AU2013201525B8 - Therapeutic and diagnostic agents - Google Patents

Description

THERAPEUTIC AND DIAGNOSTIC AGENTS FILING DATA 5 [00011 This application is associated with and claims priority from Australian Provisional Patent Application No. 2012900775, filed on 28 February 2012, entitled "therapeutic and diagnostic agents", the entire contents of which, are incorporated herein by reference. FIELD 10 [00021 The present disclosure relates generally to the field of immunointeractive molecule mediated therapy and diagnostics and agents useful for same. Enabled herein is a combinatorial, immunoglobulin-based approach to the selective treatment of disease conditions and for diagnostics. 15 BACKGROUND [0003] Bibliographic details of the publications referred to in this specification are also collected at the end of the description. 20 [0004] Reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that this prior art forms part of the common general knowledge in any country. 25 [0005] A key feature in the search for and the development of therapeutic and diagnostic agents is target discrimination and selective toxicity. The target cells in this regard include eukaryotic cells (e.g. cancer cells, virally infected host cells and certain stem cells) and prokaryotic cells (i.e. bacteria). In particular, the ability to distinguish target cells such as cancer cells or cells infected with a pathogen, or bacterial pathogen cells, amongst a 30 population of normal cells in subjects, is of paramount importance. This is particularly the case in cancer therapies where the target cancer cells have many physiological, anatomical H:\aarXntenvoven\NRPortb\DCC\AAR\4959862_ l.do-12103/2013 -2 and biochemical properties in common with surrounding normal cells. Whilst anti-cancer drugs often cause collateral damage to normal cells, their use for particularly aggressive, fast growing cancers is currently inevitable. 5 [00061 Therapeutic antibodies are one of the most rapidly growing areas of pharmaceuticals (Carter (2006) Nat. Rev. Immunol. 6:343-357; Bonavida (2007) Oncogene 26:3592-3593; Reichert and Valger-Archer (2007) Nat. Rev. Drug Discovery 6:349-356; Carter (2001) Nature Reviews 1:118-128; de Haard et al. (1998) Adv Drug Delivery Rev. 31:5-31; Chames and Batty (200) FEMS Microbiol Lett 189:1-8; Funaro et al. (2000) 10 Biotechnol Adv 18:385-401; Hudson (2000) Exp Opin Invest Drugs 9:1231-1242). Examples involving therapeutic antibodies are reviewed in Table 1 of Carter, 2001 supra and Hudson and Souriau (2003) Nat Med 9:129-134. [00071 There are many variations of engineered antibodies (e.g. mouse monoclonal, 15 chimeric, humanized and human monoclonal antibodies, single chain variable antibody fragments (scFv's), minibodies and aptamers). Diabodies have also been developed using recombinant DNA technology, which have scFv's with different binding specificities and appropriate spacing between these domains to enable both scFv's to concurrently bind antigens (Hudson and Souriau, 2003 supra). Modified antibodies of particular interest are 20 scFv's carrying the variable region sequences of the light and heavy chains linked together. scFv antibody fragments are derived from Fragment antigen binding (Fab) portions of an antibody comprising the V region of a heavy chain linked by a stretch of synthetic peptide to a V region of a light chain. Bivalent and bispecific scFv's have been formed using leucine zipper-based dimerization cassettes attached to different scFv's (de Kruif and 25 Logtenberg (1996) JBiol Chem 271:7630-7634). Bispecific antibodies with different scFv domains connected by a polypeptide chain have been designed to cross-link T-cells in tumors. The two or more interactions that such chimeric antibodies have with different surface antigens on a single cell greatly increase the strength of binding since the dissociation constants for the individual interactions are multiplicative. 30 [0008] Whilst therapeutic antibodies are important, the generation of highly-specific H:\aar\lntenoven\RPorlb\DCC\AAR\4959862_ I.doc-1210312013 -3 antibodies has faced difficulties. There is a need, therefore, to develop antibody-based therapeutic agents which are more highly selective for target cells. [00091 Naturally occurring antibodies have been proposed as highly specific targeting 5 agents (Carter, 2001 supra). However, the antigenicity of mouse monoclonal antibodies in human subjects limits therapeutic use. Such monoclonal antibodies are primarily used in immunohistochemistry and flow cytometry. [00101 The Fc domain of potential therapeutic antibodies is required for complement 10 dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC). Another useful development in the use of antibody fragments is their fusion to active agents such as radio-isotopes (Wu et al. (1996) Immunotechnology 2:21-36; Wu et a (2000), Proc. Natl. Acad Sci USA 97:8495-8500; Adams et al. (2000) Nuc Med Biol 27:330-346), enzymes (Bagshawe and Begent (1996) Adv Drug Delivery Rev 22:365-367) 15 and cytotoxins (Reiter and Pastan (1998) Tibtech 16:51-520; Kreitmann (1999) Curr Opin Immunol 11:570-578). [0011] Whilst scFv's are useful as targeting molecules, they lack the Fc domain and are unable to induce ADCC or CDC. 20 [0012] International Patent Publication Number W02007/062466, the contents of which are incorporated herein by reference, described, in one embodiment, modified scFv's referred to as "demibodies". Sets of at least two demibodies each specific for a different antigen and each comprising complementing portions of an Fc region or a reporter 25 molecule region and a complementary interactive binding domain resulted in a functional antibody-like molecule when bound together. [0013] Whereas the demibody concept is successful, it does assume the generation of a functional Fc domain or reporter molecule.

H:\aar\lntenvoven\NRPortbl\DCC\AAR\4959862 .dc12/03/2013 -4 SUMMARY [00141 The present disclosure teaches a protocol for the selective and specific targeting of cells and viruses having at least two different antigens characteristic of that cell, cell type 5 or virus. The protocol is a modification of the demibody approach as described in International Patent Publication No W02007/062466, the entire contents of which are incorporated herein by reference. The protocol enabled herein comprises at least two monomeric modified demibodies, each comprising an antigen-binding domain (ABD) and an interactive complementary binding domain (iCBD) capable of forming heterodimers but 10 not homodimers of the monomeric demibodies. Each demibody monomer is capable of binding to an antigen. Selectivity and specificity for a target cell or virus arise from a set of at least two monomeric demibodies binding to at least two antigens characteristic of the target cell or virus. Reference to a "target cell" includes eukaryotic cells, such as cancer cells or eukaryotic pathogens as well as prokaryotic cells such as bacteria. When the two 15 modified demibodies are in close proximity at the cell surface, the iCBD's facilitate the generation of a bispecific immunointeractive molecule (i.e. a demibody heterodimer). The protocol then uses an antibody specific for the demibodies dimer to provide Fc capability or reporter molecule capability or to provide a functional cytotoxic molecule or to provide other functionalities. 20 [00151 The present disclosure enables a modified form of a demibody referred to as a "demibody", a "modified demibody" or a "reduced domain demibody" or a "two-domain demibody" or a "synthetic antibody monomer". The demibody monomer taught herein obviates the need to include two complementary Fc partial domains (or complementary 25 reporter molecule domains) on each of the single demibodies. Each demibody monomer in a set comprises an ABD region and an iCBD. When together, the demibody monomers form a heterodimeric complex of two antigen binding regions via the iCBD. This is the "modified demibody dimer" or "heterodimeric binding pair" or "demibody heterodimer" or "dimeric synthetic antibody". The two iCBD's, in bound form as a heterodimer, provide a 30 unique target not present on the individual (monomeric) demibody chains. Fc function or reporter molecule function or cytotoxic agent function or other functionality is then H:\aar\ntcnvovcn\NRPortbl\DCC\AAR\4959862_ Ldoc-12/03/2013 -5 provided by an antibody specific for the dimeric iCBD's. [0016] Consequently, the present disclosure is instructional for sets of at least two modified demibodies wherein each modified demibody comprises an ABD and one or 5 other member of an iCBD pair. Two modified demibodies are designed such that, when in close proximity, each member constituting one of an iCBD pair, interacts forming a heterodimeric binding pair. This in turn permits a dimeric iCBD-specific antibody (iCBD Ab) to interact with the dimeric modified demibody to generate a functional immunointeractive molecule which provides Fc or reporter molecule capability resulting 10 in, for example, cell death, cell therapy or a reporter signal. The modified demibodies enabled herein have enhanced specificity for therapeutic, imaging and diagnostic purposes. The modified demibodies are also useful research tools such as for FACS, flow cytometry and affinity chromatography. In essence, the modified demibodies taught herein enable enhanced immunophenotypic selection of cells and viruses. For brevity, the modified 15 demibody is also referred to as a "demibody". The use of this term is contextual to a modified demibody. [00171 The ABD may be derived from an immunoglobulin such as a scFv of Fab fragment or any affinity scaffold such as a microaffinity scaffold. Examples of ABD's include 20 scFv's, domain antibodies (dAbs), nanobodies, microproteins, fibronectins, microbodies, anticalins, aptamers, darpins, avimers, afflins and Kunitz domains. The ABD may also be chemically modified such as with polyethylene glycol (PEG). In relation to the latter, an Fab fragment conjugated to PEG is generated (referred to as a "Fab' fragment"). 25 [00181 The iCBD's encompass inter alia complementary portions of a leucine zipper (LZ), a receptor-ligand (e.g. cytokine and cytokine receptor), an actin and an actin-binding protein and DNA aptamers. A LZ dimer formed by two LZ-based iCBD's is referred to as a "LZD". An antibody specific for the LZD is referred to as an "LZD-Ab" or "LZD specific Ab". The modified demibody dimer in this instance is referred to as a scFv(LZD). 30 An antibody which binds to scFv(LZD) is referred to as a scFv(LZD)-Ab.

H:\aar\Interwoven\NRPortbl\DCC\AAR4959862_ Ldoc-12/03/2013 -6 [00191 The modified demibodies enabled by the present disclosure have enhanced target specificity since each modified demibody in the set is specific for a particular antigen. Hence, by selecting cells or viruses which have an unusual (e.g. unique) pair of antigens provides a higher level of specificity and reduces the risk of non-specific binding. The use 5 of an antibody to the dimeric iCBD's enable's Fc or reporter molecule capability to be provided regardless of the target. [0020] In one embodiment, the dimeric modified demibodies are bound to a iCBD-Ab which contains a functional Fc fragment that is capable of initiating associated activities 10 such as antibody-dependent cellular cytotoxicity (ADCC) and/or complement-dependent cytotoxicity (CDC). In another embodiment, the iCBD-Ab is coupled to a reporter molecule, therapeutic agent or cytotoxic agent. In a further embodiment, the reporter molecule is a dye such as a fluorochrome which provides a particular signal. 15 [0021] Hence, the present disclosure teaches a set of modified demibody monomers each demibody monomer comprising an ABD and an iCBD such that the iCBD on each demibody enables generation of a heterodimeric form of the modified demibodies, which heterodimeric form enables a dimer-specific antibody to bind. 20 [0022] The dimeric form-specific antibody provides Fc or reporter molecule or other functionality to the heterodimeric modified demibody. In one embodiment, selective toxicity against a target cell or virus is enabled by the Fc portion which is glycosylated enabling tight binding by cytotoxic cells such as neutrophils, monocytes, natural killer cells and killer T-cells. 25 [00231 In a further embodiment, dimer-specific antibodies which bind to the modified demibody pairs (i.e. heterodimers) result in a functional cytotoxic or therapeutic immunointeractive molecule. Examples of functionalities include apoptotic, cell cycle static, lytic and cytotoxic properties. 30 [00241 In an alternative embodiment, a dimer-specific antibody may carry a functional H:\aarlnienvyen\NRPortbl\DCC\AAR\495986 2_Ldoc-12/03/2013 -7 reporter molecule such as a dye or fluorescent marker. Examples of fluorescent markers include hydroxycoumarin, aminocoumarin, methoxycumarin, cascade blue, Lucifer yellow, NBD, Phycoerythrin (PE), PerCP, allophycocyanin, hoechst 33342, DAPl5 SYTOX Blue, hoechst 33258, chromomycin A3, mithramycin, YOYO-I, SYTOX green, 5 SYTOX orange, 7-AAD, acridine orange, TOTO-I, To-PRO-I, thiazole orange, TOTO-3, TO-PRO-3, LDS 751, Alexa Fluor dyes including Alexa Fluoro-350, -430, -488, -532, 546, -555, -556, -594, - 633, -647, -660, -680, -700 and -750; BoDipy dyes, including BoDipy 630/650 and BoDipy 650/665; Cy dyes, particularly Cy2, Cy3, Cy3.5, Cy5; Cy5.5 and Cy7; 6-FAM (fluorescein); PE-Cy5, PE-Cy7, Fluorescein dT; Hexachlorofluorescein 10 (Hex); 6-carboxy- 4',5'-dichloro-2', T- dimethoxyfluorescein (JOE); Oregon green dyes, including 488-X and 514; Rhodamine dyes, including X-Rhodamine, Lissamine Rhodamine B, Rhodamine Green, Rhodamine Red and ROX; TRITC, Tetramethylrhodamine (TMR); Carboxytetramethylrhodamine (TAMRA); Tetrachlorofluorescein (TET); Red 6B, FluorX, BODIPY-FL and Texas Red. 15 [0025] The modified demibodies enabled herein are useful in the treatment of a range of conditions including cancer, infection by pathogens or the selective targeting of any cell type in a subject. They are also useful in targeting cells such as stem cells. 20 [0026] Reference to a "subject" includes a human or other primate animal, livestock animal, laboratory test animal, companion animal or avian species. A subject may also be regarded as a patient, individual, recipient and the like. [0027] The present disclosure teaches a method for treating a subject, such as a patient, 25 comprising administering to the subject at least two modified demibodies which, when bound together via iCBD's, and engaged with a dimer-specific antibody having a functional Fc domain or portion thereof, is capable of inducing cell cytotoxicity such as ADCC or CDC. Alternatively, the modified demibodies are engaged with the dimer specific antibody coupled to a cytotoxic agent or therapeutic molecule or reporter 30 molecule.

H:\aar\intenvoven\NRPortbl\DCC\AAR\4959862_ .doc-12/03/2013 -8 [0028] In an embodiment, each ABD binds to a CD antigen on a cell surface such as CD5 or CD19/CD20. When a set of modified demibodies binds to CD5 and CD19/CD20, selective cytotoxicity or diagnostic capability is provided for particular cancer cells such as chronic lymphocytic leukemia (CLL). In another embodiment, the sets of modified 5 demibodies target other pairs of surface CD antigens found on target cells but not usually present on normal cells (such as listed in Table 2). Reference to "normal" cells in this context includes non-cancer cells or non-targeted cells. [0029] Diagnostic compositions and methods for diagnosing and/or imaging and/or 10 therapy are also taught by the present disclosure. [0030] The pair of modified demibodies may be sequentially or simultaneously administered. 15 [00311 The present disclosure further teaches a method for selectively identifying a cell or virus, the method comprising contacting the cell or virus with a pair of modified demibodies wherein each modified demibody comprises first and second portions wherein each of the first portions is capable of interacting with an antigen on the cell or virus surface, the second portions are complementary iCBD's wherein upon binding of the 20 individual modified demibodies to two different antigens, the iCBD's dimerize together to provide a target for a dimer-specific antibody to bind carrying a reporter molecule. The provision of signal would be an indication of the cell or virus sought to be identified. [0032] The present disclosure further enables a method for selectively targeting a cell or 25 virus, the method comprising contacting the cell or virus with monomeric forms of modified demibodies wherein each modified demibody comprising a first portion and a second portion wherein each first portion interacts with an antigen on the cell or virus surface, and wherein each second portion is an iCBD wherein upon binding of the monomeric demibodies to two antigens, the complementary iCBDs combine to form a 30 heterodimeric demibody enabling an iCBD dimer-specific antibody to provide target signal identification, therapy or cytotoxicity to the cell. Reference to a "cell" includes a virally- H:\aar\Inten~voven\NRPoflbl\DCC\AAR\4959862_ I.doc-12/03/2013 -9 infected cell. [00331 Throughout this specification, unless the context requires otherwise, the word "comprise" or variations such as "comprises" or "comprising", will be understood to imply 5 the inclusion of a stated element or integer or method step or group of elements or integers or method steps but not the exclusion of any element or integer or method step or group of elements or integers or method steps. an" and "te"icld [00341 As used in the subject specification, the singular forms a", an" and "the" include 10 singular and plural aspects unless the context clearly dictates otherwise. Thus, for example, reference to "a demibody" includes a single demibody, as well as two or more different demibodies; reference to "an antigen or antibody" includes a single antigen or antibody, as well as two or more antigens or antibodies. Aspects disclosed herein are encompassed by the term "invention". All aspects of the invention are enabled within the width of the 15 claims. [0035] A list of abbreviations used herein is provided in Table 1.

H:\aar\lntenoven\NRPobl\DCC\AAR\4959862_.doc-I 2/)3/2013 - 10 TABLE 1 Abbreviations Abbreviation Description Ab Antibody ABD Antigen binding domain ADCC Antibody-dependent cellular cytotoxicity Ag Antigen C region Constant region CD antigen Cluster of differentiation antigen CDC Complement-dependent cytotoxicity Dimer-specific An antibody that binds to a reconstituted iCBD domain on a antibody demibody heterodimer Dimeric modified A pair of modified demibodies rendered dimeric via combinations demibody of the iCBD. DSA An antibody specific for a dimeric modified antibody Fab fragment Fragment antigen binding Fc Fragment crystalline FcR Fc receptor Fv Fragment variable H chain Heavy chain iCBD Interactive complementary binding domain Ig Immunoglobulin L chain Light chain LZD-Ab Leucine Zipper Dimer-Specific Antibody Modified demibody A two domain demibody comprising an ABD and an iCBD sc Single chain scFv Single chain variable fragment, recombinant Fab comprising V regions of heavy and light chains scFV(LZD) Dimer of modified demibodies scFv(LZD)-Ab An antibody specific for a scFV(LZD) V region Variable region H:\aar\lnnvovenNR, orbl\DCC\AARX4959862_ .doc-12/03/2013 - 11 BRIEF DESCRIPTION OF THE FIGURES [00361 Figure 1 is a diagrammatic representation of the "modified demibody" causing lysis of a cell. The heterodimeric scFv(LZD) is recognized by a leucine zipper dimer 5 (LZD)-specific antibody coupled to an intact Fc domain which induces antibody dependent cellular cytotoxicity (ADCC) or complement dependent cytotoxicity (CDC) for any combination of two antigens. [00371 Figure 2 is a diagrammatic representation of the "modified demibody" for 10 detection of specific cell types. The heterodimeric scFv(LZD) is recognized by a leucine zipper dimer (LZD)-specific antibody coupled to a fluorophore such as Alexa, Cy dyes or Quantum dots, for any combination of modified demibody pairs, for use in flow cytometry.

H:\aar\Intenvoven\NRPorbl\DCC\AAR\4959862 l.doc- 12/0312013 - 12 DETAILED DESCRIPTION [00381 The present disclosure teaches a modified demibody and its use to facilitate selective and specific targeting of cells and viruses. Pairs of modified demibodies, when 5 combined via interactive complementary binding domains (iCBD's), form an antibody like, immunointeractive molecule (i.e. reference to herein by terms such as a dimeric demibody, heterodimeric demibody, heterodimeric synthetic antibody and the like). The term "immunointeractive" in this context is used to highlight one of the principal features of an antibody, i.e. the ability for an antibody to interact specifically with an antigen. The 10 demibody is modified in the sense that it does not need to contain complementary portions of an Fc region or reporter molecule or other functionality. The modified demibody comprises an antigen binding domain (ABD) and one of a pair of iCBD's. In use, two monomeric modified demibodies form a dimer via the pair of complementary iCBD's on each monomeric modified demibody generating a bispecific immunointeractive molecule. 15 As each member of the pair of iCBD's is different, dimers formed by binding of the complementary iCBD's are necessarily heterodimeric. A dimeric modified-specific antibody (DSA), specific for a pair of iCBD's when in dimeric form, engages the dimeric modified demibody to provide an Fc region, reporter molecule or other functionality. The term "demibody" is defined in WO 2007/062466. However, expressions such as "synthetic 20 immunoglobulin", "synthetic antibody", "synthetic antibody monomer", "immunointeractive molecule" or "molecule comprising an antigen-binding domain" or "modified" forms of these molecules may be used in place of "demibody". The term "demibody" may also be used for brevity. When two demibody monomers combine via the iCBD's, the resulting heterodimer is referred to as a demibody dimer, synthetic 25 antibody dimer, immunoglobulin-like dimer and the like. [00391 Hence, an active form of the immunointeractive molecule complex taught by the present disclosure comprises two monomeric demibodies each bound to a different antigen on the same target cell or virus to form a dimer via the iCBD's, and an antibody bound to 30 the dimeric iCBD's that provides Fc or reporter signal activity or a functionality. an example of a functionality is providing a cytotoxic molecule.

H:\aar\lnvtenoven\NRPoubl\DCC\AAR\4959862_ .doc- 12/03/2013 - 13 [0040] Each modified demibody monomer enabled by the present disclosure comprises: (i) an ABD; and (ii) a member of a complementary pair of iCBD's. 5 [0041] In use, at least two modified demibodies form a dimer having: (i) ABD's directed to different antigens on the same target cell or virus; (ii) iCBD's in heterodimeric form; and (iii) an antibody specific for heterodimeric iCBD's providing Fec, reporter signal 10 activity or other functionality. [0042] The modified demibodies taught herein have a range of applications including selective cytotoxicity of target cells that include cancer cells or pathogens, selective detection of cells such as cancer cells, stem cells (embryonic or adult) or cells infected 15 with pathogens, as well as viruses. A target cell also includes prokaryotic cells (i.e. bacteria). Cell detection may be facilitated with FACS, flow cytometry, DotScan CD antibody microarrays and fluorescent microscopy. [0043] Hence, the present disclosure is instructional for a set of modified demibodies, each 20 demibody comprising an ABD and an iCBD such that the iCBD on each demibody enables generation of a heterodimeric form of the modified demibodies, which heterodimeric form enables a dimer-specific antibody to bind. [00441 In relation to an embodiment, the formation of the iCBD dimer provides a target for 25 a leucine zipper dimer (LZD)-specific antibody with a functional Fc domain, or reporter signaling activity or other functionality to bind. The dimer-specific antibody substantially does not bind to a monomeric form of the modified demibody. [0045] It will be appreciated, therefore, that in operation, a set of at least two modified 30 demibodies is required which have complementary iCBD's and which have antigen H:\aar\Intcnovcn\NRPofbl\DCC\AAR\4959862_ Ldoc-12/03/2013 - 14 binding portions directed to different antigens, which antigens are co-expressed on a target cell or virus. [0046] Accordingly, the present disclosure enables a modified demibody comprising first 5 and second portions wherein: a first portion is capable of interacting with an antigen on a target cell or virus; and a second portion is one member of an iCBD; wherein two modified demibodies form a dimer via the iCBD's, to which a dimeric specific antibody binds to provide Fc, reporter signaling activity or other functionality. 10 [0047] In an embodiment, the present disclosure teaches a modified demibody monomer comprising first and second portions wherein: a. the first portion is capable of interacting with a first antigen on a target cell or virus; 15 b. the second portion is one member of an immunointeractive complementary binding domain (iCBD) pair; wherein the modified demibody becomes part of a functional molecule when it is bound to a second modified demibody which comprises a first portion capable of interacting with another antigen on the same target cell or virus; and a second portion 20 which is the other member of the iCBD pair. [0048] In an embodiment, taught herein is an agent comprising first and second portions wherein: a. the first portion is capable of interacting with a first antigen on a target cell 25 or virus; b. the second portion is one member of an immunointeractive complementary binding domain (iCBD) pair; wherein the agent forms a dimeric complex when it is bound to a second agent which comprises a first portion capable of interacting with a second different antigen on 30 the same target cell or virus; and a second portion which is the other member of the iCBD H:aar\1ntenvoven\NRPortbl\DCC\AAR\4959862_ .doc-12/03/2013 - 15 pair. The resulting dimer formed by the two agents is a heterodimer based on the requirement for two complementary iCBD's. [0049] In an embodiment, the dimer-specific antibody which binds to the reconstitution 5 iCBD pair provides Fc capability, reporter molecule capability or a cytotoxic capability. The functional Fc domain is useful in mediating CDC or ADCC against target cells. [0050] The present disclosure is further instructional for a set of modified demibodies comprising a first modified demibody with first and second portions wherein: 10 the first portion is capable of interacting with a first antigen on a target cell or virus; and the second portion is one member of an iCBD pair; the set comprising a second modified antibody with first and second portions wherein: the first portion is capable of interacting with a second antigen on the target cell or 15 virus which is different to the first antigen; and the second portion of the other member of the iCBD pair is complementary to the iCBD of the first modified demibody. [00511 In an embodiment, enabled herein is a set of agents comprising a first agent with 20 first and second portions wherein: a. the first portion is capable of interacting with a first antigen on a target cell or virus; and b. the second portion is one member of an iCBD pair; and a second agent comprising: 25 c. a first portion which is capable of interacting with a second different antigen on the target cell or virus; and d) a second portion which is the complementary member of said iCBD pair. [0052] In a related embodiment, the present disclosure teaches a method for selectively 30 inducing a cytotoxic response on a target cell or virus in a subject, the method comprising administering to the subject first and second modified demibodies wherein the first H:aar\ntenvoven\NRorbl\DCC\AAR\4959862 1.doc-12A)3/2013 - 16 modified demibody comprises first and second portions wherein: a. the first portion is capable of interacting with a first antigen on a target cell or virus; and b. the second portion is one member of an iCBD pair; 5 and the second modified demibody comprising: c. a first portion which is capable of interacting with a different antigen on the target cell or virus; and d. a second portion which is the complementary member of the iCBD pair; wherein the modified demibodies become a functional molecule when the dimer 10 formed by the iCBD engages with a dimer-specific antibody which provides a functional Fc fragment through which complement-dependent cytotoxicity (CDC) and antibody dependent cellular cytotoxicity (ADCC) is mediated, or is coupled to a cytotoxic molecule. [00531 Taught herein is a method for selectively inducing a cytotoxic response on a target 15 cell or virus in a subject, the method comprising administering to the subject first and second agents wherein a first agent comprises first and second portions wherein: a. the first portion is capable of interacting with a first antigen on a target cell or virus; and b. the second portion is one member of an iCBD pair; 20 and a second agent comprising: c. a first portion which is capable of interacting with a second different antigen on the target cell or virus; and d. a second portion which is the complementary member of the iCBD pair; wherein the first and second agents become a dimeric complex when the iCBD's 25 dimerize the two agents together and wherein a cytotoxic response is induced when a dimer-specific antibody which provides a functional Fc fragment through which complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC) is mediated, or is coupled to a cytotoxic molecule, binds to the dimeric complex. Generally, the dimer specific antibody binds to a reconstituted iCBD domain on the dimer. 30 H:\aar\Intenvoven \NRortbl\DCC\AAR\4959862 L.doc-12/03/2013 - 17 [0054] Enabled herein is a method for selectively inducing cytotoxicity against a cell, the method comprising contacting cells with first and second modified demibodies wherein the first demibody comprises first and second portions wherein: the first portion is capable of interacting with a first antigen on a target cell or 5 virus; and the second portion is one member of an iCBD pair; and the second modified demibody comprises first and second portions wherein: the first portion is capable of interacting with an antigen on the same target cell which is different to the first antigen; and 10 the second portion, in combination with the second portion of the first mentioned modified demibody, forms a dimer of iCBD's; the modified demibodies administered for a time and under conditions sufficient for the iCBD's to engage at the cell surface to form a dimeric modified demibody to which a dimer-specific antibody with a functional Fc domain binds thereby facilitating selective 15 toxicity against the cell. [00551 The present disclosure also teaches a method for selectively inducing a cytotoxic response on a target cell or virus in a subject, the method comprising administering to the subject first and second modified demibodies wherein the first modified demibody 20 comprises first and second portions wherein: a. the first portion is capable of interacting with a first antigen on a target cell or virus; and b. the second portion is one member of an iCBD pair; and the second modified demibody comprising: 25 c. a first portion which is capable of interacting with a different antigen on the target cell or virus; and d. a second portion which is the complementary member of the iCBD pair; wherein the modified demibodies become a functional molecule when the dimer formed by the iCBD engages with a dimer-specific antibody which provides a functional 30 Fc fragment through which complement-dependent cytotoxicity (CDC) and antibody dependent cellular cytotoxicity (ADCC) is mediated, or is coupled to a cytotoxic molecule.

H:\aarnienovecn\NRPortbl\DC\AAR\4959862_1.doc-12/03/2013 - 18 [00561 As indicated above, a "target cell" can include an eukaryotic cell (e.g. cancer cell) or prokaryotic cell (i.e. bacterium) as well as cells infected with pathogens such as viruses and the malaria parasite. 5 [0057] Although the above aspects taught herein relate to the Fc portion of a dimer specific antibody, the subject invention extends to coupling such antibodies with other cytotoxic domains such as apoptotic domains or lysing domains or domains from agents which include cell cycle arrest as well as domains from therapeutic molecules. 10 [00581 Accordingly, another aspect enabled herein is a modified demibody comprising first and second portions wherein: the first portion is capable of interacting with a first antigen on a target cell or virus; 15 the second portion is one member of an iCBD pair; wherein the modified demibody is capable of forming a iCBD dimer engaged by a dimer-specific antibody coupled to a cytotoxic, therapeutic, or reporter molecule when the modified demibody is bound to a second demibody which comprises a first portion capable of interacting with another antigen on the same target cell; and 20 a second portion which is the complementary iCBD partner of the iCBD of the first modified demibody. [00591 The dimeric modified demibody is, in effect, a modular or chimeric immunointeractive molecule comprising first and second monomeric modified demibodies 25 each with first and second portions, respectively, as defined above. [00601 The present disclosure is further instructional for a set of modified demibodies comprising a first modified demibody with first and second portions wherein: the first portion is capable of interacting with a first antigen on a target cell or 30 virus; the second portion is one member of a iCBD; H:\aa\lnenoven\NRPonbl\DCC\AAR\4959862_L.doc-12A)3/2013 - 19 the set further comprising a second demibody with first and second portions wherein: the first portion is capable of interacting with a different antigen on the same target cell; and the second portion is the complementary iCBD partner of the iCBD of the first 5 modified demibody. [0061] Taught herein is a method for selectively detecting or targeting a cell or virus, the method comprising contacting cells or viruses with first and second modified demibodies wherein the first modified demibody comprises first and second portions wherein: 10 the first portion is capable of interacting with a first antigen on a target cell or virus; and the second portion is one member of an iCBD pair; the second modified demibody comprises first and second portions wherein: the first portion is capable of interacting with a different antigen on the same target 15 cell or virus; and the second portion is a complementary iCBD partner of the iCBD on the first modified demibody of the binding pair, wherein the first and second modified demibodies are capable of forming a dimer which is engaged by a dimer-specific antibody coupled to a cytotoxic, therapeutic, or 20 reporter molecule. [0062] In an embodiment, taught herein is a method of selectively identifying a cell or virus, the method comprising contacting the cell with a pair of agents wherein each agent in a pair comprises first and second portions wherein the first portions are capable of 25 interacting with two different antigens on a cell or virus, the second portions are complementary iCBD's wherein upon binding of the agents to the two antigens, the iCBD's dimerize at the cell or viral surface enabling interaction with a dimer-specific antibody coupled to a reporter molecule to provide a signal. 30 [0063] The iCBD's of the present invention include leucine zippers, a receptor-ligand, an actin and actin-binding protein and DNA aptamers. Reporter molecules include dyes such H:\aarntenvoven\NRPortbl\DCC\AAR\4959862_ .doc-12/0312013 -20 as hydroxycoumarin, aminocoumarin, methoxycumarin, cascade blue, Lucifer yellow, NBD, Phycoerythrin (PE), PerCP, allophycocyanin, hoechst 33342, DAPI, SYTOX Blue, hoechst 33258, chromomycin A3, mithramycin, YOYO-I, SYTOX green, SYTOX orange, 7-AAD, acridine orange, TOTO-I, To-PRO-I, thiazole orange, TOTO-3, TO-PRO-3, LDS 5 751, Alexa Fluor dyes including Alexa Fluro-350, -430, -488, -532, -546, -555, -556, -594, -633, -647, -660, -680, -700 and -750; BoDipy dyes, including BoDipy 630/650 and BoDipy 650/665; Cy dyes, particularly Cy2, Cy3, Cy3.5, Cy5, Cy5.5 and Cy7; 6-FAM (Fluorescein); PE-Cy5, PE-Cy7, Fluorescein dT; Hexachlorofluorescein (Hex); 6-carboxy 4',5'-dichloro-2', T-dimethoxyfluorescen (JOE); Oregon green dyes, including 488-X and 10 514; Rhodamine dyes, including X-Rhodamine, Lissamine Rhodamine B, Rhodamine Green, Rhodamine Red and ROX; TRITC, Tetramethylrhodamine (TMR); Carboxytetramethyirhodamine (TAMRA); Tetrachlorofluorescein (TET); Red 6B, FluorX, BODIPY-FL and Texas Red. 15 [00641 In the above aspects, the ABD's may be derived from an immunoglobulin or may be any affinity scaffold such as but not limited to scFv's, dAbs, nanobodies, microproteins, fibronectins, microbodies, anticalins, aptamers, darpins, avimers, afflins, and Kunitz domains. ABD's particularly include scFv and Fab, Fab' and homologous fragments as well as chemically modified fragments such as a Fab' molecule. 20 [0065] Reference to "functionalities" includes Fc capability, reporter signaling activity functions and apoptotic, cell cycle static, lytic, cytotoxic and antibiotic capability. [00661 The present disclosure further teaches a method for selectively identifying a cell or 25 virus, the method comprising contacting the cell or virus with a pair of modified demibodies wherein each modified demibody comprises first and second portions wherein each of the first portions is capable of interacting with an antigen on the cell or virus surface, the second portions are complementary iCBD's wherein upon binding of the individual modified demibodies to two different antigens the, iCBD's dimerize together to 30 provide a target for dimer-specific antibody to bind carrying a reporter molecule.

H:\aar\ntenvoven\NRPortbl\DCC\AAR\4959862_ .doc-12103/2013 -21 [00671 The present disclosure further teaches a method for selectively targeting a cell or virus, the method comprising contacting the cell or virus with monomeric forms of modified demibodies wherein each modified demibody comprising a first portion and a second portion wherein each first portion interacts with an antigen on the cell or virus 5 surface, and wherein each second portion is an iCBD wherein upon binding of the monomeric demibodies to two antigens, the complementary iCBDs combine to form a heterodimeric demibody enabling an iCBD dimer-specific antibody to provide target signal identification, therapy or cytotoxicity to the cell or virus. 10 [00681 Enabled herein is a method of selectively identifying a cell or virus, the method comprising contacting said cell with a pair of agents wherein each agent in a pair comprises first and second portions wherein said first portions are capable of interact with two different antigens on a cell or virus, said second portions are complementary iCBD's wherein upon binding of the pair of agents to the two antigens, the iCBD's dimerize at the 15 cell or virual surface enabling interaction with a dimer-specific antibody coupled to a reporter molecule to provide a signal. [00691 The modified demibodies may be administered sequentially or simultaneously. Sequential administration includes separate administration of both monomeric forms of the 20 modified demibodies within nanoseconds, seconds, minutes, hours or days. Simultaneous administration includes co-administration of the monomeric modified demibodies in a single composition or in two separate compositions. [0070] The subject may be a human or other primate animal, a livestock animal (e.g. 25 sheep, cow, pig, horse, donkey, and goat), a companion animal (e.g. dog or cat), laboratory test animal (e.g. mouse, rat, rabbit, and guinea pig), and captive wild animal or avian species (e.g. poultry birds, caged birds, aviary birds, game birds,). A subject may also be regarded as a patient, individual, recipient or the like. In an embodiment, the subject is a human. A human may be a human of any age. 30 H \aar\lntenvoven\Rorbl\DCC\AAR\4959862_ Ldoc-12A)3/2013 -22 [00711 Generally, the ABD is a scFv portion of an antibody. seFv's are generally monomeric although the extent of monomerism compared to dimerism or multivalentism may depend on the size of the linker between the VH and VL domains. The construction of scFv molecules is described in Hudson 1999 supra and Kortt et al. (2001) Biomolecular 5 Engineering 75:95-108. [00721 In another embodiment, the ABD may be a recombinant Fab fragment. This fragment corresponds to the arms of an antibody molecule which contain the complete light chains paired with the VH and CH domains of the heavy chains. 10 [00731 The present disclosure teaches other ABD's of immunoglobulins such as but not limited to domain antibodies (dAbs), nanobodies, microproteins, fibronectins, microbodies, anticalins, aptamers, darpins, avimers, afflins, and Kunitz domains or other affinity scaffolds such as but not limited to microscaffolds. 15 [00741 The ABD is specific for an antigen on the surface or sub-surface co-continuous with the external environment on a target cell or virus. The target cell or virus may be a cancer cell, a cell infected by a pathogen or parasite or another unwanted cell. A pathogen includes a eukaryotic cell such as the malaria parasite as well as a prokaryotic 20 microorganism. The present disclosure also teaches the selective targeting of a virus or a virally infected cell. A virally infected cell may express virally-derived proteins on its surface. Specific targeting of such a cell may result in loss of virus-producing cells. This is an example of an unwanted cell. 25 [00751 The present disclosure is useful in the generation of therapeutic agents to target cancer cells including leukocyte subsets. Cancer cells are generally defined by the expression of repertoires of particular CD antigens. However, targeting a single CD antigen may cause collateral damage to normal cells which carry the same CD antigen. This problem is addressed by the present invention in that a single type of modified 30 demibody alone is incapable of inducing ADCC or CDC due to lack of an Fe domain and an inability to dimerize. However, when two modified demibodies having complementary H:\aarnenvoven\NRPortbI\DCC\AAR\4959862 l.doc-12/03/2013 - 23 iCBD's are used and are directed to two different CD antigens or other antigens present on a single cancer cell, then only cells with this unusual pair of surface molecules are cytotoxically targeted after both modified demibodies have dimerized and have engaged a dimer-specific antibody. In essence, once a modified demibody has bound to its respective 5 antigen, the CD antigen moves freely in the two dimensions of the lipid bilayer and the modified demibodies eventually become in close proximity to each other. At that point, the individual iCBD's interact resulting in formation of a dimeric iCBD. For example, LZ domains come together to form a leucine zipper dimer or LZD. This is then recognized by the dimer-specific antibody which provides an Fc domain (or reporter molecule or other 10 functionality). Selective cytotoxicity is now induced for that cell. The specificity of the present invention is derived from the unusual combinations of surface molecules which correspond to a particular cancer, or unwanted cells or other target cells. Other target cells include bacteria and other prokaryotic cells. 15 [0076] With respect to cancer treatment, CD antigens are considered a particular embodiment such as two or more CD antigens selected from those listed in Table 2.

H:\aar\nienvove\,.NRPortbl\DCC\AAR\4959862_ L.do-12/0312013 - 24 TABLE 2 Summary of CD antigens CD designation Blood-related cancer CD5 + CD19/CD20 chronic lymphocytic leukemia (CLL) CD19/CD20 + K (or ?) targets clonal B-cell populations CD 1 9/CD20 + CD 10 follicular non-Hodgkins lymphoma (NHL) CD103 + CD22 (or CD25) (or CD19) hairy cell leukemia (HCL) CD4 + CD8 T-cell acute lymphocytic leukemia (T-ALL) ("cortical thymocyte") CD8 + CD57 NK cell/large granular lymphocytic leukemia CD1O + CD34 pre-B ALL CD34 + myeloid marker (i.e. CD13 or acute myeloid leukemia (AML) CD33) CD1 17 (c-kit) + myeloid marker AML CD13 or CD33 (My) + CD14 (Mo) myelomonocytic leukemias CD41 or CD61 + CD33 megakaryocytic leukemias 5 [0077] The iCBD's may constitute any of a number of entities which are capable of interacting with each other to form an association or bond. In one particularly embodiment, the iCBD is a leucine zipper domain. Examples of other iCBD's contemplated herein include a receptor-ligand (e.g. cytokine and cytokine receptor), actin and an actin-binding protein and DNA aptamers. The actual nature of the iCBD is not critical to the present 10 invention provided that upon coming together in two dimensions at the cell surface, both complementary members of iCBD's interact to form a heterodimer which is recognized by a dimer-specific antibody which provides an Fe portion, a reporter molecule and/or a therapeutic or cytotoxic moiety. 15 [00781 In an embodiment, the iCBD's comprise complementary portions of a leucine zipper. Leucine zipper amino acid sequences are shown in Table 3. Heterodimerization occurs between for example SEQ ID NOs:1 and 2, SEQ ID NOs:3 and 4 and SEQ ID NOs:3 and 5.

H\aarIntenvovcn\NRPoitbl\DCC\AAR\4959862 I.doc-12103/2013 -25 TABLE 3 Leucine zipper sequences SEQUENCE AMINO ACID SEQUENCE ID NO: 1 LEI EAAFLEQ ENTALET EVAELEQ EVQRLEN EVSQYET RYGPLGGGK 2 KGGGLEI RAAFLRR RNTALRT RVAELRQ RVQRARN RVSQYRT RYGPL 3 LEI RAAFLRQ RNTALRT EVAELEQ EVQRLEN EVSQYET RYGPLGGGK 4 KGGGLEI EAAFLER ENTALET RVAELRQ RVQRARN RVSQYRT RYGPL 5 LEI EAAFLER ENTALET RVAELRQ RVQRLRN RVSQYRT RYGPLGGGK 5 [0079] In an embodiment, the present disclosure teaches a modified demibody comprising an Fab portion (scFv) or derivative thereof, and one member of a pair of iCBD's wherein the modified demibody is bound to a second complementary demibody comprising a different Fab portion, the binding via the other member of the iCBD pair, the two modified 10 demibodies forming a target for a dimer-specific antibody providing a Fc, reporter moiety or other functional molecule. [0080] In an embodiment, the iCBD's comprise a leucine zipper. 15 [00811 In an embodiment, the Fab portion is an scFv fragment. [00821 In an embodiment, each Fab or scFv fragment has specificity for an antigen on a cancer cell such as a CD antigen. 20 [00831 The present disclosure teaches a set of modified demibodies wherein each set comprises at least two modified demibodies each having the structure: x - scFv(Ag'); or y - scFv(Ag 2 ); wherein: H:\aar\lnIe~woen\NRPorb\DCC\AAR\4959862_ Ldoc-12/03/2013 - 26 scFv(Ag') and scFv(Ag 2 ) are single chain variable fragments having specificity for two different antigens (Ag), AgI or Ag 2 ; and x and y are iCBD's capable of forming a het ero-dimer which is subsequently targeted by a dimer-specific antibody which provides Fc functionality. 5 [0084] Enabled herein is a set of agents comprising at least are agent pair each having the structure: x - scFv(Ag'); or y - scFv(Ag 2 ); 10 wherein: scFv(Ag') and scFv(Ag 2 ) are single chain variable fragments having specificity for two different antigens (Ag), AgI or Ag 2 ; and x and y are complementary iCBD's capable of dimerzing and subsequently engaging a dimer-specific antibody which provides a functional Fc portion. 15 [0085] The present disclosure further teaches a composition of matter comprising the modified demibodies: x - scFv(Ag'); and y - scFv(Ag 2 ); 20 scFv(Ag') and scFv(Ag 2 ) are single chain variable fragments having specificity for two different antigens (Ag), Agi or Ag 2 ; and x and y are iCBD's capable of forming a dimer which is subsequently targeted by a dimer-specific antibody which provides Fc functionality; the composition optionally further comprising one or more pharmaceutically acceptable diluents, excipients or 25 carriers. Alternatively, the modified demibodies may be in a freeze dried form or other dried or frozen form which is reconstituted prior to use. [0086] As indicated above, the dimer-specific antibody may be coupled with an agent such as a dye or portions of an agent such as a cytotoxic molecule, therapeutic molecule or 30 reporter molecule.

H:\aar\Intenoven\NRPor bl\DC\AR\4959862_ Idoc-12/03/2013 - 27 [0087] In relation to the above-mentioned formulae, the scFv's may be replaced by other ABD's of immunoglobulins or any affinity scaffold such as but not limited to dAbs, nanobodies, microproteins, fibronectins, microbodies, anticalins, aptamers, darpins, avimers, afflins, and Kunitz domains. 5 [0088] The modified demibodies may be maintained in different compositions or in the same composition. Multi-part pharmaceutical packs are also contemplated herein in which the modified demibodies are separately maintained but mixed prior to use. 10 [0089] The modified demibodies of the present invention may need to be deimmunized prior to administration. The term "deimmunized" includes, in relation to humans, humanization. In relation to other mammals, the term "mammalianization" may be used or in relation to birds, "avianization". Any technique of deimmunization may be used. 15 [00901 The composition taught in this aspect of the present disclosure may also include one or more pharmaceutical acceptable diluents, excipients and/or carriers. The preparation of pharmaceutical compositions is well described in the art such as Remington's Pharmaceutical Sciences, 18th Ed. (1990) (Mack Publishing Company, Easton, PA, U.S.A). As stated above, a useful embodiment is directed to modified demibodies specific 20 for CD antigens on cancer cells. Conveniently, the repertoire of the CD antigens expressed by cancer cells may be determined by a CD antibody microarray (DotScan). Notwithstanding, the target cell, may also be prokaryotic organisms or viruses or virally infected cells. 25 [0091] A useful CD antibody microarray is disclosed in International Patent Application No. PCT/AU99/01156 (WO 00/39580). [0092] Once the pattern of CD antigen expression is determined, modified demibodies specific for two CD antigens which are expressed on the target cells, for example cancer 30 cells, but not commonly on normal cells, are selected and used in therapy, for example cancer therapy.

H:\aar\lnicnoven\NRPortbl\DCC\AAR\4959862_ I.doc-I 2A)3/2013 -28 [0093] A similar approach may also be adopted in order to target other cells such as virally infected cells. 5 [0094] Still yet another embodiment provides a portion of a drug or other therapeutic agent which is coupled to a LZD-specific antibody which recognizes a dimeric complex of modified demibodies. The drug or therapeutic agent may also be internalized by the cell after binding to the cell surface. 10 [0095] The present further describes a method for diagnosing cancer in a subject, the method comprising contacting putative cancer cells with a pair of modified demibodies wherein each modified demibody comprises first and second portions wherein the first portions are capable of interacting with two different cancer specific antigens on the same cell, the second portions of each modified demibody are complementary iCBD's wherein 15 upon binding of each of the modified demibodies to their respective antigen, the iCBD's dimerize enabling a modified demibody dimer-specific antibody to bind and provide a signal through a reporter molecule. [0096] Taught herein is a method for diagnosing cancer in a subject, the method 20 comprising contacting putative cancer cells with a pair of agents wherein each agent in a pair comprises first and second portions wherein the first portions interact with two different antigens on a cancer cell, the second portions are iCBD's wherein upon binding of the pair of agents to the two antigens, the iCBD's dimerize at the cell surface enabling interaction with a dimer-specific antibody coupled to a reporter molecule to provide a 25 signal. [0097] Yet another aspect taught herein is directed to a use of a modified demibody comprising first and second portions wherein: a. the first portion is capable of interacting with a first antigen on a target cell 30 or virus; b. the second portion is one member of a pair of iCBDs; H:\aar\Intenvoven\NRPorbl\DCC\AAR\4959862_1.doc-12/03/2013 - 29 wherein the modified demibody becomes part of a functional molecule when it is bound to a second complementary modified demibody which comprises a first portion capable of interacting with another antigen on the same target cell or virus; and a second portion which is the other member of the iCBD pair, wherein the iCBD's engage with 5 dimer-specific antibody coupled to a reporter, therapeutic or cytotoxic molecule; in the manufacture of a medicament, diagnostic or affinity agent. [0098] The present disclosure further teaches a use of a set of modified demibodies comprising first and second portions wherein: 10 a. the first portion is capable of interacting with a first antigen on a target cell or virus; b. the second portion is one member of a pair of iCBDs; a second modified demibody comprising: c. a first portion which is capable of interacting with a different antigen on the 15 target cell or virus; and d. a second portion which is the complementary member of the iCBD pair; wherein the iCBD engages with a dimer-specific antibody coupled to a reporter, therapeutic or cytotoxic molecule; in the manufacture of a medicament, diagnostic or affinity agent. 20 [0099] Enabled herein is a use of an agent comprising first and second portions wherein: a. the first portion is capable of interacting with a first antigen on a target cell or virus; b. the second portion is one member of a pair of iCBDs; 25 wherein the agent forms a dimeric complex when it is bound to a second complementary agent which comprises a first portion capable of interacting with a second different antigen on the same target cell or virus; and a second portion which is the other member of the iCBD pair, wherein the iCBD's engage to form a dimeric complex and wherein a dimer-specific antibody coupled to a reporter, therapeutic or cytotoxic molecule 30 binds to the iCBD; in the manufacture of a medicament, diagnostic or affinity agent.

H:ar\nterwoven\NRPorIbl\DCC\AARX4959862_ I.doc-12/03/2013 -30 [0100] Further taught herein is a use of set of agents comprising at lest a pair of agents wherein each agent in a pair comprises first and second portions wherein: a. the first portion on each agent in a pair is capable of interacting with a 5 different antigen on a target cell or virus; b. the second portion on each agent in a pair is one member of a pair of iCBDs; wherein the iCBD's engage to form a dimeric complex and wherein a dimer specific antibody coupled to a reporter, therapeutic or cytotoxic molecule binds to the 10 iCBD; in the manufacture of a medicament, diagnostic or affinity agent. The dimeric complex is a heterodimer based on the iCBD's complementary. EXAMPLES 15 [0101] Aspects disclosed herein are further taught by the following non-limiting Examples.

H:\aarlnIenvoven\NRPortbl\DCC\AA R\4959862_1.doc-12/03/2013 -31 EXAMPLE 1 Generation of modified demibodies [0102] Monomeric modified demibodies are prepared using methods similar to those 5 described in WO 2007/062466, the contents of which are incorporated herein by reference. [0103] Leucine zipper sequences for use as iCBD's include those listed in Table 3 (SEQ ID NOs:1 through 5). Leucine zipper domains are prepared by peptide synthesis or may be purchased ready for use. Dimers comprising leucine zipper complementary pairs are 10 purified and tested. The leucine zipper domains facilitate dimerization of the monomeric modified antibodies. The operation of the demibodies is summarized in Figures 1 and 2. The term "demibody" is defined in WO 2007/062466. However, expressions such as an "synthetic immunoglobulin", "synthetic antibody", "synthetic antibody monomer", "immunointeractive molecule" or "molecule comprising an antigen-binding domain" or 15 "modified" forms of these molecules may be used in place of "demibody". When two demibody monomers combine via the iCBD's, the resulting heterodimer is referred to as a demibody dimer, synthetic antibody dimer, immunoglobulin dimer and the like, including a modified demibody heterodimer.

H:\aar\ntenvovn\NRPotb\DCC\AAR\4959862_ .doc-1210312013 - 32 EXAMPLE 2 Generation of dimer-specific antibodies [0104] Monomeric modified demibodies are capable of dimerization via their 5 complementary iCBD's to form heterodimeric modified demibodies. [0105] Polyclonal or monoclonal antibodies are raised to dimeric (or multimeric) forms of the monomeric modified demibodies. The antibodies are substantially specific for dimeric/multimeric forms of the modified demibodies and substantially do not bind to 10 monomeric forms of the demibodies. [0106] The dimer-specific antibodies provide Fc capability and/or are labeled with a reporter molecule or other functionality. 15 EXAMPLE 3 Generation of chronic lymphocytic leukemia (CLL) cell-specific demibodies [0107] Modified demibody pairs, each member of the pair having an ABD with specificity for either CD5 or CD19/CD20, are generated. The iCBD region on each modified 20 demibody comprises a leucine zipper domain. The ABD is an scFv. Pairs of demibodies target CD5 and CD19/CD20 on CLL cells. When in dimeric form, the demibodies are referred to as scFv-LZD. Antibodies specific for scFv-LZD (scFv-LZD-Abs) provide Fc capability to induce toxicity against the CLL cells.

H:\aar\ntenvoven\NRPortbl\DCC\AAR\4959862_ .doc-12/03/2013 - 33 EXAMPLE 4 Generation of clonal B-cellpopulation-specific demibodies [01081 Modified demibody pairs, each member of the pair having an ABD with specificity 5 for either CD19/CD20 + K (or k) chains, are generated. The iCBD region on each modified demibody comprises a leucine zipper domain. The ABD is an scFv. Pairs of demibodies target clonal B-cell population cells. When in dimeric form, the demibodies are referred to as scFv-LZD. Antibodies specific for scFv-LZD (scFv-LZD-Abs) provide Fc capability to induce toxicity against such cells. 10 EXAMPLE 5 Generation of non-Hodgkins lymph oma-specific demibodies [0109] Modified demibody pairs, each member of the pair having an ABD with specificity 15 for either CD19/CD20 + CD1O, are generated. The iCBD on each modified demibody region comprises a leucine zipper domain. The ABD is an scFv. Pairs of demibodies target follicular non-Hodgkins lymphoma (NHL). When in dimeric form, the demibodies are scFv-LZD. Antibodies specific for scFv-LZD (scFv-LZD-Abs) provide Fc capability to induce toxicity against the NHL cells. 20 EXAMPLE 6 Generation of hairy cell leukemia-specific demibodies [01101 Modified demibody pairs, each member of the pair having an ABD with specificity 25 for either CD103 + CD22 or CD25 or CD19, are generated. The iCBD regions on the modified demibody pairs comprise leucine zipper domains. The ABD is an scFv. Pairs of demibodies target hairy cell leukemia (HCL) cells. Antibodies specific for scFv-LZD (scFv-LZD-Abs) provide Fc capability to induce toxicity against the HCL cells.

H:\ar\nterwoven,\NRPoribl\DCC\AAR\4959862_ .doc-12/03/2013 - 34 EXAMPLE 7 Generation of T-cell acute lymphocytic leukemia-specific demibodies [01111 Modified demibody pairs, each member of the pair having specificity for either 5 CD4 + CD8, are generated. The iCBD regions comprise leucine zipper domains. The ABD is an scFv. Pairs of demibodies target T-cell acute lymphocytic leukemia (T-ALL). scFv LZD-Abs provide Fc capability to induce toxicity against the T-ALL cells. EXAMPLE 8 10 Generation of NK cell/large granular lymphocytic leukemia-specific demibodies [0112] Modified demibody pairs, each member of the pair having specificity for either CD8 + CD57, are generated. The iCBD regions comprise leucine zipper domains. The ABD is an scFv. Pairs of demibodies target NK cell/large granular lymphocytic leukemia. 15 Antibodies specific for scFv-LZD (scFv-LZD-Abs) provide Fc capability to induce toxicity against such cells. EXAMPLE 9 Generation of pre-B ALL-specific demibodies 20 [0113] Modified demibody pairs, each member of the pair having specificity for either CD1O + CD34, are generated. The iCBD regions comprise leucine zipper domains. The ABD is an scFv. Pairs of demibodies target pre-B ALL cells. scFv-LZD-Abs provide Fc capability to induce toxicity against the B ALL cells. 25 H:\aar~lntenoven\NRPoitbl\DCC\AAR\4959862_I.doc-12/03/2013 - 35 EXAMPLE 10 Generation of acute myeloid leukemia-specific demibodies [01141 Modified demibody pairs, each member of the pair having specificity for either 5 CD34 + myeloid marker (i.e. CD13 or CD33), are generated. The iCBD regions comprise leucine zipper domains. The ABD is an scFv. Pairs of demibodies target acute myeloid leukemia (AML). scFv-LZD-Abs provide Fc capability to induce toxicity against the AML cells. 10 EXAMPLE 11 Generation of AML-specific demibodies [0115] Modified demibody pairs, each member of the pair having specificity for either CD 117 (c-kit) + myeloid marker, are generated. The iCBD regions comprise leucine zipper 15 domains. The ABD is an scFv. Pairs of demibodies target AML cells. scFv-LZD-Abs provide Fc capability to induce toxicity against the AML cells. EXAMPLE 12 Generation of myelomonocytic-specific demibodies 20 [0116] Modified demibody pairs, each member of the pair having specificity for either CD13 or CD33 (My) + CD14 (Mo), are generated. The iCBD regions comprise leucine zipper domains. The ABD is an scFv. Pairs of demibodies target myelomonocytic leukemias. scFv-LZD-Abs provide Fc capability to induce toxicity against such cells. 25 H:\aar\ntenvoven\NRPortbl\DCC\AAR\4959862- .do-12/03/2013 - 36 EXAMPLE 13 Generation of megakaryocytic leukemia-specific demibodies [01171 Modified demibody pairs, each member of the pair having specificity for either 5 CD41 or CD61 + CD33, are generated. The iCBD regions comprise leucine zipper domains. The ABD is an scFv. Pairs of demibodies target megakaryocytic leukemias. scFv-LZD-Abs provide Fc capability to induce toxicity against such cells. EXAMPLE 14 10 Generation of diagnostic agents [0118] A monoclonal antibody (MAb) labeled with a fluorophore is generated to a dimeric form of the modified demibodies such as to a leucine zipper dimer (LZD). The MAb specific for the LZD labeled with a fluorophore is useful as a diagnostic agent. For 15 example, using scFv's specific for CD5 or CD19/20 enables generation of scFv-LZD demibodies which target CLL cells. MAbs labeled with the fluorophore enable detection of the CLL cells. [01191 In one embodiment, cells are isolated from a subject, and the cells incubated with 20 demibody monomers specific for either CD5 or CD19/20. Binding of the monomers to these CD antigens enables the iCBD's (e.g. leucine zippers) to bind which is then detected with a labeled MAb.

H:\aarteoen\NRPorbi\DCC\AAR4959862_ l.doc-12/03/2013 - 37 EXAMPLE 15 Generation of diagnostic agents [01201 A monoclonal antibody (MAb) labeled with a fluorophore is generated to a dimeric 5 form of the modified demibodies such as to a leucine zipper dimer (LZD). The MAb specific for the LZD labeled with a fluorophore is useful as a diagnostic agent. For example, using scFv's specific for either CD19/CD20 or K (or k) chains enables generation of scFv-LZD demibodies which target clonal B-cell populations. This is detected via the MAb labeled with the fluorophore. 10 [0121] In one embodiment, cells are isolated from a subject, and the cells incubated with demibody monomers specific for CD 1 9/CD20 or K (or k) chains. Binding of the monomers to these antigens enables the iCBD's (e.g. leucine zippers) to bind which is then detected with a labeled MAb. 15 EXAMPLE 16 Generation of diagnostic agents [01221 A monoclonal antibody (MAb) labeled with a fluorophore is generated to a dimeric 20 forn of the modified demibodies such as to a leucine zipper dimer (LZD). The MAb specific for the LZD labeled with a fluorophore is useful as a diagnostic agent. For example, using scFv's specific for either CD1 9/CD20 or CD10 enables generation of scFv LZD demibodies which target follicular non-Hodgkins lymphoma (NHL). This is detected via the MAb labeled with the fluorophore. 25 [0123] In one embodiment, cells are isolated from a subject, and the cells incubated with demibody monomers specific for CD19/20 and then CD10. Binding of the monomers to these CD antigens enables the iCBD's (e.g. leucine zippers) to bind which is then detected with a labeled MAb. 30 H:\aar\lntenvoven\NRPortbl\DCC\AAR\4959862_ .doc-12/03/2013 - 38 EXAMPLE 17 Generation of diagnostic agents [01241 A monoclonal antibody (MAb) labeled with a fluorophore is generated to a dimeric 5 form of the modified demibodies such as to a leucine zipper dimer (LZD). The MAb specific for the LZD labeled with a fluorophore is useful as a diagnostic agent. For example, using scFv's specific for CD103 or CD22 or CD25 or CD19 enables generation of scFv-LZD demibodies which target hairy cell leukemia (HCL). This is detected via the fluorophore-labeled MAb. 10 [01251 In one embodiment, cells are isolated from a subject, and the cells incubated with demibody monomers specific for CD103 or CD22 or CD25 or CD19. Binding of the monomers to these CD antigens enable the iCBD's (e.g. leucine zippers) to bind which is then detected with the labeled MAb. 15 EXAMPLE 18 Generation of diagnostic agents [01261 A monoclonal antibody (MAb) labeled with a fluorophore is generated to a dimeric 20 form of the modified demibodies such as to a leucine zipper dimer (LZD). The MAb specific for the LZD labeled with a fluorophore is useful as a diagnostic agent. For example, using scFv's specific for CD4 or CD8 enables generation of scFv-LZD demibodies which target T-cell acute lymphocytic leukemia (T-ALL). This is detected via the fluorophore-labeled MAb. 25 [01271 In one embodiment, cells are isolated from a subject, and the cells incubated with demibody monomers specific for CD4 or CD8. Binding of the monomers to these CD antigens enables the iCBD's (e.g. leucine zippers) to bind which is then detected with the labeled MAb. 30 H:\aarntenvoen\NRPorlbl\DCC\AAR4959862_ .doc-12/03/2013 - 39 EXAMPLE 19 Generation of diagnostic agents [01281 A monoclonal antibody (MAb) labeled with a fluorophore is generated to a dimeric 5 form of the modified demibodies such as to a leucine zipper dimer (LZD). The MAb specific for the LZD labeled with a fluorophore is useful as a diagnostic agent. For example, using scFv's specific for CD8 or CD57 enables generation of scFv-LZD demibodies which target NK cell/large granular lymphocytic leukemia. This is detected via the fluorophore-labeled MAb. 10 [01291 In one embodiment, cells are isolated from a subject, and the cells incubated with demibody monomers specific for CD8 or CD57. Binding of the monomers to these CD antigens enable the iCBD's (e.g. leucine zippers) to bind which is then detected with the labeled MAb. 15 EXAMPLE 20 Generation of diagnostic agents [01301 A monoclonal antibody (MAb) labeled with a fluorophore is generated to a dimeric 20 form of the modified demibodies such as to a leucine zipper dimer (LZD). The MAb specific for the LZD labeled with a fluorophore is useful as a diagnostic agent. For example, using scFv's specific for CD1O or CD34 enables generation of scFv-LZD demibodies which target pre-B ALL. This is detected via the fluorophore-labeled MAb. 25 [0131] In one embodiment, cells are isolated from a subject, and the cells incubated with demibody monomers specific for CD1O or CD34. Binding of the monomers to these CD antigens enables the iCBD's (e.g. leucine zippers) to bind which is then detected with the labeled MAb.

H:\ar\Untenvoe\NRPorlbl\DCC\AAR\4959862 Ldoc-2/03/2013 - 40 EXAMPLE 21 Generation of diagnostic agents [0132] A monoclonal antibody (MAb) labeled with a fluorophore is generated to a dimeric 5 form of the modified demibodies such as to a leucine zipper dimer (LZD). The MAb specific for the LZD labeled with a fluorophore is useful as a diagnostic agent. For example, using scFv's specific for CD34 and then myeloid marker (i.e. CD13 or CD33) enables generation of scFv-LZD demibodies which target acute myeloid leukemia (AML). This is detected via the fluorophore-labeled MAb. 10 [01331 In one embodiment, cells are isolated from a subject, and the cells incubated with demibody monomers specific for CD34 or the myeloid marker (i.e. CD13 or CD33). Binding of the monomers to these CD antigens enables the iCBD's (e.g. leucine zippers) to bind which is then detected with the labeled MAb. 15 EXAMPLE 22 Generation of diagnostic agents [0134] A monoclonal antibody (MAb) labeled with a fluorophore is generated to a dimeric 20 form of the modified demibodies such as to a leucine zipper dimer (LZD). The MAb specific for the LZD labeled with a fluorophore is useful as a diagnostic agent. For example, using scFv's specific for CD 117 (c-kit) or the myeloid marker enables generation of scFv-LZD demibodies which target AML. This is detected via the fluorophore-labeled MAb. 25 [01351 In one embodiment, cells are isolated from a subject, and the cells incubated with demibody monomers specific for CD117 (c-kit) or myeloid marker. Binding of the monomers to these CD antigens enable the iCBD's (e.g. leucine zippers) to bind which is then detected with the labeled MAb.

H:\aarnlevoven\NRPortb\DCC\AAR\4959862_ I.doc-12103/2013 -41 EXAMPLE 23 Generation of diagnostic agents [0136] A monoclonal antibody (MAb) labeled with a fluorophore is generated to a dimeric 5 form of the modified demibodies such as to a leucine zipper dimer (LZD). The MAb specific for the LZD labeled with a fluorophore is useful as a diagnostic agent. For example, using scFv's specific for CD13 or CD33 (My) or CD14 (Mo) enables generation of scFv-LZD demibodies which target myelomonocytic leukemias. This is detected via the fluorophore-labeled MAb. 10 [0137] In one embodiment, cells are isolated from a subject, and the cells incubated with demibody monomers specific for either CD13 or CD33 (My) or CD14 (Mo). Binding of the monomers to these CD antigens enable the iCBD's (e.g. leucine zippers) to bind which is then detected with the labeled MAb. 15 EXAMPLE 24 Generation of diagnostic agents [0138] A monoclonal antibody (MAb) labeled with a fluorophore is generated to a dimeric 20 form of the modified demibodies such as to a leucine zipper dimer (LZD). The MAb specific for the LZD labeled with a fluorophore is useful as a diagnostic agent. For example, using scFv's specific for CD41 or CD61 or CD33 enables generation of scFv LZD demibodies which target megakaryocytic leukemias. This is detected via the fluorophore. 25 [0139] In one embodiment, cells are isolated from a subject, and the cells incubated with demibody monomers specific for CD41 or CD61 and then CD33. Binding of the monomers to these CD antigens enable the iCBD's (e.g. leucine zippers) to bind which is then detected with the labeled MAb. 30 H:\aar\Intenvoven\NRPortbl\DCC\AAR\4959862_ .doc-12/03/2013 - 42 [01401 Those skilled in the art will appreciate that aspects of aspects described herein are susceptible to variations and modifications other than those specifically described. It is to be understood that these aspects include all such variations and modifications. These aspects also include all of the steps, features, compositions and compounds referred to or 5 indicated in this specification, individually or collectively, and any and all combinations of any two or more of the steps or features.

H \aarnenvoven\NRortb\DCC\AAR4959862_1 doc-12A)3/2013 -43 BIBLIOGRAPHY Adams et al. (200) Nuc. Med Biol. 27: 3 30-346 Bagshawe and Begent (1996) Adv. Drug Delivery Rev. 22: 365-367 Bonavida (2007) Oncogene 26:3592-3593 Carter (2006) Nat. Rev. Immunol. 6:343-357 Chames and Baty (2000) FEMS Microbiol. Lett. 189: 1-8 de Haard et al. (1998) Adv. Drug Delivery Rev. 31: 5-31 de Kruif and Logtenberg (1996) JBiol Chem 271:7630-7634 Funaro et al. (2000) Biotechnol. Adv. 18: 3 85-401 Hudson (2000) Exp. Opin. Invest Drugs 9: 1231-1242 Hudson and Souriau (2003) Nat Med 9:129-134 Kortt et al. (2001) Biomolecular Engineering 75:95-108 Kreitman (1999) Curr. Opin. Immunol. 1]. 570-578 Reichert and Valger-Archer (2007) Nat. Rev. Drug Discovery 6:349-3 56 Reiter and Pastan (1998) Tibtech 16: 51-520 Wu et al. (1996) Immunotechnology 2: 21-36 H.\aarlntenvoven\RPortb\CC\AR\4959862_1.doe-12/03/2013 - 44 Wu et al. (1997) Proc. Natl. Acad. Sci. USA 97. 8495-8500

Claims (14)

1. A method for selectively inducing a cytotoxic response on a target cell or virus in a subject, said method comprising administering to said subject first and second demibody monomers wherein said first demibody monomer comprises first and second portions wherein: a. said first portion interacts with a first antigen on a target cell or virus; and b. said second portion is one member of an immunointeractive complementary binding domain ( iCBD) pair; and said second demibody monomer comprising: c. a first portion which interacts with a second different antigen on said target cell or virus; and d. a second portion which is the complementary member of said iCBD pair; wherein the demibody monomers become a heterodimeric demibody when the iCBD's dimerize two demibody monomers and wherein a cytotoxic response is induced when a dimer-specific antibody which provides a functional Fc fragment through which complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC) is mediated, or is coupled to a cytotoxic molecule, binds to the dimeric demibody at a target site formed by the iCBD pair.

2. The method of Claim 1 wherein the first portion is an scFv or another antigen binding fragment derived from an immunoglobulin.

3. The method of Claim 1 wherein the first portion is an affinity scaffold.

4. The method of Claim 3 wherein the affinity scaffold is selected from dAbs, nanobodies, microproteins, fibronectins, microbodies, anticalins, aptamers darpins, avimers, afflins, and Kunitz domains.

5. The method of Claim 1, wherein the subject is a human, primate, livestock animal, companion animal, laboratory test animal, captive wild animal or avian species.

6. The method of Claim 5 wherein the subject is human. H:\nti\\licawoven\NRPontbl\DCC\M'lh6391360-I.doc-5/)6/2014 - 46

7. The method of any one of Claims 1 to 6 wherein one or both demibody monomers is or are specific for a CD antigen.

8. The method of any one of Claims 1 to 7 wherein the iCBD pair comprises complementary portions of a leucine zipper.

9. The method of Claim 1 wherein the dimer-specific antibody is a leucine zipper domain dimer-specific antibody (LZD-Ab).

10. A modified demibody monomer when used in the method of any one of Claims I to 9 comprising first and second portions wherein: a. said first portion interacts with a first antigen on a target cell or virus; b. said second portion is one member of an immunointeractive complementary binding domain (iCBD) pair; wherein said demibody monomer forms a heterodimeric demibody when bound to a second demibody monomer which comprises a first portion which interacts with a second different antigen on the same target cell or virus; and a second portion which is the other member of said iCBD pair wherein the iCBD pair forms a target site to which a dimer specific antibody binds.

11. A set of demibody monomers when used in accordance with any one of Claims 1 to 9 comprising a first demibody monomer with first and second portions wherein: a. said first portion interacts with a first antigen on a target cell or virus; and b. said second portion is one member of an iCBD pair; and a second demibody monomer comprising: c. a first portion which is capable of interacting with a second different antigen on said target cell or virus; and d. a second portion which is the complementary member of said iCBD pair; wherein said demibody monomers form a heterodimeric antibody when each is bound to two different antigens on a same cell or virus wherein the iCBD pair forms a target site to which a dimer-specific antibody binds. H:\fmtr\lnteoven\NRPtblDCC\FMT\6391360_lIdoc-5/06/2014 - 47

12. A modified demibody monomer when used in the method of any one of Claims 1 to 9 to manufacture a medicament, the demibody monomer comprising first and second portions wherein: a. said first portion interacts with a first antigen on a target cell or virus; b. said second portion is one member of an immunointeractive complementary binding domain (iCBD) pair; wherein said demibody monomer forms a heterodimeric demibody when bound to a second demibody monomer which comprises a first portion which interacts with a second different antigen on the same target cell or virus; and a second portion which is the other member of said iCBD pair wherein the iCBD pair forms a target site to which a dimer specific antibody binds.

13. A set of demibody monomers when used in accordance with any one of Claims 1 to 9 to manufacture a medicament, the set of demibody monomers comprising a first demibody monomer with first and second portions wherein: a. said first portion interacts with a first antigen on a target cell or virus; and b. said second portion is one member of an iCBD pair; and a second demibody monomer comprising: c. a first portion which is capable of interacting with a second different antigen on said target cell or virus; and d. a second portion which is the complementary member of said iCBD pair; wherein said demibody monomers form a heterodimeric antibody when each is bound to two different antigens on a same cell or virus wherein the iCBD pair forms a target site to which a dimer-specific antibody binds.

14. The method of any one of Claims 1 to 9 or a modified demibody monomer of Claim 10 or 12 or set of demibody monomers of Claim 11 or 13 substantially as hereinbefore described with reference to the Examples and/or Figures.