} - linker 2 - heterodimer-promoter domain.

Preferably, the second of such three polypeptide chains will comprise, N-terminus to C-terminus, the N-terminus, the second antigen variable light chain (VL) antigen-binding domain, the heavy chain variable domain (VH) antigen-binding domain ) of the first antigen, heterodimer-promoter domain and C-terminus. 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. Preferably, the antigen binding domain of the heavy chain variable domain is linked to the heterodimer promoter domain by an intermediate linker peptide (linker 2). Most preferably, the second of the three polypeptide chains will thus contain, in N-terminal to C-terminal direction, VL _{second antigen} - linker 1 - VH _{first antigen} - linker 2 - heterodimer-promoter domain.

Preferably, the third of these three polypeptide chains will contain a linker peptide (linker 3) and Fc region CH2-CH3 domains (Fc 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 functional antigen binding site that is specific for the first antigen (i.e. either CD19 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

- 16 040028 to the second antigen (ie either CD3 or CD19, depending on the identity of the first antigen). Thus, the choice 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 is coordinated such that the two polypeptide chains together contain antigen-binding domains of the light and heavy chain variable domains capable of binding to CD19 and CD3.

1. Preferred linkers.

Most preferably, the length of the linker 1 that separates such VL and VH domains of the polypeptide chain is chosen so as to largely or completely prevent such VL and VH domains from linking to each other. Thus, the VL and VH domains of the first polypeptide chain are unable to significantly or completely bind to each other. Similarly, the VL and VH domains of the second polypeptide chain are unable to substantially or completely bind to each other. A preferred intermediate spacer peptide (linker 1) has the sequence (SEQ ID NO: 1): GGGSGGGG.

The task of linker 2 is to separate the VH domain of the polypeptide chain from the heterodimer promoter domain of this polypeptide chain. Any of a number of linkers can be used as linker 2. A preferred sequence for such linker 2 is the amino acid sequence ASTKG (SEQ ID NO: 2) which is derived from the CH1 domain of IgG, or GGCGGG (SEQ ID NO: 3) having a cysteine residue, which can be used to covalently link the first and second polypeptide chains to each other via a disulfide bond. Since the ASTKG linker 2 (SEQ ID NO: 2) does not contain such cysteine, the use of such linker 2 is preferably associated with the use of a cysteine-containing heterodimer promoter domain such as the E-helix of SEQ ID NO: 12 or the K-helix of SEQ ID NO: 13 (see . below).

The task of linker 3 is to separate the heterodimer-promoter domain of the polypeptide chain from the Fc domain of this polypeptide chain. Any of a number of linkers can be used as linker 3. A preferred sequence for such linker 3 is the amino acid sequence DKTHTCPPCP (SEQ ID NO: 4). The preferred sequence for spacerlinker 3 is the amino acid sequence GGGDKTHTCPPCP (SEQ ID NO: 5).

2. Preferred heterodimer promoter domains.

The formation of heterodimers of the first and second polypeptide chains can be driven by the inclusion of heterodimer promoter domains. Such domains include GVEPKSC (SEQ ID NO: 6) or VEPKSC (SEQ ID NO: 7) in one polypeptide chain and GFNRGEC (SEQ ID NO: 8) or FNRGEC (SEQ ID NO: 9) in another polypeptide chain (US 2007/0004909).

More preferably, however, the heterodimer-promoter domains of the present invention are formed from one, two, three, or four tandem-repeating helical domains of opposite charge, which contain a sequence of at least six, at least seven, or at least eight charged amino acid residues. (Apostolovic, B. et al. (2008) pH-Sensitivity of the E3/K3 Heterodimeric Coiled Coil, Biomacromolecules 9:3173-3180; Arndt, K. M. et al. (2001) Helix-stabilized Fv (hsFv) Antibody Fragments: Substituting the Constant Domains of a Fab Fragment for a Heterodimeric Coiled-coil Domain, J. Molec Biol 312:221-228 Amdt, K. M. et al (2002) Comparison of In Vivo Selection and Rational Design of Heterodimeric Coiled Coils, Structure 10:1235-1248 Boucher, C. et al (2010) Protein Detection By Western Blot Via Coiled-Coil Interactions, Analytical Biochemistry 399:138-140 Cachia, P. J. et al (2004) Synthetic Peptide Vaccine Development: Measurem ent Of Polyclonal Antibody Affinity And Cross-Reactivity Using A New Peptide Capture And Release System For Surface Plasmon Resonance Spectroscopy J. Mol. Recognize. 17:540-557; De Crescenzo, G.D. et al. (2003) RealTime Monitoring of the Interactions of Two-Stranded de novo Designed Coiled-Coils: Effect of Chain Length on the Kinetic and Thermodynamic Constants of Binding, Biochemistry 42:1754-1763; Fernandez-Rodriquez, J. et al. (2012) Induced Heterodimerization And Purification Of Two Target Proteins By A Synthetic CoiledCoil Tag, Protein Science 21:511-519; Ghosh, T.S. et al. (2009) End-To-End And End-To-Middle Interhelical Interactions: New Classes Of Interacting Helix Pairs In Protein Structures, Acta Crystallographica D65:10321041; Grigoryan, G. et al. (2008) Structural Specificity In Coiled-Coil Interactions, Curr. Opin. struc. Biol. 18:477-483; Litowski, J.R. et al. (2002) Designing Heterodimeric Two-Stranded α-Helical Coiled-Coils: The Effects Of Hydrophobicity And α-Helical Propensity On Protein Folding, Stability, And Specificity, J. Biol. Chem. 277:37272-37279; Steinkruger, J.D. et al. (2012) The d'--d--d' Vertical Triad is Less Discriminating Than the a'--a--a' Vertical Triad in the Antiparallel Coiled-coil Dimer Motif, J. Amer. Chem. soc. 134(5):26262633; Straussman, R. et al. (2007) Kinking the Coiled Coil - Negatively Charged Residues at the Coiled-coil Interface, J. Molec. Biol. 366:1232-1242; Tripet, B. et al. (2002) Kinetic Analysis of the Interactions between Troponin C and the C-terminal Troponin I Regulatory Region and Validation of a New Peptide Delivery/Capture System used for Surface Plasmon Resonance, J. Molec. Biol. 323:345-362; Woolfson, D.N. (2005) The Design Of Coiled-Coil Structures And Assemblies, Adv. Prot. Chem. 70:79-112; Zeng, Y. et al. (2008) A LigandPseudoreceptor System Based On de novo Designed Peptides For The Generation Of Adenoviral Vectors With Altered Tropism, J. Gene Med. 10:355-367).

- 17 040028

Such repeating helical domains may be exact repeats or may have substitutions. For example, the heterodimer promoter domain of the first polypeptide chain may contain a sequence of eight negatively charged amino acid residues, and the heterodimer promoter domain of the second polypeptide chain may contain a sequence of eight positively charged amino acid residues (or vice versa). It does not matter which helix is provided for the first or second polypeptide chains, provided that a helix of opposite charge is used for the other polypeptide chain. However, a preferred CD19 x CD3 bispecific monovalent diabody according to the present invention has a first negatively charged helix polypeptide chain. The positively charged amino acid may be lysine, arginine, histidine, etc., and/or the negatively charged amino acid may be glutamic acid, aspartic acid, etc. The positively charged amino acid is preferably lysine and/or the negatively charged amino acid is preferably glutamic acid. It is possible to use only one heterodimer promoter domain (because such a domain will inhibit homodimerization and thereby allow heterodimerization), however, it is preferred that both the first and second diabody polypeptide chains in accordance with the present invention contain heterodimer promoter domains.

In a preferred embodiment, one of the heterodimer promoter domains will contain four tandem E-helical domains (SEQ ID NO: 10: EVAALEK-EVAALEK-EVAALEK-EVAALEK) whose glutamate residues will form a negative charge at pH 7, while the other heterodimers will the promoter domains will contain four tandem K-helical domains (SEQ ID NO: 11: KVAALKE-KVAALKE-KVAALKE-KVAALKE ) whose lysine residues will form a positive charge at pH 7. The presence of such charged domains provides a link between the first and second polypeptides and, thus promoting heterodimerization. Particularly preferred is a heterodimer promoter domain in which one of the four tandem E-helical domains of SEQ ID NO: 10 has been modified to contain a cysteine residue: EVAACEK-EVAALEK-EVAALEK-EVAALEK (SEQ ID NO: 12). Similarly, a heterodimer promoter domain is particularly preferred in which one of the four tandem K-helical domains of SEQ ID NO: 11 has been modified to contain a cysteine residue: I^AACKE-KVAALKE-KVAALKE-KVAALKE (seq id NO: 13).

3. Covalent binding of polypeptide chains.

Bispecific anti-CD19 x CD3 monovalent diabodies of the present invention are designed such that their first and second polypeptide chains are covalently linked to each other via one or more 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, linker 2 may contain a cysteine residue. Additionally or alternatively, linker 3 may contain a cysteine residue as in SEQ ID NO: 4 or SEQ ID NO: 5. Most preferably, one or more helical domains of the heterodimer promoter domain will be substituted with a cysteine residue as in SEQ ID NO: 12 or in SEQ ID NO: 13.

In specific embodiments, the anti-CD19 x CD3 bispecific monovalent diabodies of the present invention may further have an albumin-binding domain to extend the in vivo half-life.

4. Preferred Fc domains.

The Fc domain of the anti-CD19 x CD3 bispecific monovalent Fc diabodies of the present invention may be either the entire Fc region (eg, the complete Fc region of IgG) or only a fragment of the entire Fc region. Although the Fc domain of the bispecific monovalent Fc diabodies of the present invention may have the ability to bind to one or more Fc receptors (e.g. FcyR), more preferably such an Fc domain will cause reduced binding to FcyRIA (CD64), FcyRIIA (CD32A) , FcyRIIB (CD32B), FcyRIIIA (CD16a) or FcyRIIIB (CD16b) (relative to the binding exhibited by the wild-type Fc region) or will substantially abolish the ability of such an Fc domain to bind to such receptor(s). The Fc domain of the bispecific monovalent Fc diabodies of the present invention may include part or all of the CH2 domain and/or part or all of the CH3 domain of the entire Fc region, or may contain a CH2 variant and/or a CH3 variant sequence (which may include itself, for example, one or more insertions and/or one or more deletions in relation to the CH2 or CH3 domains of the entire Fc region). The Fc domain of the bispecific monovalent Fc diabodies of the present invention may contain non-Fc polypeptide portions, or may contain portions of non-naturally occurring complete Fc regions, or may contain non-naturally occurring CH2 and/or CH3 domain orientations (such such as two CH2 domains or two CH3 domains, or in an N-terminal to C-terminal direction a CH3 domain linked to a CH2 domain, etc.).

According to a preferred embodiment, each of the first and third polypeptide

- 18 040028 chains of bispecific monovalent Fc diabodies to CD19 x CD3 according to the present invention contain CH2-CH3 domains which bind together to form an immunoglobulin (IgG) Fc domain. The amino acid sequence of the CH2-CH3 domain of human IgG1 is (SEQ

ID NO: 14):

APELLGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVKFNWYVD

GVEVHNAKTK PREEQYNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAK GQPREPQVYT LPPSREEMTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE ALHNHYTQKS LSLSPGK.

Thus, both the CH2 and/or CH3 domains of the first and third polypeptide chains may consist of SEQ ID NO: 14 or a variant thereof.

It is particularly preferred that the CH2-CH3 domains of the first and third polypeptide chains of the anti-CD19 x CD3 bispecific monovalent Fc diabodies according to the present invention show reduced binding (or virtually no binding) to FcyRIA (CD64), FcyRIIA (CD32A), FcyRIIB (CD32B). ), FcyRIIIA (CD16a) or FcyRIIIB (CD16b) (relative to the binding shown by the wild-type Fc region (SEQ ID NO: 14)). Fc variants and mutants capable of mediating such altered binding are well known in the art and include amino acid substitutions at positions 234 and 235, a substitution at position 265, or a substitution at position 297 (see, for example, US Patent No. 5,624,821, incorporated herein by reference). In a preferred embodiment, the CH2-CH3 domain of the first and/or third polypeptide chains of the anti-CD19 x CD3 bispecific monovalent Fc diabodies of the present invention comprises a substitution at position 234 with alanine and at position 235 with alanine.

The CH2 and/or CH3 domains of the first and third polypeptide chains need not be identical in sequence and are advantageously modified to allow complex formation between the two polypeptide chains. For example, an amino acid substitution (preferably a substitution with an amino acid containing a bulky bulge-forming side group, e.g., tryptophan) can be introduced into the CH2 or CH3 domain such that a spatial interaction prevents interaction with a similarly mutated domain and forces the mutated domain to pair with the domain in which the complementary or adaptive mutation has been engineered, ie. depression (for example, replacement with glycine). Such a set of mutations may be constructed in any pair of polypeptides containing a bispecific monovalent Fc diabody molecule, and in addition may be constructed in any part of the polypeptide chains of said pair. Methods for constructing proteins to promote heterodimerization, rather than homodimerization, are well known in the art, in particular with regard to the construction of immunoglobulin-like molecules, and are covered by this document (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 hereby incorporated herein by reference in its entirety). Preferably, a ridge is designed in the CH2-CH3 domains of the first polypeptide chain and a trough is designed in the CH2-CH3 domains of the third polypeptide chain. Thus, the overhang will assist in preventing homodimerization of the first polypeptide through its CH2 and/or CH3 domains. Because the third polypeptide chain preferably contains a pit substitution, it will heterodimerize with the first polypeptide chain and will also homodimerize with itself. The preferred overhang is created by modifying the Fc domain of native IgG with modification T366W. The preferred pit is created by modifying the Fc domain of native IgG to contain modification T366S, L368A and Y407V. To help purify the homodimer of the third polypeptide chain from the final bispecific monovalent Fc diabody 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 mutated, preferably with an amino acid substitution at position 435 (H435R). Thus, the homodimer of the third polypeptide chain will not bind protein A, while the bispecific monovalent Fc diabody will retain its ability to bind protein A via the protein A binding site in the first polypeptide chain.

A preferred sequence for the CH2 and CH3 domains of the first polypeptide chain of the anti-CD19 x CD3 bispecific monovalent Fc diabodies according to the present invention would have the overhang sequence (SEQ ID NO: 15):

- 19 040028

APEAAGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVKFNWYVD GVEVHNAKTK PREEQ YNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAK GQPREPQVYT LPPSREEMTK NQVSLWCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE ALHNHYTQKS LSLSPGK.

A preferred sequence for the CH2 and CH3 domains of the third polypeptide chain of the anti-CD 19 x CD3 bispecific monovalent Fc diabodies according to the present invention would have a recess bearing sequence (SEQ ID NO: 16): APEAAGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVKFNWYVD

GVEVHNAKTK PREEQYNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAK GQPREPQVYT LPPSREEMTK NQVSLSCAVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLVSKL TVDKSRWQQG NVFSCSVMHE ALHNRYTQKS LSLSPGK

It should be noted that the CH2-CH3 domains in SEQ ID NO: 15 and SEQ ID NO: 16 include a substitution at position 234 with alanine and at position 235 with alanine and thus form an Fc domain showing reduced binding (or little or no binding ) with FcyRIA (CD64), FcyRIIA (CD32A), FcyRIIB (CD32B), FcyRIIIA (CD16a) or FcyRIIIB (CD16b) (relative to the binding shown by the wild-type Fc region (SEQ ID NO: 14).

It is preferred that the first polypeptide chain will have a CH2-CH3 overhang-bearing sequence, such as the sequence of SEQ ID NO: 15. However, it should be appreciated that a CH2-CH3 trough-bearing domain (e.g., SEQ ID NO: 16) can be used in of the first polypeptide chain, in which case a CH2-CH3 overhang domain (eg, SEQ ID NO: 15) in the third polypeptide chain should be used.

5. Preferred variable domains for CD 19.

The antigen-binding domain of the light chain variable domain of any anti-CD19 antibody can be used in accordance with the present invention. Preferably, however, such a _CD i9 VL domain will have the amino acid sequence (SEQ ID NO: 17):

ENVLTQSPAT LSVTPGEKAT ITCRASQSVS YMHWYQQKPG QAPRLLIYDA

SNRASGVPSR FSGSGSGTDH TLTISSLEAE DAATYYCFQG SVYPFTFGQG TKLEIK, with the underlined sequences representing, respectively, their CDR1, CDR2 nCDR3:

VLcdi9CDR1: RASQSVSYMH (SEQ ID NO: 18), VLCD19 CDR2: DASNRAS (SEQ ID NO: 19),

VL _C di9 CDR3: FQGSVYPFT (SEQ ID NO: 20).

Similarly, the antigen-binding domain of the heavy chain variable domain of any anti-CD19 antibody can be used in accordance with the present invention. Preferably, however, such a _C di9 VH domain will have the amino acid sequence (SEQ ID NO: 21):

QVTLRESGPA LVKPTQTLTL TCTFSGFSLS TSGMGVGWIR QPPGKALEWL

AHIWWDDDDKR YNPALKSRLT ISKDTSKNQV FLTMTNMDPV DTATYYCARM

ELWSYYFDYW GQGTTVTVSS, with the underlined sequences representing respectively their CDR1, CDR2 HCDR3:

VHCD19 CDR1: TSGMGVG (SEQ ID NO: 22)

VH _C Di9 CDR2: HIWWDDDKRYNPALKS (SEQ ID NO: 23),

VH _C di9CDR3: MELWSYYFDY (SEQ ID NO: 24).

6. Preferred CD3 variable domains.

The antigen-binding domain of the light chain variable domain of any anti-CD3 antibody can be used in accordance with the present invention. Preferably, however, such a _cd3 VL domain will have the amino acid sequence (SEQ ID NO: 25):

QAVVTQEPSL TVSPGGTVTL TCRSSTGAVT TSNYANWVOQ KPGQAPRGLI

GGTNKRAPWT PARFSGSLLG GKAALTITGA QAEDEADYYC ALWYSNLWVF

GGGTKLTVLG, with the underlined sequences representing, respectively, their CDR1, CDR2 HCDR3:

VL _CD 3 CDR1: RSSTGAVTTSNYAN (SEQ ID NO:26),

VLcd3 CDR2: GTNKRAP (SEQ ID NO:27)

VL _C d3 CDR3: ALWYSNLWV (SEQ ID NO:28).

Similarly, the antigen-binding domain of the heavy chain variable domain of any anti-CD3 antibody can be used in accordance with the present invention. Preferred is 20040028 but, however, such a VHCD3 domain would have the amino acid sequence (SEQ ID NO: 29):

EVQLVESGGG LVQPGGSLRL SCAASGFTFS TYAMNWVRQA

PGKGLEWVGR IRSKYNNYAT YYADSVKGRF TISRDDSKNS LYLQMNSLKT

EDTAVYYCVR HGNFGNSYVS WFAYWGOGTL VTVSS, with the underlined sequences representing their CDR1, CDR2 and CDR3, respectively:

UNCOZ CDRE TYAMN (SEQ ID NO:30),

VH _CD 3 CDR2: RIRSKYNNYATYYADSVKG (SEQ ID NO:31),

VH _C D3 CDR3: HGNFGNSYVSWFAY (SEQ ID NO:32).

7. Optional albumin-binding domain.

As disclosed in WO 2012/018687, to further improve the in vivo pharmacokinetic properties of CD19 x CD3 bispecific monovalent diabodies and CD 19 x CD3 bispecific monovalent Fc diabodies according to the present invention, such diabodies can be modified to contain the polypeptide portion of the serum binding protein at one or more ends of the diabody. Most preferably, such a serum binding protein polypeptide portion will be located at the C-terminus of the diabody. A particularly preferred polypeptide portion of the serum binding protein for this purpose is the albumin binding domain (ABD) from the G protein of streptococcus. Albumin binding domain 3 (ABD3) of the G protein of Streptococcus strain G148 is particularly preferred.

Albumin-binding domain 3 (ABD3) of the G protein of Streptococcus strain G148 consists of 46 amino acid residues forming a stable three-stranded bundle and has a wide albumin-binding specificity (Johansson, M.U. 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 abundant protein in plasma and has a half-life in humans of 19 days. Albumin has several low molecular weight binding sites that allow it to bind non-covalently to other proteins and thus prolong its serum half-lives.

Thus, the second polypeptide chain of such a CD19 x CD3 bispecific monovalent diabody or an albumin-binding domain bispecific Fc monovalent diabody contains a linker (linker 4) that separates the E-helix (or K-helix) of such a polypeptide chain from the albumin-binding domain. domain. The preferred sequence for such linker 4 is SEQ ID NO: 33: GGGS. A preferred albumin binding domain (ABD) has the sequence (SEQ ID NO: 34): LAEAKVLANRELDKYGVSDYYKNLIDNAKSAEGVKALIDEILAALP.

C. Illustrative monovalent anti-CD19 x CD3 DART-A bispecific Fc diabody.

The present invention provides an exemplary anti-CD19 x CD3 bispecific monovalent Fc diabody capable of simultaneously and specifically binding to CD19 and CD3. Such an exemplary diabody is referred to as DART-A. The diabodies according to the present invention have been found to exhibit enhanced functional activity relative to other diabodies against CD19 x CD3.

As indicated above, the CD19 x CD3 bispecific monovalent Fc diabodies of the present invention contain three polypeptide chains. The first DART-A polypeptide chain contains, N-terminus to C-terminus, N-terminus, a VL domain of a monoclonal antibody capable of binding to CD19 (VL _CD19 ) (SEQ ID NO: 17), an intermediate linker peptide (Linker 1 ; GGGSGGGG (SEQ ID NO: 1)), VH domain of a monoclonal antibody capable of binding to CD3 (VH _CD3 ) (SEQ ID NO: 29), intermediate linker peptide (linker 2; ASTKG (SEQ ID NO: 2)), heterodimer -promoter (E-helical) domain (EVAA£EK-EVAALEK-EVAALEK-EVAALEK (SEQ ID NO: 12)), intermediate linker peptide (spacer-linker 3; GGGDKTHTCPPCP (SEQ ID NO: 5)), a polypeptide that contains overhang domains CH2 and CH3 (Fc domain sequence, SEQ ID NO: 15), and the C-terminus.

Thus, the first DART-A polypeptide chain consists of SEQ ID NO: 17 - SEQ ID NO: 1 SEQ ID NO: 29 - SEQ ID NO: 2 - SEQ ID NO: 12 - SEQ ID NO: 5 - SEQ ID NO: 15.

The amino acid sequence of the first DART-A polypeptide is (SEQ ID NO: 35):

- 21 040028

ENVLTQSPATLSVTPGEKATITCRASQSVSYMHWYQQKPGQAPRLLIYDASNR ASGVPSRFSGSGSGTDHTLTISSLEAEDAAIYYCFQGSVYPFTFGQGTKLEIKGGGSG GGGEVQLVESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVGRIRS KYNNYATYYADSVKGRFTISRDDSKNSLYLQMNSLKTEDTAVYYCVRHGNFGNSYV SWFAYWGQGTLVTVSSASTKGEVAACEKEVAALEKEVAALEKEVAALEKGGGDKT HTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD

GVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTI SKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYK TTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

A preferred polynucleotide encoding such a polypeptide is (SEQ ID NO: 36):

gagaatgtgctcacacagtcccctgcaactctgagcgtaactccaggggagaaggccaccatcacgtgtagagcctccca gagtgtgagctacatgcactggtatcagcagaaacctggacaagctcccaggttgctgatctatgacgcgagcaaccgggctagtggc gttccatcccggttttctggctcaggatctggcactgaccacaccctcaccatatccagccttgaagccgaagatgccgcaacctactac tgctttcaggggagtgtgtatcccttcacattcggtcagggtacaaagctggagattaagggtggaggatccggcggcggaggcgag gtgcagctggtggagtctgggggaggcttggtccagcctggagggtccctgagactctcctgtgcagcctctggattcaccttcagcac atacgctatgaattgggtccgccaggctccagggaaggggctggagtgggttggaaggatcaggtccaagtacaacaattatgcaac ctactatgccgactctgtgaagggtagattcaccatctcaagagatgattcaaagaactcactgtatctgcaaatgaacagcctgaaaac cgaggacacggccgtgtattactgtgtgagacacggtaacttcggcaattcttacgtgtcttggtttgcttattggggacaggggacactg gtgactgtgtcttccgcctccaccaagggcgaagtggccgcatgtgagaaagaggttgctgctttggagaaggaggtcgctgcacttg aaaaggaggtcgcagccctggagaaaggcggcggggacaaaactcacacatgcccaccgtgcccagcacctgaagccgcgggg ggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgt gagccacgaagaccctgaggtcaagtt caactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggag cagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtc tccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcc cccatcccgggaggagatgaccaagaaccaggtcagcctgtggtgcctggtcaaaggcttctatcccagcgacatcgccgtggagtg ggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagct caccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaa gagcctctccctgtctccgggtaaa.

The second DART-A polypeptide chain contains, N-terminus to C-terminus, the N-terminus, the VL domain of a monoclonal antibody capable of binding to CD3 (VL _CD3 ) (SEQ ID NO: 25), an intermediate linker peptide (linker 1 ; GGGSGGGG (SEQ ID NO: 1)), VH domain of a monoclonal antibody capable of binding to CD19 (VH _CD19 ) (SEQ ID NO: 21), intermediate linker peptide (linker 2; ASTKG (SEQ ID NO: 2)), heterodimer -promoter (K-helical) domain (KVAACKE-KVAALKE-KVAALKE-KVAALKE (SEQ ID NO: 13) and C-terminus.

Thus, the second DART-A polypeptide consists of SEQ ID NO: 25 - SEQ ID NO: 1 - SEQ ID NO: 21 - SEQ ID NO: 2 - SEQ ID NO: 13.

The amino acid sequence of the second DART-A polypeptide is (SEQ ID

NO:37):

QAVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGG TNKRAPWTPARFSGSLLGGKAALTITGAQAEDEADYYCALWYSNLWVFGGGTKLT VLGGGGSGGGGQVTLRESGPALVKPTQTLTLTCTFSGFSLSTSGMGVGWIRQPPGKA LEWLAHIWWDDDKRYNPALKSRLTISKDTSKNQVFLTMTNMDPVDTAΊYΎCARME LWSYYFDYWGQGTTVTVSSASTKGKVAACKEKVAALKEKVAALKEKVAALKE.

A preferred polynucleotide encoding such a polypeptide has the sequence (SEQ ID NO: 38):

- 22 040028 caggctgtggtgactcaggagccttcactgaccgtgtccccaggcggaactgtgaccctgacatgcagatccagcacagg cgcagtgaccacatctaactacgccaattgggtgcagcagaagccaggacaggcaccaaggggcctgatcgggggtacaaacaaa agggctccctggacccctgcacggttttctggaagtctgctgggcggaaaggccgctctgactattaccggggcacaggccgaggac gaagccgattactattgtgctctgtggtatagcaatctgtgggtgttcgggggtggcacaaaactgactgtgctgggagggggtggatc cggcggaggtggacaggtgacactgagggaatctggtccagctctggtgaaacccactcagacgctcactctcacttgcacctttagt gggttctcactgtccacatctggcatgggagtaggctggattcgacagccacctgggaaagccttggagtggcttgcccacatctggtg ggatgacgacaagcggtataatcccgcactgaagagcagactgaccatcagcaaggatacatccaagaaccaggtgtttctgaccatg accaacatggaccctgtcgatacagccacctactattgtgctcgcatggagttgtggtcctactacttcgactattggggacaaggcaca accgtgactgtctcatccgcctccaccaagggcaaagtggccgcatgtaaggagaaagttgctgctttgaaagagaaggtcgccgca cttaaggaaaaggtcgcagccctgaaagag.

The third DART-A polypeptide chain contains, in the N-terminal to C-terminal direction, the N-terminus, a peptide (linker 3; DKTHTCPPCP (SEQ ID NO: 4)), a polypeptide that contains the CH2 and CH3 recess-bearing domains (sequence Fc domain, SEQ ID NO: 16), and C-terminus.

Thus, the third DART-A polypeptide consists of SEQ ID NO: 4 - SEQ ID NO: 16.

The amino acid sequence of the third DART-A polypeptide is (SEQ ID NO: 39):

DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVK FNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP APIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQP ENNYKTTVMTPVLDSDGSFFLVSKLTVLSDNCSWKHEGN

A preferred polynucleotide that encodes such a polypeptide has the sequence (SEQ ID NO: 40):

gacaaaactcacacatgcccaccgtgcccagcacctgaagccgcggggggaccgtcagtcttcctcttccccccaaaacc caaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaa ctggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagc gtcctcaccgtc ctgcaccaggactggctgaatggc aaggagtacaagtgcaaggtctc caac aaagccctccc agcccc catcgag aaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaacc aggtcagcctgagttgcgcagtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaac tacaagaccacgcctcccgtgctggactccgacggctccttcttcctcgtcagcaagctcaccgtggacaagagcaggtggcagcag gggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccgctacacgcagaagagcctctccctgtctccgggtaaa.

D. Control DARTs.

To more convincingly demonstrate the properties of bispecific monovalent diabodies to CD19 x CD3, two control DARTs were constructed in accordance with the present invention. The first control DART (control DART 1) was able to bind to fluorescein and CD3. The second DART control (DART control 2) was able to bind to fluorescein and CD19.

The anti-fluorescein antibody used to generate the control DART 1 and 2 was the 4-4-20 antibody (Gruber, M. et al. (1994) Efficient Tumor Cell Lysis Mediated By A Bispecific Single Chain Antibody Expressed In Escherichia coli, J. Immunol. 152(11):5368-5374; Bedzyk, W. D. et al. (1989) Comparison Of Variable Region Primary Structures Within An Anti-Fluorescein Idiotype Family J. Biol. Chem. 264(3):1565-1569), which used in control diabodies. The amino acid sequences of the variable light and variable heavy domains of an anti-fluorescein antibody 4-4-20 are as follows: the amino acid sequence of the light chain variable domain of an anti-fluorescein antibody 4-4-20 (SEQ ID NO: 41)

DWMTQTPFS LPVSLGDQAS

ISCRSSQSLV HSNGNTYLRW YLQKPGQSPK VLIYKVSNRF SGVPDRFSGS GSGTDFTLKI SRVEAEDLGV YFCSQSTHVP WTFGGGTKLE IK;

amino acid sequence of the heavy chain variable domain of an anti-fluorescein antibody 4-4-20 (SEQ ID NO: 42)

EVKLDETGGG LVQPGRPMKL SCVASGFTFS DYWMNWVRQS PEKGLEWVAQ IRNKPYNYET YYSDSVKGRF TISRDDSSKSS VYLQMNNLRV EDMGIYYCTG SYYGMDYWGQ GTSVTVSS.

1. Control DART 1 (fluorescein x CD3).

The first polypeptide chain of the control _DART 1 contains, in the N-terminal to C-terminal direction, the N-terminus, the VL domain of a monoclonal antibody capable of binding to fluorescein (VL _4.4.20 ) (SEQ ID NO: ₄₁ ), an intermediate linker peptide (linker 1; GGGSGGGG (SEQ ID NO: 1)), VH domain of a monoclonal antibody capable of binding to CD3 (VH _CD3 ) (SEQ ID NO: 29), intermediate linker peptide (linker 2; GGCGGG (SEQ ID NO: 3)), heterodimer-promoter (E-helical) domain (EVAALEK-EVAALEK-EVAAALEK-EVAALEK (SEQ ID NO: 10)), intermediate linker peptide (spacer-linker 3; GGGDKTHTCPPCP (SEQ ID NO: 5) ), a polypeptide that contains overhang CH2 and CH3 domains (Fc domain sequence, SEQ ID NO: 15), and a C-terminus.

Thus, the first polypeptide chain of the control DART 1 consists of SEQ ID NO: 41 - SEQ ID NO: 1 - SEQ ID NO: 29 - SEQ ID NO: 3 - SEQ ID NO: 10 - SEQ ID NO: 5 - SEQ ID NO : 15.

The amino acid sequence of the first polypeptide chain of the control DART 1 is (SEQ ID NO: 43):

DVVMTQTPFSLPVSLGDQASISCRSSQSLVHSNGNIYLRWYLQKPGQSPKVLI

YKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHVPWTFGGGTKLEI KGGGSGGGGEVQLVESGGGLVQPGGSLRLSCAASGFTFSIYAMNWVRQAPGKGLE WVGRIRSKYNNYATYYADSVKGRFTISRDDSKNSLYLQMNSLKTEDTAVYYCVRHG NFGNSYVSWFAYWGQGTLVTVSSASTKGEVAACEKEVAALEKEVAALEKEVAALE KGGGDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVK FNWYVDGVEVHNAKTKPREEQYNSIYRVVSVLTVLHQDWLNGKEYKCKVSNKALP APIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQP ENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS PGK.

The second polypeptide chain of control DART 1 contains, N-terminus to C-terminus, the N-terminus, the VL domain of a monoclonal antibody capable of binding to CD3 (VL _CD3 ) (SEQ ID NO: 25), an intermediate linker peptide (linker 1 ; GGGSGGGG (SEQ ID NO: 1)), monoclonal antibody VH domain capable of binding to fluorescein (VH _Fluor ) (SEQ ID NO: 42), intermediate linker peptide (linker 2; GGCGGG (SEQ ID NO: 3)), heterodimer -promoter (K-helical) domain (KVAALKE-KVAALKE-KVAALKE-KVAALKE (SEQ ID NO: 11) and C-terminus.

Thus, the second polypeptide chain of the control DART 1 consists of SEQ ID NO: 25 - SEQ ID NO: 1 - SEQ ID NO: 42 - SEQ ID N03 - SEQ ID NO: 11.

The amino acid sequence of the second polypeptide chain of the control DART 1 is (SEQ ID NO: 44):

QAVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGG

TNKRAPWTPARFSGSLLGGKAALTITGAQAEDEADYYCALWYSNLWVFGGGTKLT

VLGGGGSGGGGEVKLDETGGGLVQPGRPMKLSCVASGFTFSDYWMNWVRQSPEKG

LEWVAQIRNKPYNYETYYSDSVKGRFTISRDDSKSSVYLQMNNLRVEDMGIYYCTGS YYGMDYWGQGTSVTVSSASTKGKVAACKEKVAALKEKVAALKEKVAALKE.

The third polypeptide chain of control DART 1 contains, N-terminus to C-terminus, the N-terminus, a peptide (linker 3; DKTHTCPPCP (SEQ ID NO: 4)), a polypeptide that contains the CH2 and CH3 recess-bearing domains (sequence Fc domain, SEQ ID NO: 16), and C-terminus.

Thus, the third polypeptide chain of the control DART 1 consists of SEQ ID NO: 4 - SEQ ID NO: 16.

The amino acid sequence of the third polypeptide chain of the control DART 1 is (SEQ ID NO: 39):

DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVK

PGK.

2. Control DART 2 (fluorescein x CD19).

The first polypeptide chain of the control DART 2 contains, N-terminus to C-terminus, N-terminus, the VL domain of a monoclonal antibody capable of binding to CD19 (VL _CD19 ) (SEQ ID NO: 17), an intermediate linker peptide (linker 1 ; GGGSGGGG (SEQ ID NO: 1)), monoclonal antibody VH domain capable of binding to fluorescein (VH _Fluor ) (SEQ ID NO: 42), intermediate linker peptide (linker 2; GGCGGG (SEQ ID NO: 3)), heterodimer -promoter (E-helical) domain (EVAALEK-EVAALEK-EVAALEK-EVAALEK (SEQ ID NO: 10)), intermediate linker peptide (spacer-linker 3; GGGDKTHTCPPCP (SEQ ID NO: 5)), a polypeptide that contains a bearing protrusion the CH2 and CH3 domains (Fc domain sequence, SEQ ID NO: 15), and the C-terminus.

Thus, the first polypeptide chain of the control DART 2 consists of SEQ ID NO: 17 - SEQ ID NO: 1 - SEQ ID NO: 42 - SEQ ID NO: 3 - SEQ ID NO: 10 - SEQ ID NO: 5 - SEQ ID NO : 15.

The amino acid sequence of the first polypeptide chain of the control DART 2 is (SEQ ID NO: 45):

- 24 040028

ENVLTQSPATLSVTPGEKATITCRASQSVSYMHWYQQKPGQAPRLLIYDASNR

ASGVPSRFSGSGSGTDHTLTISSLEAEDATYYCFQGSVYPFTFGQGTKLEIKGGGSG

GGGEVKLDETGGGLVQPGRPMKLSCVASGFTFSDYWMNWVRQSPEKGLEWVAQIR NKPYNYEIYYSDSVKGRFTISRDDSKSSVYLQMNNLRVEDMGIYYCTGSYYGMDY

WGQGTSVTVSSASTKGEVAACEKEVAALEKEVAALEKEVAALEKGGGDKTHTCPPC PAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHN AKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQP REPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

The second polypeptide chain of the control DART 2 contains, N-terminus to C-terminus, N-terminus, the VL domain of a monoclonal antibody capable of binding to fluorescein (VLFluor) (SEQ ID NO: 41), an intermediate linker peptide (linker 1; GGGSGGGG (SEQ ID NO: 1)), VH domain of a monoclonal antibody capable of binding to CD3 (VH _CD3 ) (SEQ ID NO: 29), intermediate linker peptide (linker 2; GGCGGG (SEQ ID NO: 3)), heterodimer- a promoter (K-helical) domain (KVAALKE-KVAALKE-KVAALKE-KVAALKE (SEQ ID NO: 11) and a C-terminus.

Thus, the second polypeptide chain of the control DART 2 consists of SEQ ID NO: 41 - SEQ ID NO: 1 - SEQ ID NO: 29 - SEQ ID NO: 3 - SEQ ID NO: 11.

The amino acid sequence of the second polypeptide chain of the control DART 2 is (SEQ ID NO: 46):

QAVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGG

TNKRAPWTPARFSGSLLGKAALTITGAQAEDEADYYCALWYSNLWVFGGGTKLT VLGGGGSGGGGEVKLDETGGGLVQPGRPMKLSCVASGFTFSDYWMNWVRQSPEKG LEWVAQIRNKPYNYETYYSDSVKGRFTISRDDSKSSVYLQMNNLRVEDMGIYYCTGS YYKEGMDYWGQGTSVAKETVAKASTKAL

The third polypeptide chain of the control DART 2 contains, in the N-terminal to C-terminal direction, the N-terminus, a peptide (linker 3; DKTHTCPPCP (SEQ ID NO: 4)), a polypeptide that contains the CH2 and CH3 recess-bearing domains (sequence Fc domain, SEQ ID NO: 16), and C-terminus.

Thus, the third polypeptide chain of the control DART 2 consists of SEQ ID NO: 4 - SEQ ID NO: 16.

The amino acid sequence of the third control DART 2 polypeptide is (SEQ ID NO: 39):

DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVK

FN W Υ V DG V E VH N A KTK P RE EQ YN STYRVV S VLTVLHQDWLNGKEYKCKV SNKALP

APIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQP ENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNRYTQKSLSLS

PGK.

E. Pharmaceutical compositions.

Compositions in accordance with the present invention include bulk drug compositions useful in the manufacture of pharmaceutical compositions (i.e., crude or non-sterile compositions) and pharmaceutical compositions (i.e., compositions that are acceptable for administration to a subject or patient) that can be used in the preparation of unit dosage forms. Such compositions comprise a prophylactically or therapeutically effective amount of the anti-CD19 x CD3 bispecific monovalent diabodies of the present invention, or a combination of such agents, and a pharmaceutically acceptable carrier. Preferably, the compositions of the present invention comprise a prophylactically or therapeutically effective amount of a CD19 x CD3 bispecific monovalent diabody of the present invention and a pharmaceutically acceptable carrier.

The present invention also encompasses pharmaceutical compositions comprising an anti-CD19 x CD3 bispecific monovalent diabody according to the present invention (and in particular an anti-CD19 x CD3 monovalent Fc diabody) 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 specific embodiment, the term pharmaceutically acceptable means approved by a federal or state government regulatory agency or listed in the USP or other generally accepted pharmacopoeia for use in animals, and more specifically, in humans. The term carrier refers to the diluent, adjuvant (e.g., Freund's adjuvant (complete and incomplete), adjuvant, or vehicle with which the therapeutic agent is administered. Such pharmaceutical carriers may be sterile liquids such as water and oils, including oils from petroleum, animal, vegetable or synthetic origin

- 25 040028 denium, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. Water is the preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous solutions of dextrose and glycerol can also be used as liquid carriers, especially for injectable solutions. Acceptable 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 etc. The composition may also optionally contain minor amounts of wetting or emulsifying agents or pH buffering agents. Such compositions may take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained release formulations, and the like.

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

Compositions in accordance with the present invention may be formulated in neutral form or in salt form. Pharmaceutically acceptable salts include, but are not limited to, salts derived from anions such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., as well as salts derived from cations such as cations derived from sodium, potassium, ammonium, calcium, iron hydroxides, isopropylamine, triethylamine, 2ethylaminoethanol, histidine, procaine, etc.

The present invention also relates to a pharmaceutical package or kit containing one or more containers filled with a CD19 x CD3 bispecific monovalent diabody according to the present invention (and in particular an anti-CD19 x CD3 bispecific monovalent Fc diabody) alone or with such a pharmaceutically acceptable carrier. . In addition, one or more other prophylactic or therapeutic agents useful in the treatment of a disease may also be included in a pharmaceutical package or kit. The present invention also relates to a pharmaceutical package or kit containing one or more containers filled with one or more of the ingredients of the pharmaceutical compositions in accordance with the present invention. Optionally, such container(s) may be associated with an explanation in the form prescribed by a government agency regulating the manufacture, use, or sale of pharmaceuticals or biological products, the explanation reflecting that authority's approval of the manufacture, use, or sale for administration to humans.

The present invention relates to kits that can be used in the above methods. The kit may contain bispecific CD19 x CD3 monovalent diabodies and, more preferably, a CD19 x CD3 bispecific monovalent Fc diabody according to the present invention. The kit may further comprise one or more other prophylactic and/or therapeutic agents useful in the treatment of cancer in one or more containers; and/or the kit may further comprise one or more cytotoxic antibodies that bind one or more cancer antigens associated with the cancer. In some 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.

F. Methods of administration.

Compositions in accordance with the present invention may be provided for the treatment, prevention and alleviation of one or more symptoms associated with a disease, disorder or infection by administering to a subject an effective amount of a fusion protein or conjugate molecule in accordance with the present invention or a pharmaceutical composition containing a fusion protein or a conjugated molecule according to the present invention. In a preferred aspect, such compositions are substantially purified (ie substantially free of substances that limit their effect or cause unwanted side effects). In a specific embodiment, the subject is an animal, preferably a mammal, such as a non-primate (e.g., bovine, equine, feline, canine, rodent, etc.) or a primate (e.g., a monkey such as a cynomolgus macaque, human, etc.). d.). In 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, receptor-mediated endocytosis (see, for example, Wu et al (1987) Receptor-Mediated In Vitro Gene Transformation By A Soluble DNA Carrier System, J Biol Chem 262:4429-4432), construction of a nucleic acid as part of a retroviral or other vector, etc.

Methods for administering a molecule according to the present invention include, without limitation, parenteral administration (eg, intradermal, intramuscular, intraperitoneal, intravenous, and subcutaneous), epidural, and mucosal (eg, intranasal and oral routes). In a specific embodiment, the CD19 x CD3 bispecific monovalent diabodies or CD19 x CD3 bispecific monovalent Fc diabodies of the present invention are administered intramuscularly, intravenously or subcutaneously. The compositions may be administered by any conventional route, such as infusion or bolus injection, absorption through epithelial or mucocutaneous membranes (eg, oral mucosa, rectal mucosa and intestinal mucosa, etc.), as well as can be administered together with other biologically active agents. The introduction can be systemic or local. In addition, pulmonary administration may also be used, for example by use of an inhaler or nebulizer and an aerosol formulation. See, for example, US Pat. 99/66903, each of which is incorporated herein by reference in its entirety.

The present invention also relates to such CD19 x CD3 bispecific monovalent diabodies (and in particular to CD19 x CD3 bispecific monovalent Fc diabodies) according to the present invention, which are packaged in a sealed container, such as an ampoule or sachet, with indicating the number of molecules. In one embodiment, the anti-CD19 x CD3 bispecific monovalent diabodies of the present invention are supplied as a dry, sterilized, lyophilized powder or anhydrous concentrate in a sealed container and can be reconstituted, for example, with water or saline, to a concentration suitable for administration to a subject. Preferably, the anti-CD19 x CD3 bispecific monovalent diabodies of the present invention are supplied as a dry, sterilized, lyophilized powder in a sealed container at a unit dosage of at least 5 µg, more preferably at least 10 µg, at least 15 µg, at least at least 25 µg, at least 50 µg, at least 100 µg, or at least 200 µg.

Lyophilized anti-CD19 x CD3 bispecific monovalent diabodies or anti-CD19 x CD3 bispecific monovalent Fc diabodies according to the present invention should be stored at 2-8° C. in their original container and the molecules should be administered within 12 hours, preferably within 6 hours , within 5 hours, within 3 hours, or within 1 hour after recovery. In an alternative embodiment, the CD19 x CD3 bispecific monovalent diabodies of the present invention are supplied in liquid form in a sealed container, indicating the amount and concentration of the molecule, fusion protein, or conjugated molecule. Preferably, the liquid form of the anti-CD19 x CD3 bispecific monovalent diabodies of the present invention is supplied in a sealed container in which the molecules are present at a concentration of at least 1 µg/mL, more preferably at least 2.5 µg/mL, at least at least 5 µg/ml, at least 10 µg/ml, at least 50 µg/ml or at least 100 µg/ml.

The amount of a composition according to the present invention that will be effective in treating, preventing or alleviating one or more of the symptoms associated with the disorder can be determined by standard clinical procedures. The precise dosage used in the formulation will also depend on the route of administration and the severity of the condition, and will also be determined by the judgment of the practitioner and the circumstances of each patient. Effective doses can be extrapolated from dose-response curves obtained using in vitro test systems or animal models.

For CD19 x CD3 bispecific monovalent diabodies or CD19 x CD3 bispecific monovalent Fc diabodies encompassed 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 at least about 0.3 ng/kg per day to about 0.9 ng/kg per day, at least about 1 ng/kg per day to about 3 ng/kg per day , from at least about 3 ng/kg per day to about 9 ng/kg per day, from at least about 10 ng/kg per day to about 30 ng/kg per day, from at least about 30 ng/kg per day to about 90 ng/kg per day, from at least about 100 ng/kg per day to about 300 ng/kg per day, from at least about 200 ng/kg per day to about 600 ng/kg per day , from at least about 300 ng/kg per day to about 900 ng/kg per day, from at least about 400 ng/kg per day to about 800 ng/kg per day, from at least about 500 ng/kg per day to about 1000 ng/kg per day, from at least about 600 ng/kg per day to about 1000 ng /kg per day, from to

- 27 040028 at least about 700 ng/kg per day to about 1000 ng/kg per day, from at least about 800 ng/kg per day to about 1000 ng/kg per day, from at least about 900 ng/kg per day to about 1000 ng/kg per day, or at least about 1000 ng/kg per day.

In another embodiment, the patient is administered according to a treatment regimen comprising one or more doses of such a prophylactically or therapeutically effective amount of CD19 x CD3 bispecific monovalent diabodies (and in particular CD19 x CD3 bispecific monovalent Fc diabodies) encompassed by the present invention, while the treatment regimen is carried out for 2, 3, 4, 5, 6 or 7 days. In some embodiments, the treatment regimen comprises intermittent dosing of a prophylactically or therapeutically effective amount of the anti-CD19 x CD3 bispecific monovalent diabodies encompassed by the present invention (e.g., dosing on Day 1, Day 2, Day 3, and Day 4 of a given week and not dosing prophylactically). or a therapeutically effective amount of the anti-CD19 x CD3 bispecific monovalent diabodies (and in particular the anti-CD19 x CD3 bispecific monovalent Fc diabodies) encompassed by the present invention on day 5, day 6 and day 7 of the same week). As a rule, 1, 2, 3, 4, 5 or more courses of treatment are carried out. Each course may have the same regimen or a different regimen.

In another embodiment, the administered dose is increased during the first quarter, first half, or first two-thirds or three-quarters of the regimen(s) (e.g., during the first, second, or third 4-course regimens) until a daily prophylactically or therapeutically effective amount is reached. bispecific anti-CD19 x CD3 monovalent diabodies (and in particular anti-CD19 x CD3 bispecific monovalent Fc diabodies) encompassed by the present invention.

In table. 2 shows 5 examples of the various dosing regimens described above for a typical course of treatment.

table 2

Mode Day Dosage of diabody (i.e. diabody per kg of subject's weight per day) 1 1,2,3,4 100 100 100 100 100 5,6,7 absent absent absent absent absent 2 1,2,3,4 300 500 700 900 1,000 5,6,7 absent absent absent absent absent 3 1,2,3,4 300 500 700 900 1,000 5,6,7 absent absent absent absent absent 4 1,2,3,4 300 500 700 900 1,000 5,6,7 absent absent absent absent absent

The dosage and frequency of administration of CD19 x CD3 bispecific monovalent diabodies or CD19 x CD3 bispecific monovalent Fc diabodies according to the present invention can be reduced or altered by enhancing tissue uptake and permeability of the CD19 x CD3 bispecific monovalent diabodies using modifications such as such as lipidization.

The dosage of CD19 x CD3 bispecific monovalent diabodies or CD19 x CD3 bispecific monovalent Fc diabodies according to the present invention administered to a patient can be calculated for use as monotherapy. Alternatively, the CD19 x CD3 bispecific monovalent diabodies or CD19 x CD3 bispecific monovalent Fc diabodies of the present invention are used in combination with other therapeutic compositions and the dosage administered to the patient is lower than when these molecules are used as monotherapy. .

Pharmaceutical compositions in accordance with the present invention may be administered locally at the site 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 a porous, non-porous or gelatinous material, including membranes such as elastic membranes or fibers. Preferably, when administering a molecule in accordance with the present invention, care should be taken when using materials that the molecule does not absorb.

Compositions in accordance with the present invention can be delivered in a vesicle, in particular in 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, p. 353-365 (1989); Lopez-Berestein, ibid., p. 317-327).

Compositions in accordance with the present invention can be delivered in a controlled release or sustained release system. Any technique known to the skilled person

- 28 040028 in the art can be used to prepare sustained release formulations containing one or more anti-CD19 x CD3 bispecific monovalent diabodies in accordance with the present invention. See, for example, US Pat. No. 4,526,938, PCT Publication WO 91/05548; PCT publication WO 96/20698; Ning et al. (1996) Intratumoral Radioimmunotheraphy Of A Human Colon Cancer Xenograft Using A Sustained-Release Gel, Radiotherapy & Oncology 39:179-189, Song et al. (1995) Antibody Mediated Lung Targeting Of Long-Circulating Emulsions, PDA Journal of Pharmaceutical Science & Technology 50:372-397; Cleek et al. (1997) Biodegradable Polymeric Carriers For A bFGF Antibody For Cardiovascular Application, Pro. Int'l. Symp. control. Rel. Bioact. mater. 24:853-854; and Lam et al. (1997) Microencapsulation Of Recombinant Humanized Monoclonal Antibody For Local Delivery, Proc. Int'l. Symp. Control Rel. Bioact. mater. 24:759-760, each of which is hereby incorporated by reference in their entirety. In one embodiment, a pump can be used in a controlled release system (see Langer, supra; Sefton, (1987) Implantable Pumps, CRC Crit. Rev. Biomed. Eng. 14:201-240; Buchwald et al. (1980) Long -Term, Continuous Intravenous Heparin Administration By An Implantable Infusion Pump In Ambulatory Patients With Recurrent Venous Thrombosis, Surgery 88:507-516 and Saudek et al. Engl. J. Med. 321:574-579). According to 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 Neurol 25:351-356 Howard et al (1989) Intracerebral Drug Delivery In Rats With Lesion-Induced Memory Deficits, J Neurosurg 7(1):105-112); US patent No. 5679377; US patent No. 5916597; US patent No. 5912015; US patent No. 5989463; US patent No. 5128326; PCT Publication No. WO 99/15154 and PCT Publication No. WO 99/20253). Examples of polymers used in sustained release formulations include, but are not limited to, poly(2-hydroxyethyl methacrylate), poly(methyl methacrylate), poly(acrylic acid), ethylene vinyl acetate, poly(methacrylic acid), polyglycolides (PLG), polyanhydrides, poly(N-vinylpyrrolidone), poly(vinyl alcohol), polyacrylamide, poly(ethylene glycol), polylactides (PLA), lactide-glycolide copolymer (PLGA), and polyorthoesters. The controlled release system can be placed close to the therapeutic target (eg, in the lungs), thus requiring only a fraction of the systemic dose (see, for example, Goodson, in Medical Applications of Controlled Release, supra, vol. 2, p. 115-138 (1984)). Polymer compositions useful as controlled release implants can be used according to Dunn et al. (see US patent No. 5945155). This particular method is based on the therapeutic effect of the in situ controlled release of the bioactive material from the polymer system. Implantation, as a rule, can be carried out in any place in the body of the patient, if necessary, therapeutic treatment. You can use a non-polymeric continuous delivery system, which uses a non-polymeric implant in the body of the subject as a drug delivery system. Upon implantation into the body, the organic solvent of the implant will disperse, disperse, or be carried out of the composition into the surrounding tissue fluid, and the non-polymeric material will gradually coagulate or precipitate to form a solid microporous matrix (see US Pat. No. 5,888,533).

Controlled release systems are discussed in a review by Langer (1990, New Methods Of Drug Delivery, Science 249:1527-1533). Any technique known to the person skilled in the art may be used to prepare sustained release formulations containing one or more therapeutic agents in accordance with the present invention. See, for example, US patent No. 4526938; International Application Publication Nos. 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 & Oncology 39:179-189, Song et al. (1995) Antibody Mediated Lung Targeting Of LongCirculating Emulsions, PDA Journal of Pharmaceutical Science & Technology 50:372-397; Cleek et al. (1997) Biodegradable Polymeric Carriers For A bFGF Antibody For Cardiovascular Application, Pro. Int'l. Symp. control. Rel. Bioact. mater. 24:853-854; and Lam et al. (1997) Microencapsulation Of Recombinant Humanized Monoclonal Antibody For Local Delivery, Proc. Int'l. Symp. Control Rel. Bioact. mater. 24:759-760, each of which is hereby incorporated by reference in their entirety.

If the composition according to the present invention is a nucleic acid encoding a CD19 x CD3 bispecific monovalent diabody according to the present invention, the nucleic acid may be administered in vivo to allow expression of the CD19 x CD3 bispecific monovalent diabody or the bispecific monovalent Fc diabody it encodes. CD19 x CD3 by constructing it as part of a suitable nucleic acid expression vector and introducing it in such a way that it becomes intracellular, for example, by using a retroviral vector (see US patent No. 4980286), or by direct injection

- 29 040028 injection, or by applying bombardment with microparticles (for example, gene gun, Biolistic, Dupont), or by coating with lipids, or by using cell surface receptors or transfecting agents, or by introducing it into association with a homeobox-like peptide, which, as known to be in the nucleus (see, e.g., Joliot et al. (1991) Antennapedia Homeobox Peptide Regulates Neural Morphogenesis, Proc. Natl. Acad. Sci. (U.S.A.) 88:1864-1868), etc. Alternatively, the nucleic acid can be introduced intracellularly and incorporated into the DNA of the host cell for expression by homologous recombination.

Treatment of a subject with a therapeutically or prophylactically effective amount of an anti-CD19 x CD3 bispecific monovalent diabody (and in particular an anti-CD19 x CD3 bispecific monovalent Fc diabody) according to the present invention may involve treatment with a single dose or, preferably, may involve treatment with a series of doses. In a preferred example, the subject is treated with such a diabody once a week for about 1 to 10 weeks, preferably 2 to 8 weeks, more preferably about 3 to 7 weeks, and even more preferably for about 4, 5, or 6 weeks. weeks. Pharmaceutical compositions according to the present invention may 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, once every six weeks, once every two months, twice a year, or once every year. It will also be understood that the effective dosage of the molecules used for treatment may increase or decrease over the course of a particular treatment.

G. Uses of compositions according to the present invention.

The CD19 x CD3 bispecific monovalent diabodies of the present invention (and in particular the CD19 x CD3 bispecific monovalent Fc diabodies of the present invention) are capable of treating any disease or condition associated with or characterized by CD19 expression. Thus, without limitation, such molecules can be used in the diagnosis or treatment of acute myeloid leukemia (AML), chronic myelogenous leukemia (CML), including CML blast crisis and CML-associated Abelson oncogene (Bcr-ABL translocation), myelodysplastic syndrome. (MDS), acute B-cell lymphoblastic leukemia (B-ALL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, chronic lymphocytic leukemia (CLL), including Richter's syndrome or Richter's transformation in CLL, hairy cell leukemia ( HCL), neoplasms from blast plasmacytoid dendritic cells (BPDCN), non-Hodgkin's lymphomas (NHL), including mantle cell leukemia (MCL) and small cell lymphocytic lymphoma (SLL), Hodgkin's lymphoma, systemic mastocytosis and Burkitt's lymphoma; autoimmune lupus (SLE), allergies, asthma and rheumatoid arthritis. The bispecific monovalent diabodies of the present invention, as an adjunct, can be used in the manufacture of medical devices for the treatment of the conditions described above.

Examples

The present invention described as a whole will be better understood by reference to the following examples, which are presented for the purpose of illustration and are not intended to limit the present invention unless otherwise indicated.

Example 1

Quantification of CD19 expression on the cell surface.

To identify acceptable target cell lines when evaluating the biological activity of bispecific diabodies to CD19 x CD3, the level of CD19 expression on the cell surface was first confirmed using quantitative FACS (QFACS) in a panel of human B-cell lymphoma/leukemia cell lines, including Nalm-6 ( acute lymphoblastic leukemia), Raji (Burkitt's lymphoma), Daudi (Burkitt's lymphoma), HBL-2 (mantle cell lymphoma), MEC-1 (chronic lymphocytic leukemia) and Jeko-1 (mantle cell lymphoma), MOLM- 13 (acute myeloid leukemia cell line), JIMT-1 (breast cancer) and Colo205 (colon cancer). The absolute number of CD19 antibody binding sites on the surface was calculated using the QFACS kit. As shown in Table. 3, the absolute number of CD19 binding sites in cell lines ranked in the following order: Raji (high) > Nalm-6 (medium) > Daudi (medium) > HBL2 (medium) > MEC-1 (low) > Jeko-1 (low). As expected, CD19 expression in MOLM-3, JIMT-1 and Colo205 CD19 was absent, consistent with CD19 being a B cell specific antigen.

- 30 040028

Table 3

Target cell line Expression of CD 19 on the surface (antibody binding sites) Raji 670322 Nalm-6 442730 Daudi 369860 HBL-2 334725 MEC-1 177025 Jeko-1 124396

Example 2

Binding affinities.

To quantify the extent of binding of a bispecific monovalent diabody against human or cynomolgus CD19 x CD3 and CD3, BIACORE™ assays were performed using an exemplary anti-CD19 x CD3 bispecific monovalent Fc diabody, DART-A. In BIACORE™ assays, the dissociation rate kd was measured. The binding affinity (KD) between an antibody and its target is a function of the rate constants of the association reaction (association rate ka) and dissociation (dissociation rate kd) according to the formula: KD=[kd]/[ka]. The BIACORE™ assay used surface plasmon resonance to directly measure these kinetic parameters.

Binding results of DART-A (0-100 nM) to soluble human or cynomolgus CD3 and CD19 were analyzed using Surface Plasmon Resonance (SPR) technology (BIAcore) as shown in Table 1. 4.

The binding affinity of DART-A is similar for human or cynomolgus CD3 (KD=21.2 and 21.9 nM, respectively). However, DART-A has an affinity for cynomolgus CD19 that is approximately 10-fold lower than that for human CD19 (KD=20.3 and 2.0 nM, respectively) due to an increased dissociation rate constant (kd) for cynomolgus CD19. toque. The cynomolgus monkey remains a suitable species for toxicological assessments, although differences in CD19 binding affinity should be considered.

Table 4

ka (± SD) kd (±SD) KD (± SD) Antigens (M-ls-1) (s-1) (pM) CD3e/6 human 2.2 x 10 ⁵ 4.5 (±0.1)x10' ³ 21.2 (± 1.7) CD3e/5 cynomolgus macaque 2.0 (± 0.1) x 10 ⁵ 4.3 (±0.2) x ΙΟ' ³ 21.9 (± 1.2) human CD19-His 2.8 (± 0.3) x 10 ⁵ 5.6 (± 0.6) x 10 ⁴ 2.0 (± 0.2) CD19-His cynomolgus macaque 2.4 (± 0.1) x 10 ⁵ 4.7 (± 1.1) x10' ³ 20.3 (± 1.3)

Example 3

Characteristics of cell binding.

Binding of DART-A to blood leukocytes (purified from whole blood) from mice, rats, rabbits, cynomolgus monkeys and humans was assessed in vitro by flow cytometry. Leukocytes were stained with DART-A and control DART 1 and control DART 2 at concentrations of 25 or 100 nM for approximately 1 hour. Control DART 2 contains the same CD19 binding component as DART-A, while control DART 1 contains the same CD3 binding component as DART-A. After incubation, DART proteins bound to leukocytes were detected with an anti-E-helix/K-helix (EK) mAb that recognizes the region of heterodimerization of the EK helix of DART proteins. He observed DART-A binding in mouse, rat or rabbit blood leukocytes. Similarly, none of the DART control diabodies showed any binding to mouse, rat, or rabbit leukocytes. As expected, both concentrations of test DART-A showed specific binding to both human and cynomolgus leukocytes.

A bifunctional ELISA assay was used to demonstrate the simultaneous interaction of both target antigens with DART-A. ELISA plates were coated with soluble human CD3ε/δ heterodimer and incubated at 4° C. overnight and then blocked with BSA. Various concentrations of DART-A were added and then soluble human CD19-6uotuh was added. The plates were washed and the immune complex was detected with a chemiluminescent substrate conjugated with streptavidin. As shown in FIG. 3, DART-A is able to bind to both CD19 and CD3 simultaneously.

Further, the bispecific binding of DART-A in blood leukocytes (purified from whole blood) of both cynomolgus monkey and human was evaluated by flow cytometry. Leukocytes stain

- 31 040028 Valid with DART-A, DART 1 control, or DART 2 control at concentrations ranging from 0.005 to 80 nM for approximately 1 hour. After incubation, binding to leukocytes was detected using a biotinylated anti-E-helix/K-helix mAb (EK), which recognizes the heterodimerization region of the EK helix of the DART protein and streptavidin-PE. Because DART-A binds to CD3, a combination of CD4 and CD8 was used to determine the T cell population. Similarly, CD20 was used as a B cell marker instead of CD19. Therefore, in this study, events gated as CD4+ and CD8+ represent the T cell population, and events gated as CD20+ represent the B cell population. DART-A exhibited bispecific binding to both human and cynomolgus B cells and T cells. In contrast, control DART diabodies showed only monospecific binding to B cells (control DART 2) or T cells (control DART 1) according to their respective binding specificity. Binding titer curves of diabodies DART-A and control DART for human and cynomolgus B and T cells are shown in FIG. 4A-4B and FIG. 5A-5B.

Example 4

DART-A mediated redirected killing of target tumor cells using human T cells.

The ability of DART-A to mediate redirected killing of target cells was assessed on 3 target cell lines of B-cell lymphoma Raji/GF (Burkitt's lymphoma), HBL-2 (mantle cell lymphoma) and Jeko-1 (mantle cell lymphoma) demonstrating CD19 expression range using purified human primary T cells as effector cells. Raji/GF cells showed the highest levels of CD19 expression, followed by average levels of CD19 expression in HBL-2 cells and lower levels of CD19 expression in Jeko-1 cells. Killing of target tumor cells was measured using a lactate dehydrogenase (LDH) release assay, in which the enzymatic activity of LDH released from cells as a result of cell death was quantified, or using a luciferase assay, in which the relative light unit (RLU) of luciferase is an indicator for determining relative viability of Raji/GF target cells that were engineered to express both green fluorescent protein (GFP) and luciferase reporter genes.

DART-A demonstrated potent redirected cell killing in all 3 cell lines using purified T cells from several independent human donors at an effector to target cell ratio (E:T) of 10:1 (FIGS. 6A-6C). The mean effective concentration at 50% maximal activity (EC ₅₀ ) values was 1.05/10 ^-1 pM for HBL-2 cells, 3.07x10 ^-1 pM for Raji/GF cells and 1.46x10 ^-1 pM for Jeko- cells. 1 (Table 5). Control DART 1 did not mediate T-cell redirected killing of target cells. Most importantly, no cell killing activity was observed in cell lines (Molm-13 and Colo205) that do not express CD19 (Fig. 6D-6F).

Table 5

Summary of EC ₅₀ values from typical DART-A mediated redirected killing of Raji/GF target cells at varying E:T cell ratios

E:T= 10:1 E:T = 5:1 E:T = 2.5:1 Cytotoxicity ECso (pM) Yeshah (%) ECso (pM) Yeshah (%) EU so (pM) Yeshah (%) 24 hours 0.14 63 0.67 24 No data 4 48 hours 0.0441 73 0.38 48 1.08 eleven

To assess DART-A activity in the presence of a lower number of effector cells, redirected cell killing was tested at lower E:T cell ratios of 5:1, 2.5:1, and 1:1 on Raji/GF cells after incubation with DART-A. within 24-96 hours. Cytotoxic activity observed at a ratio of 5:1 was approximately half of the maximum activity observed at a ratio of 10:1; lower specific activity was also observed with the ratio

E:T cells 2.5:1 at both 24 and 48 hours (FIGS. 7A-7D and Table 5). After incubation for 72 or 96 hours, cytotoxicity against Raji/GF cells increased significantly at ratios of 2.5:1 and 1:1 (see FIGS. 8A-8D and Table 6). Of note, efficient cell lysis at lower E:T cell ratios increased in Raji/GF cells as a function of time, indicating serial killing of target cells by activated DART-A T cells. The kinetics confirm that time is the limiting factor at low T cell counts to efficiently eliminate more target cells. In conclusion, data from several experiments using human T cells from different donors show that DART-A mediated redirected cell killing activity is both E:T cell ratio and time dependent, particularly at lower E: T cells.

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Table 6

Summary of EC50 values from typical DART-A mediated redirected killing of Raji/GF target cells at lower E:T cell ratios

E:T = 2.5:1 E:T=1:1 Cytotoxicity EU ₅ about (pM) Yeshah (%) EU ₅ about (pM) Yeshah (%) 72 hours 1.54 64 2.79 33 96 hours 0.422 87 0.440 56

Example 5

DART-A mediated depletion of autologous B cells in human and cynomolgus PBMCs.

Because normal B cells express CD19, and since DART-A has been shown to cross-react with cynomolgus monkey CD19 and CD3 (Example 2, Figures 4A-4B and Figures 5A-5B), DART-A mediated cytotoxicity was investigated on peripheral blood mononuclear cells (PBMC) of both humans and cynomolgus monkeys. A dose-dependent depletion of CD20+ B cells was observed in both human and cynomolgus PBMCs following treatment with DART-A (FIGS. 9A-9B). As expected, no B cell depletion was observed after treatment with control DART 1.

To further confirm DART-A activity against PBMCs, several independent human or cynomolgus PBMC donors were used with varying ratios of T cells (effectors) to B cells (targets) E:T cells to evaluate efficacy. In the summary (Table 7), EC50 values for human B cell depletion ranged from 1.84 to 6.27 pM; while EC50 values for cynomolgus monkey B cell depletion ranged from 106.8 to 859.1 pM. The efficacy of DART-A in depleting autologous human B cells was higher than in depleting cynomolgus macaque B cells (approximately 100-fold higher on average; range 19-312-fold). However, it should be noted that the E:T cell ratio for cynomolgus monkey PBMCs (average approximately 4:1) was lower than that for human PBMCs (average approximately 8:1), which probably contributed to the greater difference in EC values. ₅₀ considering the fact that the activity of DART-A mediated redirected killing depends on the E:T cell ratio. Potential differences in CD19 expression levels between human and cynomolgus B cells may represent another variable factor contributing to the apparent difference in potency.

Table 7

Summary of EC ₅₀ values for autologous B cell depletion

ECso values (pM) ECso values (pM) DART- A Control DART DART-A Control DART Man 5 (E:T=8:1) 6.27 Lack of activity Javanese macaque 5 (E:T=3:1) 166.2 Lack of activity Person 6 (E:T=8:1) 2.75 Lack of activity Javanese macaque 6 (E:T=2:1) 859.1 Lack of activity Man 7 (E:T=5:1) 5.52 Lack of activity Javan macaque 7 (E:T=3:1) 106.8 Lack of activity Person 8 (E:T=9:1) 1.84 Lack of activity Javanese macaque 8 (E:T=7:1) 239.1 Lack of activity Average 4.20 Not applicable Average 342.8 Not applicable

Importantly, DART-A mediated effective cytotoxicity against Raji/GF target cells using cynomolgus monkey PBMC as effector cells with an EC ₅₀ of 1.30x10 ^-2 pM (FIG. 10) was similar to that observed using human T cells as effector cells (mean EC ₅₀ =3.07x10 ^-1 pM), indicating that human and cynomolgus monkey T cells have comparable activity against the same target cell line. These data further support the use of the cynomolgus monkey as a suitable species for evaluating DART-A in non-clinical toxicology studies.

Example 6

Evaluation of DART-A mediated cytokine release using human or cynomolgus effector cells.

DART-A mediated B cell depletion in human and cynomolgus PBMCs was associated with cytokine release in both human and cynomolgus PBMCs. As briefly described below in Table. 8, EC ₅₀ values for cytokine release are equivalent to those

- 33 040028 or exceed those for autologous human or cynomolgus B cell depletion, indicating that DART-A is more effective in mediating B cell depletion than in inducing cytokine release in vitro. Cytokine release profiles between humans and cynomolgus monkeys in vitro show some similarities and differences. IFN-γ and TNF-α were the predominant cytokines released in both species after treatment with DART-A. However, IL-6 was the third most predominant cytokine produced in humans based on Emax values, while IL-2 was only produced in significant amounts in cynomolgus monkey PBMC. Comparison of mean EC50s for the most sensitive cytokines produced in PBMC after incubation with DART-A (IFN-γ, TNF-α and IL-6 in humans) with mean EC50s for depletion of autologous B cells showed the presence of at least 6 -fold detectable margin of safety, which is reflected in the difference between B-cell depletion and cytokine production in humans (Table 9).

Table 8

Brief description of EC50 values for autologous B-cell depletion and cytokine production after human or cynomolgus PBMC with DART-A

human RVMS Analysis (number of donors tested) Mean ECso (pM) Depletion of human B cells (n = 4) 4.20 Human cytokine release (n = 2) IFN-γ 25.21 TNF-a 31.83 IL-10 70.11 IL-6 23.55 IL-4 No data* IL-2 56.77 RVMS cynomolgus monkey Depletion of cynomolgus macaque B cells (n = 4) 342.8 Cytokine release from cynomolgus monkey (n = 2) IFN-γ 925.24 TNF-a 262.22 IL-6 306.68 IL-5 486.22 IL-4 1718.63 IL-2 2930.19

* Below detection limit.

Table 9

Summary of EC50 values for cytotoxicity versus cytokine production following in vitro evaluation of DART-A in human cells

CTL ¹ Analysis Human RVMS ² Analysis (number of donors) Average EC ₅₀ (pM) Analysis (number of donors) Mean ECso (pM) Cytotoxicity exhaustion autologous B cells HBL-2 (n=5) 0.11 RVMS (n = 7) 4.20 Raji/GF (η = 5) 0.31 Jeko-1 (n=3) 0.15 Cytokine release (η = 2) Cytokine release (n = 2) IFN-γ 5.51 IFN-γ 25.21

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TNF-a 3.71 TNF-a 31.83 IL-2 6.32 IL-6 23.55 Multiplicity of difference EU ₅₀ (indicative 12-58 Multiplicity of difference EU ₅ o (approximate 6-8 safety margin) safety margin)

¹ Activity was assessed using CTL assay with CD19 expressing tumor cells (HBL-2, Raji/GF or Jeko-1) in the presence of purified human T cells (E:T=10:1). Cytokine release was assessed using Raji/GF tumor cells.

² Activity was assessed in normal human PBMCs.

DART-A mediated cytokine release by T cells in the presence of target cells was also investigated. Dose dependent DART cytokine production TNF-α, IFN-γ and IL-2 was observed from human T cells with the highest levels (Emax) observed for IL-2 (>7000 pg/mL) after co-incubation with Raji target cells /GF (FIGS. 11A-11C; Table 10). Also received significant levels of TNF-α and IFN-γ (~2000-4000 pg/ml). As expected, incubation with the DART diabody control did not result in any detectable cytokine production (FIGS. 11A-11C). When analyzing the mean _EC50 for the most sensitive cytokine produced by purified human T cells (TNF-α) after incubation with DART-A in the presence of CD19-expressing target cells (Raji/GF), compared to the mean _EC50 for cytotoxicity in against target cells of at least sensitive target cell line (Raji/GF), at least a 12-fold detectable margin of safety between target cell killing and cytokine production was observed (see Table 9 above).

Table 10

_EC50 values for DART-A mediated cytokine production by human T cells in the presence of Raji/GF target cells from two independent donors at E:T=10:1

D47067 D44284 Cytokine EC50 (pM) Yesah (pg/ml) EU ₅ about (pM) Yesah (pg/ml) IFN-γ 8.58 3885 2.44 2219 TNF-a 6.79 3722 0.626 2361 IL-2 11.31 8496 1.33 7492

Example 7

Evaluation of DART-A mediated activation of human and cynomolgus macaque T cells.

The effect on T cells during DART-A mediated redirected lysis of target cells (Raji/GF cells) was characterized by assessing the expression of T cell activation markers CD69 and CD25. Both CD25 and CD69 were upregulated in human and cynomolgus monkey CD4+ and CD8+ T cells in a dose dependent manner (FIGS. 12A-12D and FIGS. 13A-13D), indicating that DART-A mediated redirected cell killing is associated with simultaneous activation of T-cells. No T cell activation was observed in the presence of CD19-negative JIMT-1 cells (FIG. 14A14D). These data confirm that T-cell activation depends on the cooperative interaction of target cells. Further confirming this conclusion, control DART 1 did not mediate the induction of T cell activation markers (FIGS. 12A-12D and FIGS. 13A-13D).

Example 8

Evaluation of the mechanism of action of CD19 x CD3.

To confirm that the putative mechanism of CD19 x CD3 mediated redirected cell killing by T cells is mediated by granzyme B and perforin, intracellular levels of granzyme B and perforin in T cells were measured after exposure to DART-A or control DART 1 in the CTL assay. Dose-dependent activation of granzyme B and perforin levels in both CD8+ and CD4+ T cells was observed after 24-hour incubation of human T cells with DART-A in the presence of Raji/GF target cells at an E:T cell ratio of 10: 1 (FIGS. 15A-15B). In contrast, no activation of granzyme B or perforin was observed when human T cells were incubated with Raji/GF target cells and control DART 1. These data suggest that DART-A mediated killing of target cells can be mediated through the granzyme B and perforin pathways. Of note, activation of granzyme B (FIG. 15A) and perforin (FIG. 15B) was higher in CD8+ T cells compared to CD4+ T cells, indicating that CD8+ T cells are expected to be more effective against CTL.

We also assessed the expansion of T cells in the joint effector:target cell interaction using CD9 x CD3, taking into account the fact that proliferation simultaneously with T-cell activity

- 35 040028 vation was registered earlier. CFSE-labeled human T cells were co-cultured with HBL-2 target cells at an E:T cell ratio of 10:1 in the presence of DART-A or control DART 1 at a concentration of 200 ng/ml. Proliferation of CFSE-labeled T cells was monitored by CFSE dilution levels versus time using FACS analysis (FIGS. 16A-16B). In FIG. 16A shows CFSE staining profiles in the presence of DART-A or control DART1 and target cells. The addition of DART-A resulted in proliferation (dilution with CFSE) with approximately 75% cell proliferation after 72 hours of incubation (FIG. 16B). In contrast, no proliferation of CFSE-labeled T cells was observed in the presence of control DART 1.

Example 9

Efficacy in mixed xenografts.

Inhibition of human B-cell lymphoma tumor growth by DART-A was evaluated in female NOD/SCID mice (n=8/group) injected with either HBL-2 (human mantle cell lymphoma) or Raji (Burkitt's) tumor cells, mixed with activated human T cells. In both studies, human T cells and tumor cells (HBL-2 or Raji) were pooled at a ratio of 1:5 (1x106 and 5x106 cells, respectively) and injected into SC mice on day 0. Medium was then injected as a control (tumor cells only), DART-A or control DART 1 IV on days 0, 1, 2 and 3.

As shown in FIG. 17, growth of HBL-2 tumor cells was completely inhibited after IV treatment with DART-A at dose levels >0.2 μg/kg. Although treatment with 0.02 μg/kg DARTA appeared to slow the growth of HBL-2 tumors, the response was not statistically significant. HBL2 tumors in the medium and control DART treated groups reached an average tumor volume of approximately 1000 mm ³ on day 13.

As shown in FIG. 18, no significant growth of Raji tumor cells was observed until the end of the study (day 28) in mice treated with DART-A at all doses evaluated (0.8-100 μg/kg). Treatment with 0.8 μg/kg DART-A resulted in a complete response in 7 out of 8 mice. Raji tumors in the medium and control DART 1 treated groups showed tumor growth in vivo with mean tumor volumes reaching 2449.4±421.1 mm ³ and 2968.2±200.0 mm ³ respectively at the end of the study (Day 28) .

Example 10

Efficacy in established tumor models.

The antitumor activity of DART-A was evaluated in a xenograft model of HBL-2 (human mantle cell lymphoma) in NSG B2m -/- female mice (n=8/group). Mice were implanted with HBL-2 tumor cells (5x10 6 cells) intradermally (ID) on day 0 followed by intraperitoneal (IP) injection of PBMCs (5x10 ⁷ cells) on day 4. Treatment with medium, control DART 1 or DART-A administered IV was started. on day 17, upon reaching the average tumor volumes for the groups receiving the medium, control DART 1 or DART-A, 438.4±80.2, 433.7±63.8 and 531.7±122.5 mm ³ respectively. On day 21 (2nd dose), mean tumor volumes increased to 1306.1±240.4, 1185.6±138.7 and 958.5±216.1 mm ³ in the medium, control DART 1 and DART-A, respectively. On day 24 (3rd dose), the mean tumor volume in the media and control DART 1 groups increased to 2401.3±397.4 and 2623.7±351.5 mm ³ , respectively. In contrast, the group that received DART-A showed a 45% reduction in volume to 527.3±148.3 mm ³ . Tumors in the DART-A treated group continued to decrease in size, eventually reaching a final volume of 111.4 ± 38.9 mm ³ on day 42, an 88.3% reduction from the maximum observed tumor volume (FIG. 19A). Briefly, treatment with 0.5 mg/kg DART-A resulted in the reduction of large established lymphoma tumors from HBL-2 mantle zone cells. No relapse was noted until day 42, when the study was terminated.

Another group of tumor-bearing mice with a mean tumor size of 541 mm ³ (n=4 mice) was scheduled to be treated with 500 μg/kg DART-A on study day 14. During the first dose (study day 17), tumors reached a mean volume of 2279±61 mm ³ (FIG. 19B). However, prior to the third (study day 24) and fourth (study day 28) doses, tumor volumes were 1469±162 and 848±74 mm ³ , respectively, corresponding to a reduction of 36% and 63%, respectively.

Example 11.

Toxicokinetic study of DART-A in cynomolgus monkeys.

A non-GLP (Good Laboratory Practice) toxicology study was performed on cynomolgus monkeys to evaluate different doses and regimens of DART-A. Phase A included 1 of the 2-hour IV infusions of animal-specific escalating doses of DART-A performed for evaluation each week thereafter for 4 weeks, as summarized in Table 1. 11 (phase A). Half of the phase A cynomolgus monkeys in each group (1M/1F) were euthanized on day 36 or day 64. A phase B study was then performed to evaluate 2-hour IV infusions of DART-A given as fixed doses in each group (Table 11, phase B). For groups 3, 4, and 5, the same doses (5, 50, or 500 ng/kg, respectively) were administered every subsequent week for a total of

- 36 040028 doses per animal. Group 6 received 500 ng/kg on study days 1, 4, 8, 11, and 15 as a 2-hour

IV infusion for a total of 5 doses per animal. All phase B animals were sacrificed on day 18.

Table 11

Scheme No. groups 1 2 exp male 2 2 erimen Female 2 2 ha (phase A) Test material Wednesday DART-A DART-A Dose 1 2 3 4 5 1 2 3 4 5 Study Dose Day 1 8 15 22 29 1 8 15 22 29 Dose level (ng/kg) 0 500 5000 50000 50000 0 2000 10000 100000 100000 Dose volume (ml/kg) 0.3 0.3 0.3 Dose concentration (ng/ml) 0 1700 16700 166700 166700 0 7000 33300 333300 333300 Experimental scheme (phase B) No. groups Myself ec Female Test material Dose Day study dose administration Actually ii dose level* (ng/kg) Dose volume (ml/kg) Actual dose concentration (ng/ml) 3 1 1 DART-A 1 1 5 0.3 150 2 8 5 oh, s 150 3 15 5 oh, s 150 4 1 1 DART-A 1 1 50 oh, s 1500 2 8 50 oh, s 1500 3 15 50 oh, s 1500 5 1 1 DART-A 1 1 500 oh, s 15000 2 8 500 oh, s 15000 3 15 500 oh, s 15000 6 1 1 DART-A 1 1 500 0.3 15000 2 4 500 0.3 15000 3 8 500 0.3 15000 4 eleven 500 0.3 15000 5 15 500 0.3 15000

* Shown is the actual dose level administered based on analysis of dosage formulation concentrations. It should be noted that the actual doses were 10 times less than the protocol-defined dose levels for phase B.

1. DART-A toxicokinetic study in cynomolgus monkeys (Phase A).

A non-GLP compliant, pilot, exploratory dose escalation study of DART-A was performed in cynomolgus monkeys. In the phase A study, two groups of cynomolgus monkeys (n=4/group; 2M/2F) were administered individually escalating doses of medium (day 1) or DART-A as 2 hour IV infusions on days 1, 8, 15, 22, and 29. macaques in group 1 received medium as a control ^ 500 ^ 5000 ^ 50000 ^ 50000 ng/kg DART-A. Cynomolgus macaques in group 2 received medium as a control ^ 2000 ^ 10000 ^ 100,000 ^ 100,000 ng/kg DART-A. Half of the phase A cynomolgus monkeys in each group (1M/1F) were euthanized on day 36 or day 64.

No DART-A related mortality was observed during Phase A of the study, and no DART-A related macroscopic or organ weight changes were observed. However, DART-A related effects were observed at dose levels >500 ng/kg, as detailed below. These effects were consistent with the pharmacology of the test product and were not considered toxicologically significant.

In phase A on day 36, DART-A associated microscopic changes were limited to lymph nodes (inguinal, submandibular, and mesenteric), spleen, and gut-associated lymphoid tissue (GALT) for all groups 1 and 2 animals in the absence of recovery microscopic changes by day 64. At day 36, microscopic changes in the lymph nodes and spleen were similar and consisted of a moderate to marked decrease in the number and size of the identified lymphoid follicular germinal centers; in GALT, a minimal decrease in the number and size of lymphoid follicular germinal centers was observed. By day 64, test product-related changes were still present in lymph nodes, spleen, and GALT; in the lymph nodes and spleen, microscopic changes consisted of a moderate to marked decrease in the number and size of lymphoid follicular germinal centers, and in GALT, a minimal decrease in the number and size of lymphoid follicular germinal centers was observed.

Immunohistochemistry (IHC) assessment of bone marrow (sternum), lymph nodes (inguinal, submandibular, and mesenteric), and spleen showed a significant decrease in CD20-positive cells in phase A animals at day 36, with only partial recovery of CD20 staining by day 64. On day 36 generally did not show CD20-positive staining in the bone marrow or lymph nodes of animals in phase A, with the exception of one animal from group 1, which had very weak, weak to moderate CD20-positive staining in the germinal centers and lymphocytic corona, respectively, only the inguinal lymph node. In the spleen, CD20-positive cells were identified only in the red pulp of 3 animals (1 animal from group 1; 2 animals from group 2) with a very rare to rare CD20-positive cell frequency and minimal or mild to moderate intensity.

In recovered animals (Day 64), moderately stained CD20-positive cells, from very rare to single, were noted in the bone marrow of animals of only group 1. In the lymph nodes (inguinal, submandibular and mesenteric), CD20-positive staining was identified in only one animal from the group 1; the frequency of CD20-positive cells tended to decrease significantly in all lymph nodes, while the staining intensity of positive cells was moderate to noticeable. CD20-positive cells were identified in the spleen of all animals of groups 1 and 2. In animals of group 1, CD20-positive cells were identified in lymphoid follicles, periarteriolar lymphoid clutches (PALS) and red pulp in one animal and in PALS and red pulp in another animal. In animals of group 2, CD20-positive cells were identified very rarely or rarely only in the red pulp with a weak staining intensity.

The FACS-based assessment of B-cell, cytokine, and ADA depletion in the Phase A portion of this study is briefly described below.

a. FACS assessment of B-, T-, NK-cells and monocytes.

Whole blood samples were collected for flow cytometry evaluation of a range of lymphocyte subgroups and monocytic cell populations. Animals in phase A were evaluated at week -1; prior to dosing on days 1, 8, 15, 22 and 29; 24 hours after start of infusion (SOI) on days 2, 9, 16, 23 and 30; 72 hours after SOI on days 4, 11, 18, 25 and 32; on day 35; and also on days 43, 50, 57, and 63 (applied to only half of the phase A cynomolgus monkeys from each group, killing after a recovery period on day 64).

Dose-dependent decreases in the absolute number of B-lymphocytes (CD20+) were observed starting from day 9 (24 hours after SOI) for both groups (groups 1 and 2). In B cell populations, animals treated with 500 ng/kg/dose (group 1) tended to return to pre-study levels on days 11 and 15, while values for animals treated with 2000 ng/kg/dose (group 2) remained low. B lymphocytes were nearly depleted on day 16 (24 hours post SOI) after the second dose of DART-A for both dosed groups and remained depleted for all subsequent time points (FIGS. 20A-20B). B-lymphocyte depletion has been a putative pharmacological effect of DART-A.

Other immune cell populations, including monocytes, natural killer (NK) cells, and T cells, were also assessed by flow cytometry.

A transient, dose-dependent decrease in CD14+ monocyte counts was noted starting on day 9, 24 hours after SOI, for most animals treated with a dose of 2000 ng/kg (Group 2). Decreases in the number of CD14+ monocytes were also noted on day 16 and day 23, 24 hours after SOI, for most animals treated with a dose of 5000 and 50,000 ng/kg, respectively (group 1) and for all animals treated with a dose of 10,000 and 100,000 ng/kg respectively, (group 2). CD14+ monocyte populations recovered rapidly after the end of each infusion and generally reached pre-study baseline values or exceeded those at the time point before the next dose, declining again 24 hours after subsequent doses. At the end of the dosing phase, CD14+ monocytes fluctuated with values that deviated from pre-study levels and maintained values that were at or above baseline pre-study levels.

Temporary, dose-dependent declines were observed for all T-lymphocytes, CD4+ T-lymphocytes,

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CD8+ T-lymphocytes, regulatory T-lymphocytes (CD4+/CD25+/FoxP3+), CD159a+ NK cells, CD16+ NK cells and CD16+/CD159a+ NK cells, with the largest reductions observed in animals treated with 2000 ng/kg/dose, (group 2) on day 9 (24 hours after SOI) and after 5000 ng/kg/dose in animals of group 1 on day 16 (24 hours after SOI). T-lymphocyte and NK cell subgroups recovered rapidly after the end of each infusion and generally reached pre-study baseline values or exceeded those before the next dose on days 15 and 22, declining again 24 hours after each dose. No additional reductions were noted for group 1 or 2 on day 30 (24 hours after SOI) after doses administered on day 29.

Populations of CD4+ and CD8+ T lymphocytes were also assessed for activation of activation markers PD-1, TIM-3, CD25 and CD69 to determine the activation status of cells after a dose of DART-A. Various increases in the frequency of PD-1+, CD69+ and CD25+ in CD4+ T lymphocytes, as well as PD-1+ and CD69+ in CD8+ T lymphocytes were observed after dosing, indicating an increase in the frequency of activated CD4+ and CD8+ T lymphocytes in whole blood after dosing. The frequency of CD69+, PD1+ and CD25+ (to a lesser extent, observed in some, but not all animals) in CD4+ T lymphocytes, as well as PD-1+ and CD69+ in CD8+ T lymphocytes increased after doses of 500 ng/kg (group 1) and 2000 ng/kg (group 2) starting on day 9 (24 hours after SOI). These values, as a rule, remained above the initial levels and were very different for all subsequent points in time. Relative percentages of CD25+/CD69+, TIM-3+, and PD-1+/TIM-3+ lymphocyte populations tended to be <1% of the total CD4+ and CD8+ T-lymphocyte population, making it difficult to evaluate any DART-A-related changes . Taken together, these data suggest that DART-A administration tended to upregulate PD-1 and CD69 expression in both CD4+ and CD8+ T lymphocytes, along with a more modest increase in CD25+ in CD4+ T lymphocytes in some but not all animals.

b. Assessment of cytokines.

Serum levels of IL-2, IL-4, IL-5, IL-6, IL-10, TNF-α, and IFN-γ were determined (EMD Millipore assay) before dosing and 4 and 24 hours after the start of each infusion . Transient, dose-correlated increases in IL-10 levels (up to 1918 pg/mL) were observed 4 h after the start of the infusion at which cynomolgus monkeys were administered escalating doses ranging from 500 to 50,000 ng/kg in group 1 and from 2000 up to 100,000 ng/kg in group 2, by IV infusion over a 2-hour period for the first 3 doses; levels generally returned to baseline within 24 hours of starting the infusion. Generally, no IL-10 was detected 4 hours after the start of the last DART-A infusion in either group (50,000 ng/kg in group 1 or 100,000 ng/kg in group 2) with one exception (animal 2502). Animal 2502 experienced a transient increase in IL-10 to 332 pg/ml after the last dose (100,000 ng/kg on day 29), but this increase was lower in absolute magnitude than the increase in IL-10 after the first dose of 100 μg/kg on day 22 (570 pg/ml). One cynomolgus macaque (animal 1001) at a dose level of 50,000 ng/kg, which experienced the highest levels of IL-10 (1918 pg/ml on day 22), also simultaneously experienced transient increases in IL-2 (32 pg/ml), IL 5 ( 45 pg/ml) and IL-6 (357 pg/ml). Transient increases in IL-6 levels were observed in most cynomolgus monkeys 4 h after DART-A infusion; however, elevations were generally similar in absolute magnitude to or less than (with the exception of animal 1001 described above) compared to levels observed after control medium infusion on day 1 (up to 190 pg/mL) with no clear response per dose. No clear clinical indications associated with elevated levels of cytokines were observed.

No sustained change in IL-5, IL-4, IL-2, IFN-γ, or TNF-α levels was observed following DART-A infusion. IL-5 levels varied, with approximately half of the animals in each group experiencing slight and transient increases (~10-20 pg/mL; except for animal 1001 discussed above) following DART-A infusion on days 15 or 22. IFN levels -γ in some animals was also variable during the course of the study with small (<~10 pg/mL) and transient increases after infusion.

2. DART-A toxicokinetic study in cynomolgus monkeys (Phase B).

In the phase B study, 4 groups of cynomolgus monkeys (n=2/group; 1M/1F) were given fixed doses of DART-A as 2-hour IV infusions, with 3 doses given on days 1, 8, and 15 for group 3 (5 ng/kg), group 4 (50 ng/kg) and group 5 (500 ng/kg) and 5 doses were administered on days 1, 4, 8, 11 and 15 for group 6 (500 ng/kg). All animals in phase B were euthanized on day 18.

No DART-A related mortality was observed during the Phase B study, and no DART-A related gross or organ weight changes were observed.

At day 18, DART-A associated microscopic changes were limited to lymph nodes (inguinal, submandibular, and mesenteric) and spleen in Groups 4, 5, and 6 animals and consisted of a minimal to moderate reduction in the number and size of identified lymphoid follicular germinal centers, with it did not observe a clear change in the size and/or cell density of lymphoid tissue containing PALS in the spleen. No microscopic changes were recorded in group 3 (5 ng/kg).

Additional formalin-fixed, paraffin-embedded bone marrow sections were evaluated

- 39 040028 (sternum), lymph nodes (inguinal, submandibular and mesenteric), and spleen by IHC for cell populations that express CD20 (Mordenti, J. et al. (1991) Interspecies Scaling Of Clearance And Volume Of Distribution. Data For Five Therapeutic Proteins, Pharm. Res. 8(11):1351-1359). No significant difference was observed in the distribution and/or intensity of staining of CD20-positive cells in the bone marrow (sternum), lymph nodes (inguinal, submandibular and mesenteric), or spleen in groups 3, 4, 5, or 6. In all tissues, positive staining was associated with cell membrane. Typically, in the bone marrow, single CD20-positive cells were localized throughout the bone marrow stroma, and the staining intensity of the positive cells was moderate to marked. In the lymph nodes, CD20-positive cells were mainly associated with the germinal centers and lymphocytic crown of follicles, with a smaller number of CD20-positive cells located in the paracortex, medullary ligaments, as well as in the medullary and subcapsular sinuses. Staining intensity in the germinal centers, lymphocytic corona, medullary ligaments, and medullary and subcapsular sinuses was moderate to marked. The intensity of staining in the paracortex was weak to noticeable. In the spleen, CD20-positive cells were mainly associated with germinal centers, follicular mantle, and marginal zone of lymphoid follicles, with fewer CD20-positive cells located in PALS and red pulp, and staining intensity was moderate to prominent.

The following briefly describes the FACS-based assessment of B cell depletion, cytokines, and ADA in the Phase B portion of this study.

a. FACS evaluation of B cells, T cells, NK cells and monocytes.

Whole blood samples were collected for flow cytometry evaluation of a range of lymphocyte subgroups and monocytic cell populations. Animals in phase B were evaluated at week -1; before dosing on days 1, 4 (Group 6 only), 8, 11 (Group 6 only), and 15; 24 hours after SOI on days 2, 9 and 16; and also 72 hours after SOI on day 4 and day 11 (groups 3-5 only).

Marked dose-dependent reductions in total B-lymphocytes (CD20+) were observed 24 hours after the start of the infusion for doses administered on days 1, 8, and 15. kg (group 4) and 500 ng/kg (groups 5 and 6) were observed on day 2 (24 hours after SOI). B-lymphocyte populations recovered rapidly after each infusion in groups 3 and 4 and were observed to fluctuate near baseline pre-study levels at all other time points. Groups 5 and 6 saw minimal recovery between doses, and generally decreased values were maintained at all time points after day 2 (FIGS. 21A-21D). Permanent depletion of B lymphocytes has been a putative pharmacological effect of DART-A at higher doses.

Other immune cell populations, including monocytes, NK cells, and T cells, were also assessed by flow cytometry.

Temporary, dose-dependent reductions in total CD14+ monocyte count were noted starting on day 2 (24 hours after SOI) in most animals in all dosed groups (groups 3-6). Additional changes in CD14+ monocytes were also observed in the majority of animals in all dosed groups on days 9 and 16, and the values were highly variable.

Transient, dose-dependent decreases in the number of all T-lymphocytes, regulatory T-lymphocytes (CD4+/CD25+/FoxP3+), CD4+ T-lymphocytes, CD8+ T-lymphocytes and NK cell subgroups were observed, with the largest reductions for all groups (groups 3-6 ) was generally noted on day 2 (24 hours after SOI). T cell subsets recovered rapidly after each infusion and at all doses tended to reach pre-study levels or exceed pre-study levels in all groups. Additional reductions in CD4+, CD8+ and regulatory T lymphocytes were also observed on days 9 and 16 (24 hours after SOI) in those treated with doses of 50 ng/kg (group 4), 500 ng/kg (group 5) and multiple doses of 500 ng/kg (group 6) animals, however, the reductions observed at these time points were lower in absolute magnitude compared to those observed on day 2 after the first dose of DART-A. In subsets of NK cells, as a rule, there was a tendency to recover after the first day of infusion and to be present with variable values at all other time points.

Populations of CD4+ and CD8+ T lymphocytes were assessed for activation of the activation markers PD1, TIM-3, CD25 and CD69 to determine the activation status of the cells after dosing with DART-A. The relative percentages of CD25+/CD69+ (for CD4+ T cells only), TIM-3+, and PD1+/TIM3+ in CD4+ and CD8+ T cell populations tended to be <1% of all CD4+ and CD8+ T cell populations, making it difficult to evaluation of any changes related to DART-A. Increases in the frequency of PD-1+ and CD69+ in CD4+ and CD8+ T lymphocytes were observed over several time points in the 500 ng/kg/dose animals in groups 5 and 6 starting on day 2 (24 hours after SOI). In addition, more modest increases in CD25+ frequency in CD4+ and CD8+ T lymphocytes were observed in 500 ng/kg/dose treated animals (groups 5 and 6) starting on day 2 (24 hours after SOI), as well as increases in relative percent the proportion of CD25+/CD69+ in CD8+ T-lymphocytes in animals treated with multiple doses of 500 ng/kg/dose (group 6). Increases in absolute value compared to those observed on day 2 were also observed, usually on days 9 and 16 (24 hours after

SOI) after successive doses. Taken together, these data indicate that administration of DART-A resulted in a trend towards increased expression of PD-1, CD69 and CD25 in CD4+ and CD8+ T lymphocytes.

b. Assessment of cytokines.

Serum levels of IL-2, IL-4, IL-5, IL-6, IL-10, TNF-α and IFN-γ were determined (EMD Millipore assay) pre-dose on days -7, 8 and 15 and after 4 and 24 hours after the start of infusions on days 1, 8 and 15. Transient, dose-correlated increases in IL-10 levels (up to 173 pg/mL) were observed 4 hours after the start of the 1st DART-A infusion, in which cynomolgus macaques administered doses of DART-A 5, 50 or 500 ng/kg (groups 3-6); levels generally returned to baseline within 24 hours of starting infusions. Increases in IL-10 were more pronounced in all groups after the 1st dose of DART-A compared to increases in IL-10 after the 2nd and ^3rd doses (<22 pg/ml in all animals except for animal 5001, described below ), which confirms either a desensitization of cytokine release and/or a rapid decrease/elimination of the target cell population responsible for T-cell activation after the 1st dose. Temporary increases in IL-6 levels (up to 162 pg/ml) were alternately observed in some animals in all dosed groups 4 hours after the start of each DART-A infusion, with levels generally returning to baseline levels or approaching those in within 24 hours. However, based on the data obtained with the introduction of medium in phase A, which observed an increase in IL-6 levels up to 190 pg / ml, it can be concluded that the levels of IL-6 fluctuation in phase B (<162 pg / ml) do not necessarily reflect drug-dependent responses. No sustained changes in IL-5, IL-4, IL-2, IFNy or TNF-α levels were observed throughout the study.

One cynomolgus monkey (animal 5001) experienced elevated levels of IFN-γ, TNF-α, IL-2, IL-4, IL5 and IL-10 on day 8 prior to the 2nd DART-A infusion. Cytokine levels further increased (IL-4, IL-5, IL-6, IL-10, IFN-γ and TNF-α) or remained stable (IL-2) 4 hours after the 2nd infusion on day 8. Levels cytokines began to decline 24 hours after the 2nd dose, with variable increases in some cytokines after the 3rd DART-A infusion on day 15. Although all cytokine levels fell below their peak levels, cytokine levels continued to exceed baseline levels at the last time point of assessment ( day 16) in this cynomolgus macaque. No clear clinical indications associated with elevated levels of cytokines were observed.

Example 12.

Ig-like half-life in human FcRn-transgenic mice.

A pharmacokinetic (PK) study was performed on a human FcRn transgenic mouse using a human FcRn transgenic mouse strain B6.Cg-Fcgrtt ^mIDcr (CAGFCGRT)276Dcr/DcrJ (Stock Number 004919) available from Jackson Laboratories, Bar Harbor, Maine, USA, for evaluation of DART-A and control DART 1 (Fig. 22). The terminal half-life was 85.6 and 92.1 hours, respectively. Also, three human mAbs were evaluated on the same test system for comparison, which were characterized by terminal half-lives ranging from 58.7 to 102.7 hours.

Example 13

Efficacy in disseminated Raji leukemia/lymphoma cell models in human PBMC-reconstituted mice.

Disseminated tumor models were created using luciferase-transduced Raji lymphoma cells (Raji.luc) in female NSG B2m-/- mice reconstituted with human PBMC, with mice treated with vehicle, control DART 1 or DART-A, or one day after tumor cell injections (early treatment or leukemia paradigm) or after at least two weeks of in vivo growth (advanced tumor or lymphoma model).

Early processing model.

After reconstitution with human PBMC on study day -14, Raji.luc lymphoma cells were injected on study day 0 and treatment started the next day (study day 1). Medium was injected as a control, 0.5 mg/kg control DART 1 or 0.5 mg/kg DART-A IV once every 3-4 days for a total of 12 doses (i.e. on study days 1, 4, 6, 8, 11, 13, 15, 18, 20, 22, 25 and 27). Mice were observed for survival on study day 54 or study day 56.

In the medium or control DART 1 groups, diffuse profiles of disseminated tumor cells rapidly confluent and expanded in most mice over weeks 2 and 3. Animal deaths in these two control groups were noted as early as two weeks after tumor cell implantation. All mice in the medium or control DART 1 treated groups died or were humanely euthanized (ie if the animals were agonized or lost more than 15% of their body weight) on study days 40 or 53, respectively. The survival curves are shown in Fig. 23. In contrast, the DART-A treated group did not show tumor cell growth at any time point, and no animal death was observed at the end of the study. The survival curve is shown in Fig. 23.

- 41 040028

A dose ranging study was performed in a follow-up experiment under the same experimental conditions as described above, except that 0.16 µg/kg, 0.8 µg/kg, 4 µg/kg, 20 µg/kg, 0.1 mg/kg or 0.5 mg/kg DART-A IV once every 3–4 days for a total of 12 doses (i.e. study days 1, 4, 6, 8, 11, 13, 15 , 18, 20, 22, 25 and 27). Medium and control DART 1 were administered as described above on the same study days. Rapid progression of Raji.luc tumor growth was observed in mice treated with vehicle or control DART 1. DART-A was effective at all doses tested, with most mice surviving (>8%) and tumor free at study end (Day 56) at doses below 20 μg/kg (Fig. 24).

Model of the established tumor.

The ability of CD19 x CD3 to control the tumor burden of Raji.luc cells during treatment delay was tested in a separate set of experiments. For this established tumor model, human PBMC reconstituted mice were inoculated IV with Raji.luc cells on study day 0 (approximately one week after PBMC injection) and allowed to grow for 14 days prior to treatment.

Mice were randomized on study day 12 into 3 groups based on tumor burden and then treated with medium, 0.1 mg/kg DART-A or 0.5 mg/kg DART-A starting on study day 14 (2 weeks after inoculation of Raji.luc cells) with a total of 10 doses (i.e. on study days 14, 15, 16, 19, 21, 23, 30, 33, 35 and 37). In the medium treated group, the initial lesions markedly increased in size by the end of the first week of treatment, and 5/7 mice died within 2 weeks of treatment (FIG. 25). In contrast, the tumor burden in most animals in both DART-A treated groups was greatly reduced or disappeared during the first week of treatment. Only one animal in each DART-A treated group, each with a relatively high tumor burden present at randomization, died during the first week of treatment (FIG. 25). By study day 50, only 2 mice in the 0.1 mg/kg DART-A treated group had peripheral tumors.

A dose-ranging study in a treatment delay model was performed under the usual experimental conditions described above. In this experiment, human PBMC reconstituted mice were inoculated with Raji.luc IV cells on study day 0 (5 days after PBMC injection) and allowed to grow until study day 14 before randomization. Treatment started on study day 15 with medium, 0.1 mg/kg control DART 1 or 0.16 µg/kg, 0.8 µg/kg, 4 µg/kg, 20 µg/kg, 0.1 mg/kg or 0.5 mg/kg DART-A for a total of 9 doses (i.e. study days 15, 17, 21, 23, 25, 28, 30, 32 and 35).

In the medium treated group, the tumor burden gradually increased and all mice died or were euthanized by the end of the second week of treatment (FIG. 26). Mice treated with 0.1 mg/kg control DART 1 showed slightly slower tumor progression compared to media treated mice (FIG. 26). In contrast, tumor burden in animals treated with DART-A generally showed a dose-dependent decrease over time (FIG. 26).

Example 14

Pharmacokinetic parameters of DART-A in cynomolgus monkeys.

The cynomolgus monkey was chosen as an appropriate pharmacological model for the DARTA assay based on the equivalent distribution of both target antigens in this species compared to humans based on immunohistochemistry with the previous mAbs.

The study carried out in accordance with the present invention included 5 treatment groups consisting of 10 cynomolgus monkeys per group (5 males, 5 females). Groups were treated by IV infusion over 2 hours with medium as control for the first infusion on day 1 followed by treatment with medium as control (group 1) or DART-A (groups 2-5) once a week for four weeks. While group 1 animals continued to receive medium as control for all 4 subsequent infusions, animals from groups 2-5 received DART-A at 0.2 µg/kg, 2 µg/kg, 5 µg/kg, or 10 µg/kg at all subsequent infusions on days 8, 15, 22, and 29 (Table 12). Three males and 3 females were euthanized and necropsied on day 37, while the rest of the recovered monkeys in the group (2 males and 2 females) were euthanized and necropsied on day 121 after a 12 week recovery period.

- 42 040028

Table 12

No. of infusion examined Infusion No. DART-A (groups 2-5 only) Research Day DART-A (mcg/kg) given as a 2-hour IV infusion Group 1 Group 2 Group 3 Group 4 Group 5 1 Not applicable 1 0 0 0 0 0 2 1 8 0 0.2 2 5 10 3 2 15 0 0.2 2 5 10 4 3 22 0 0.2 2 5 10 5 4 29 0 0.2 2 5 10

Analysis of the concentration of DART-A in serum collected at different time points was performed using ELISA. The serum DART-A concentration versus time profile for a typical animal from Group 5 is shown in FIG. 27. PK parameters (two-component model) were evaluated according to doses of 0.2, 2, 5 and 10 µg/kg (Table 13). As a rule, dose-proportional increases in maximum serum concentrations (Cmax) and AUC _inf were observed over the entire evaluated dose range. Despite differences in primary PK parameters in some pairwise comparisons of doses, in general, there were no consistent trends in clearance (CL), intercameral clearance (CLD2) and volumes of distribution (V1 and V ₂ ) for DART-A at the estimated doses, which indicates linear PK. CL values in animals of groups 2-5 are below the glomerular filtration rate (GFR) for cynomolgus monkeys (~125 ml/h/kg), indicating that practically no elimination occurs by renal filtration, as expected for a protein with this molecular weight (109.3 kDa). Mean initial volumes of distribution (V1) for animals from groups 2, 3, 4 and 5 are similar to or greater than plasma volume in cynomolgus monkeys (~45 ml/kg), indicating a slight depletion of DART-A in the central compartment as a result of binding with target cells. Mean beta half-lives and MRTs tended to increase with increasing dose, with the mean beta half-life (t1/2, β) being 161 hours (6.7 days) and the mean retention time (MRT) was 191 hours (8 days). Of note, only three animals in the GLP-compliant study (n=1 in the 2 µg/kg dose group; n=2 in the 5 µg/kg dose group) had aberrant PK profiles after the 2nd and subsequent infusions of DART- A (n=2) or after the fourth DART-A infusion (n=1) and were subsequently confirmed as ADA positive. Therefore, serum concentration data after affected infusion cycles were excluded from PK modeling for these 3 animals.

Table 13

Two-component analysis of PK parameters of DART-A in cynomolgus monkeys sign 0.2 2 mcg/kg/day 5 mcg/kg/day 10 mcg/kg/day mcg/kg/day (average ± (average ± (mean ± SD) (mean ± SD) SD) SD) Stach (NG/ML) 3.18±0.46 31.9±3.0 82.9 ± 5.6 166.6 ± 11.0 AUCinf 155±37 2258±634 6714± 1846 13756±2534 (h*ng/ml) CL (ml/h/kg) 0.95±0.17 0.80±0.22 0.73 ± 0.24 0.70±0.13 CLD2 (ml/hour/kg) 0.87±0.15 1.65±0.36 1.70±0.74 1.40±0.40 Vi (ml/kg) 43.4±5.8 50.9±5.1 52.3 ± 3.5 54.5 ± 4.5 V2 (ml/kg) 65.3 ± 17.5 77.9 ± 17.9 93.4 ± 33.3 105.3±33.0 Vss (ml/kg) 108.7 ± 16.6 128.8 ± 17.2 145.8±34.6 159.8±36.0 ti/2 (hours) 14.0±1.5 11.0±2.2 12.3±3.8 15.0±4.4 ίι/2,β (hours) 119.1±27.6 143.4±43.4 184.9±72.0 205.6 ± 66.9 MRT (hours) 116.3±20.3 173.0±54.5 221.2 ± 86.5 235.9 ± 72.0

Cytokine release in DART-A-treated cynomolgus monkeys.

Serum samples were collected to assess cytokine levels before infusion and 2 and 22 hours after the end of infusion on days 1, 8, 15, 22 and 29. Cytokines assessed were TNF-α, IFN-γ, IL-2, IL-4, IL-5, IL-6 and IL-10.

An increase in IL-6, IL-10, IFN-γ and TNF-α levels was observed after the first DART-A infusion (Day 8), with the greatest increase in cytokine levels occurring 2 hours after the end of the infusion. A dose-dependent increase in DART-A was observed for IL-10 and IFN-γ at dose levels >2 μg/kg, while an increase in IL-6 was observed at dose levels >5 μg/kg. Changes in TNF-α levels were small

- 43 040028 shimi and were only observed at the highest dose level (10 µg/kg). Changes in cytokine levels were transient and returned to baseline or approached within 24 hours of the start of the infusion, and there were no clinical indications associated with these elevated cytokine levels. Subsequent DART-A infusions on days 15, 22, and 29 resulted in smaller increases in cytokine levels, generally comparable to those observed after control product infusion. Mean levels of IL-2, IL-4 and IL-5 showed variable and isolated changes in individual animals, but remained within <20 pg/ml in all groups throughout the study and were not considered toxicologically significant.

The levels of cytokines in animals treated with 0.2 μg/kg DART-A were comparable to the variations observed after infusion of the control product (group 1).

DART-A mediated depletion of circulating B cells in cynomolgus monkeys.

Circulating absolute levels of B cells, T cells, including subgroups of T cells (CD4 vs. CD8), activation markers (CD25, CD69, PD-1 and TIM-3), and NK cells were measured in the study as a pharmacodynamic endpoint. points. Samples were collected before infusion and 2, 22, and 70 hours after the end of the infusion on days 1, 8, 22, and 29 before final necropsy on day 37, and then once a week or twice a week during the recovery period on days 36. 43, 50, 57, 64, 78, 92, 106 and 119.

In FIG. 28 shows that DART-A treatment resulted in a dose-dependent depletion of CD20+ B cells, a putative target population. Complete or near complete depletion of CD20+ cells was observed 24 hours after the start of the first DART-A infusion at dose levels >2 μg/kg. The depletion of CD20+ B cells was sustained throughout the treatment period and lasted for 3-5 weeks after the final dose. A dose-dependent return to pre-dose levels was observed in CD20+ B cells during the last few weeks of the 12-week recovery period.

Example 15

Autologous depletion of malignant B cells in primary samples from CLL patients.

To evaluate DART-A mediated cytolytic activity in primary CLL PBMC patient samples, patients were incubated with DART-A or control DART 1. Due to limited patient sample availability, only 2 concentrations (50 ng/mL and 200 ng/mL) were tested. The percentages of malignant CLL B cells (CD19+/CD5+ or CD20+/CD5+) and T cells (CD19-/CD5+ or CD4+ and CD8+) were measured by flow cytometry before treatment and after treatment for 3 and 6 days. DART-A partially blocks anti-CD19 antibody staining in the tested CLLs, therefore, it was not practical to use CD19+/CD5+ to gate malignant B cells after treatment with DART-A. Thus, depletion of malignant B cells was determined by gating in CD20+/CD5+ cells after treatment with DART-A.

The E:T ratio, as measured by the incidence of malignant CLL B cells and T cells, was typically <1:10. In a representative CLL donor experiment shown, the ratio of E:T cells was approximately 1:23 before treatment based on CD19-/CD5+ T cells, accounting for only 4% of all PBMCs and approximately 90% of PBMCs identified as CD19+/CD5+ malignant B- cells. In FIG. 29A-29I and in the table. 14 shows the effects of treatment with DART-A compared to a CLL patient sample. Incubation of DART-A with CLL PBMC resulted in a time-dependent depletion of malignant B cells at both DART-A concentrations assessed, accompanied by a concomitant increase in T cells (CD4 and CD8) observed at day 6. In addition, a marker of T cell activation CD25 was upregulated after treatment with DART-A for 6 days, indicating that DART-A induces T cell activation during redirected killing of malignant B cells. In contrast, B cell killing or CD25 activation was not observed with control DART 1 by day 6.

Of note, DART-A mediated the same level of killing at both concentrations evaluated, but the level of killing increased with time, with approximately 40% and 85% of malignant B cells dying on day 3 and day 6, respectively, indicating that that time is a critical factor for effective elimination of target cells at low ratios of T cells to targets in the presence of sufficient DART-A.

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Table 14

Percentages of malignant B cells (CD20+/CD5+), T cells (CD4, CD8+) and activated

T cells (CD25+/CD4+ and CD25+/CD8+) in CLL PBMCs (E:T=1:23) after treatment with DART-A or _____ control DART 1 for 3 and 6 days ____________

Moment Percentage (%) of time CD20+/CD5+ CD4+ CD8+ CD25+/CD4+ CD25+/CD8+ Day 0 CD4+ and CD8+ T cell subgroup not measured on day 0 Preliminary CD19-/CD5+ used 70.7 n/ ^{a 1} treatment to determine T- cell population on day 0 (4%) as described above. Day 3 DART-A (50 40.5 1.01 1.78 ng/ml) Control Total percentages of DART 1 (50 73.5 3.08 1.81 T cells were too ng/ml) low (< 5 %) for DART-A (200 T- 42.3 1.61 1.68 ng/mL) cellular activation of CD25 on Control Day 0 or Day 3. DART 1 (200 72.9 3.23 1.62 ng /ml) Day 6 DART-A (50 ng/ml) 11.3 57.4 22.2 87.7 82.8 Control DART 1 (50 ng/ml) 74.9 3.54 2.26 1.4 20.6 DART-A (200 ng/ml) 12.3 65.2 20.9 91.6 90.7 Control DART 1 (200 ng/ml) 73.4 2.79 1.82 1.4 14.3

All publications and patents referred to in this specification are incorporated herein by reference to the same extent as if it were indicated that each individual publication or patent application was expressly and individually incorporated by reference in its entirety. Although the present invention has been described using specific embodiments, it should be understood that further modifications are possible, and this application is intended to cover any variations, applications, or variations of the present invention, generally consistent with the principles of the present invention and including such departures. from the present disclosure, which are within the known or accepted practice in the field to which the present invention pertains, and which may be applicable to the essential features set forth above.

Claims (23)

CLAIM 1. Bispecific monovalent Fc-diabody to CD19 x CD3, capable of specifically binding to CD19 and CD3, while the diabody contains the first, second and third polypeptide chains, while these polypeptide chains form a covalently linked complex, and also: I) the specified first polypeptide chain contains in the direction from the N-terminus to the C-terminus: A) an IA domain containing: (1) a subdomain (IA1) that contains a VL domain capable of binding to either CD19 (VL _CD i ₉ ) or CD3 (VLcd3); and (2) a subdomain (IA2) that contains a VH domain capable of binding to either CD19 (VH _Cd19 ) or CD3 (VHcd3); wherein said subdomains IA1 and IA2 are separated from each other by a polypeptide linker and are either (a) VLcdi9 and VHcd3; or ^{(b) VL} CD3 ^{and VH} CD19 ^; B) an IB domain containing a positively or negatively charged helical domain, said IB domain being separated from said IA domain by a polypeptide linker; C) an IC domain containing the CH2-CH3 domain of the antibody; And II) the specified second polypeptide chain contains in the direction from the N-terminus to the C-terminus: A) an IIA domain containing: (1) a subdomain (IIA1) that contains a VL domain capable of binding to either CD19 (VL _Cd19 ) or Cd3 (VL _Cd3 ); and (2) a subdomain (IIA2) that contains a VH domain capable of binding to either CD19 (VH _Cd19 ) or CD3 (VH _Cd3 ); while these subdomains IIA1 and IIA2 are separated from each other by a polypeptide linker and are: (a) VL _Cd19 and VH _Cd3 if the indicated subdomains of IA1 and IA2 are VL _Cd3 and VH _Cd19 ; or (b) VL _Cd3 and VH _Cd19 if said subdomains of IA1 and IA2 are VL _Cd19 and VH _Cd3 ; B) an IIB domain containing a positively or negatively charged helical domain, wherein said IIB domain is separated from said IIA domain by a polypeptide linker, and wherein said charged helical domain of said IB domain and said charged helical domain of said IIB domain have opposite charges, wherein one of said charged helical domains of said IB domain and said IIB domain contains the amino acid sequence of SEQ ID NO: 12, and the other of said charged helical domains of said IB domain and said IIB domain contains the amino acid sequence of SEQ ID NO: 13; And iii) said third polypeptide chain contains in the direction from the N-terminus to the C-terminus the IIIC domain, which contains the CH2-CH3 domain of the antibody; wherein said _Cd19 VL domain and said _Cd19 VH domain form a CD19 binding domain, and said _Cd3 VL and _Cd3 VH domains form a CD3 binding domain; and said CH2-CH3 domains of said first and third polypeptide chains form an Fc domain capable of binding to an Fc receptor, thereby forming said CD19 x CD3 bispecific monovalent Fc diabody. 2. Bispecific monovalent Fc-diabody to CD19 x CD3 according to claim 1, in which: (A) each of said IB and IIB domains contains a cysteine residue that covalently links said first polypeptide chain to said second polypeptide chain via a disulfide bond; and (B) each of said IC and IIIC domains contains a cysteine residue that covalently links said first polypeptide chain to said third polypeptide chain via a disulfide bond. 3. An anti-CD19 x CD3 bispecific monovalent Fc diabody according to any one of claims 1 and 2, wherein said _Cd19 VL contains the amino acid sequence of SEQ ID NO: 17 and said _Cd19 VH contains the amino acid sequence of SEQ ID NO: 21. 4. An anti-CD19 x CD3 bispecific monovalent Fc diabody according to any one of claims 1 to 3, wherein said _Cd3 VL contains the amino acid sequence of SEQ ID NO: 25 and said _Cd3 VH contains the amino acid sequence of SEQ ID NO: 29. 5. An anti-CD19 x CD3 bispecific monovalent Fc diabody according to any one of claims 1 to 4, wherein said CH2-CH3 domain of said IC domain comprises the amino acid sequence of SEQ ID NO: 15, and said CH2-CH3 domain of said IIIC domain contains the amino acid the sequence of SEQ ID NO: 16. 6. A bispecific anti-CD19 x CD3 monovalent Fc diabody according to any one of claims 1 to 5, wherein said polypeptide linker that separates said IB domain from said IA domain contains - 46 040028 contains the amino acid sequence of SEQ ID NO: 2, and said polypeptide linker that separates said IIB domain from said IIA domain contains the amino acid sequence SEQ ID NO: 2. 7. An anti-CD19 x CD3 bispecific monovalent Fc diabody according to any one of claims 1 to 5, wherein said polypeptide linker that separates said IB domain from said IA domain contains the amino acid sequence of SEQ ID NO: 3, and said polypeptide linker, which separates said IIB domain from said IIA domain, contains the amino acid sequence of SEQ ID NO: 3. 8. An anti-CD19 x CD3 bispecific monovalent Fc diabody according to any one of claims 1-6, wherein: (A) said first polypeptide chain contains the amino acid sequence of SEQ ID NO: 35; (B) said second polypeptide chain contains the amino acid sequence of SEQ ID NO: 37; and (C) said third polypeptide chain contains the amino acid sequence of SEQ ID NO: 39. 9. An anti-CD19 x CD3 bispecific monovalent Fc diabody according to any one of claims 1 to 8, which is capable of cross-reacting with both human and primate CD19 and with both human and primate CD3. 10. Pharmaceutical composition for the treatment of a disease or condition characterized by the expression of CD19, containing a bispecific monovalent Fc-diabody to CD19 x CD3 according to any one of claims 1 to 9 and a physiologically acceptable carrier. 11. The use of a bispecific anti-CD19 x CD3 monovalent Fc diabody according to any one of claims 1 to 9 as a pharmaceutical agent for the treatment of a disease or condition characterized by CD19 expression. 12. Use of an anti-CD19 x CD3 bispecific monovalent Fc diabody according to any one of claims 1 to 9 in the treatment of a disease or condition characterized by CD19 expression. 13. The use of a pharmaceutical composition according to claim 10 in the treatment of a disease or condition characterized by the expression of CD19. 14. Use according to any one of claims 12, 13, wherein said disease or condition characterized by CD19 expression is a malignant tumor. 15. Use according to claim 14, wherein said cancer is selected from the group consisting of acute myeloid leukemia (AML), chronic myelogenous leukemia (CML), CML blast crisis, CML-associated Abelson oncogene (Bcr-ABL translocation), myelodysplastic syndrome (MDS), acute B-cell lymphoblastic leukemia (B-ALL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, chronic lymphocytic leukemia (CLL), Richter's syndrome, Richter's transformation CLL, hairy cell leukemia (HCL) , neoplasms of blast plasmacytoid dendritic cells (BPDCN), non-Hodgkin's lymphomas (NHL), mantle cell leukemia (MCL), small cell lymphocytic lymphoma (SLL), Hodgkin's lymphoma, systemic mastocytosis, and Burkitt's lymphoma. 16. A diabody containing a first polypeptide chain containing a negatively charged helical domain and a second polypeptide chain containing a positively charged helical domain, said negatively and positively charged helical domains to ensure heterodimerization, wherein said first and second polypeptide chains form a covalently linked complex, wherein: (A) the specified first polypeptide chain contains a polypeptide containing the amino acid sequence of SEQ ID NO: 2 associated with the specified negatively charged helical domain containing the amino acid sequence of SEQ ID NO: 12; and (B) the specified second polypeptide chain contains a polypeptide containing the amino acid sequence of SEQ ID NO: 2 associated with the specified positively charged helical domain containing the amino acid sequence of SEQ ID NO: 13; wherein said negatively charged helical domain of said first polypeptide chain and said positively charged helical domain of said second polypeptide chain are covalently linked to each other via a disulfide bond. 17. A diabody containing a first polypeptide chain containing a negatively charged helical domain and a second polypeptide chain containing a positively charged helical domain, said negatively and positively charged helical domains to ensure heterodimerization, wherein said first and second polypeptide chains form a covalently linked complex, wherein: (A) the specified first polypeptide chain contains a polypeptide containing the amino acid sequence of SEQ ID NO: 3 associated with the specified negatively charged helical domain containing the amino acid sequence of SEQ ID NO: 12; and (B) said second polypeptide chain comprises a polypeptide comprising the amino acid sequence of SEQ ID NO: 3 linked to said positively charged helical domain. - 47 040028 nom containing the amino acid sequence of SEQ ID NO: 13; wherein said negatively charged helical domain of said first polypeptide chain and said positively charged helical domain of said second polypeptide chain are covalently linked to each other via a disulfide bond. 18. A pharmaceutical composition for the treatment of a disease or condition characterized by CD19 expression, comprising a diabody according to any one of claims 16, 17 and a physiologically acceptable carrier. 19. A polynucleotide encoding the first polypeptide chain of a CD19 x CD3 bispecific monovalent Fc diabody according to any one of claims 1 to 6, which contains the amino acid sequence of SEQ ID NO: 35. 20. A polynucleotide encoding a second polypeptide chain of a bispecific anti-CD19 x CD3 monovalent Fc diabody according to any one of claims 1 to 6, which contains the amino acid sequence of SEQ ID NO: 37. 21. An expression vector containing a polynucleotide according to any one of claims 19, 20. 22. A polynucleotide according to claim 19, which contains the sequence of SEQ ID NO: 36. 23. A polynucleotide according to claim 20, which contains the sequence of SEQ ID NO: 38.