The assignee for this patent, patent number 8623356, is The
Reporters obtained the following quote from the background information supplied by the inventors: "The present invention relates generally to a set of synthetic immunointeractive molecules referred to herein as 'demibodies' which are useful in targeting particular cells in a subject. More particularly, the present invention provides a set of demibodies wherein at least two molecules from within the set, each specific for a different antigen on a target cell, are required to interact together at the cell surface in order to form an active complex which directs demibody-mediated cellular or complement mediated cytotoxicity and/or reporter function and/or therapeutic activity. The demibodies of the present invention are useful in the targeting of particular cells such as cancer cells including leukemic cells, pathogens including malarial, bacterial and viral agents, and stem cells including embryonic and adult stem cells and pathogen cells. The present invention provides, therefore, methods of treatment, diagnosis and undertaking research and compositions comprising demibodies useful for same.
"Bibliographic details of the publications referred to by author in this specification are collected at the end of the description.
"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.
"A key feature in the search for and development of therapeutic agents is target discrimination or selective toxicity. In particular, the ability to distinguish target cells such as cancer cells or cells infected with pathogen cells amongst a population of normal cells in a subject is of paramount importance. This is particularly the case in cancer therapies where the target cancer cells have many physiological, anatomical and biochemical properties in common with surrounding normal cells. Whilst some anti-cancer drugs do cause collateral damage to normal cells, their use may be indicated or at least justified for particularly aggressive, fast growing cancers.
"Therapeutic antibodies are the most rapidly growing area of pharmaceuticals, with more than 30 antibodies in late-phase clinical trials (Hudson and
"Whilst therapeutic antibodies are, important, multi-specific antibodies have not been as successful. There is a need, therefore, to develop antibody-based drugs and other therapeutic agents which are more highly selective for target cells.
"Whole antibodies have been proposed as highly specific targeting agents (Carter, Nature Reviews 1:118-128, 2001). In one proposal, cytotoxic agents are linked to an antibody specific for an antigen on a target cell. However, although antibodies have a high degree of target specificity, they have not achieved wide pathological therapeutic use and are primarily used in clinical imaging applications. This may be due to their relatively long circulating half-lives and their associated effector functions.
"Modified antibodies, however, have achieved some level of acceptance in immuno-therapeutic applications (Carter (2001) supra;
"Examples involving therapeutic antibodies are reviewed in Table 1 of Carter (2001) supra.
"These antibodies all contain the Fc domain which is required for complement-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 radioactive isotypes (Wu et al, Immunotechnology 2:21-36, 1996; Wu et al, Proc. Natl. Acad. Sci.
"Modified antibodies of particular interest are single chain variable fragments (scFv) 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.
"Whilst scFv antibody fragments have achieved a reasonable level of utility as targeting molecules, they lack the Fc domain and are unable to induce ADCC or CDC.
"The present invention enables modified forms of scFv antibody fragments to be used in targeted cell therapy and/or diagnosis and/or research."
In addition to obtaining background information on this patent, VerticalNews editors also obtained the inventors' summary information for this patent: "Throughout this specification, unless the context requires otherwise, the word 'comprise', or variations such as 'comprises' or 'comprising', will be understood to imply the inclusion of a stated element or integer or group of elements or integers but not the exclusion of any other element or integer or group of elements or integers.
"The present invention combines the specificity of antibody-antigen interactions to generate immunointeractive molecules referred to herein as 'demibodies' which are capable of targeting particular cells and inducing cytotoxicity and/or reporter function and/or facilitating cell therapy. More particularly, one aspect of the present invention provides sets of at least two demibodies wherein each demibody comprises an antigen-binding portion, an agent or portion thereof and one or other member of a binding pair. Two demibodies are designed such that when in close proximity, each member constituting one of a complementary binding pair, interacts forming a binding pair. This in turn permits interaction of portions of the agent to generate a functional agent or interaction of two agents which agent or agents have properties resulting in for example, cell death, cell therapy or providing a reporter signal. Hence, the agent on the demibodies can act as or form a reporter molecule, therapeutic molecule or cytotoxic molecule. Preferably, the demibodies comprise non-functional portions of the agent. When in close proximity, the reporter or therapeutic or cytotoxic molecule portions come together and a functional molecule capable of providing a signal or inducing cytotherapy or cytotoxicity is reconstituted. This increases the specificity for imaging, diagnostic and therapeutic purposes. The demibodies are also useful research tools such as for FACS, Flow cytometry and affinity chromatography. The demibodies may also be used to detect products of cells such as proteins or different phosphorylated or glycosylated or other post-translational modifications thereof. In essence, the demibodies of the present invention enable enhanced immunophenotypic selection of cells, viruses or products thereof such as proteins.
"The antigen binding portion may be derived from an immunoglobulin such as a scFv (or F'ab fragment) or any affinity scaffold such as a microaffinity scaffold. Examples include dAbs, nanobodies, microproteins, fibronectins, microbodies, anticalins, aptamers, darpins, avimers, afflins, and Kunitz domains.
"The demibodies have enhanced target specificity since each demibody in the set is specific for a particular antigen. Hence, by selecting cells or virus having an unusual pair of antigens reduces the risk of non-specific binding.
"In one embodiment, each demibody carries an incomplete Fc domain but upon binding of the pair of demibodies, the two incomplete Fc domains now form a functional Fc domain or functional portion thereof. The bound demibodies together have a biologically functional Fc domain and can initiate associated activities such as antibody-dependent cellular cytotoxicity (ADCC) and/or complement-dependent cytotoxicity (CDC). In another embodiment, the two incomplete domains form, when reconstituted, an agent such as a reporter molecule, therapeutic agent or cytotoxic agent. In a further embodiment, the agents are dyes such as fluorchromes which when together provide a particular signal.
"Hence, one aspect of the present invention provides a composition comprising a set of pairs of demibodies which, together, are able to induce highly selective cytotoxicity. The selective toxicity follows the demibodies interacting with at least two different antigens on the surface of a target cell. The two different antigens being predominantly only expressed simultaneously on a target cell population. Even if one of these antigens (or analogous antigens) is represented on normal cells, unless both antigens are present on the one cell, the pair of demibodies will not come together on the surface of the cell via the complementary binding pairs to form a functional cytotoxic domain such as a functional Fc domain, therapeutic molecule, cytotoxic molecule or reporter molecule.
"In a further embodiment, the reconstitution of the demibody pairs results in a functional cytotoxic or therapeutic molecule. Examples of such reconstituted functional molecules are apoptotic, cell cycle static, lytic or cytotoxic molecules, antibiotics, peptides and cytokines.
"In an alternative embodiment, each demibody carries a functional reporter molecule such as a dye or fluorescent marker and when the demibodies reconstitute together, the combined signal (or combined dye) or combined fluorescent signal is different from the individual signals (or dyes). Examples of fluorescent markers include hydroxycoumarin, aminocoumarin, methoxycumarin, cascade blue, Lucifer yellow, NBD, Phycoerythrin (PE), PerCP, allophycocyanin, hoechst 33342, DAP1, SYTOX Blue, hoechst 33258, chromomycin A3, mithramycin, YOYO-1, SYTOX green, SYTOX orange, 7-AAD, acridine orange,
"The demibodies of the present invention are useful in the treatment of a range of conditions including cancer, infection by pathogenic agents or the selective targeting of any cell type in a subject. They are also useful in targeting cells such as stem cells.
"Reference to a subject includes a human or other primate, livestock animal, laboratory test animal, companion animal or avian species. A subject may also be regarded as a patient.
"The present invention contemplates, therefore, a method of treating in a subject, such as a patient, comprising administering to the subject at least two demibodies which, when bound together via binding pairs, forms a functional cytotoxic domain such as an Fc domain or functional portion thereof capable of inducing cell cytotoxicity such as ADCC or CDC. Alternatively, the demibodies reconstitute a cytotoxic agent or therapeutic molecule.
"Diagnostic compositions and methods for diagnosing and/or imaging and/or therapy also form part of the present invention.
"The pair of immunointeractive molecules may be simultaneously or sequentially administered.
"The present invention further provides a method for selectively identifying a cell said method comprising contacting said cell with a pair of demibodies wherein each demibody comprises first, second and third portions wherein said first portions are capable of interacting with one or two antigens on the cell, said second portions comprise distinct reporter molecules or complementary non-functional portions of a single reporter molecule and said third portions are complementary binding pairs wherein upon binding of the individual demibodies to the two antigens, the binding pairs combine enabling the reporter molecules to provide a combined signal or to reconstitute a single reporter molecule.
"A list of abbreviations used herein is provided in Table 1.
"TABLE-US-00001 TABLE 1 Abbreviations ABBREVIATION DESCRIPTION Ab antibody ADCC antibody-dependent cellular cytotoxicity Ag antigen C region constant region CD antigen cluster of differentiation antigen CDC complement-dependent cytotoxicity Fab fragment fragment antigen binding Fc fragment crystalline FcR Fc receptor Fv fragment variable H chain heavy chain Ig immunoglobulin L chain light chain sc single chain scFv single chain variable fragment, recombinant Fab comprising V region of heavy and light chains V region variable region
BRIEF DESCRIPTION OF THE FIGURES
"FIGS. 1A and B are a representations of the amino acid sequences for the thioredoxin-Demibody conjugates. (A) Trx-CD45-mOrange-E3 (Demibody A, DBA; SEQ ID NO: 9) and (B) Trx-CD20-T-Sapphire-K3 (Demibody B5 DBB5; SEQ ID NO: 10).
"FIGS. 2A through 2D are photographic representations of protein gels. (A and B) Presence of .about.78 kDa band (marked 'target') from an induced sample showing an expression of pET32a-30103s1 and pET32a-30103s2, (C) The samples in (A) and (B) were purified by metal-affinity chromatography under denaturing conditions, refolded and run on SDS-PAGE, which showed .about.80 purity. (D) Demonstration of the expression of Trx-DBA and His.sup.6-GST-DBB in soluble fraction with .about.50% soluble protein.
"FIG. 3 is a graphical representation showing fluorescence excitation spectra of the refolded TRX Demibodies. The emission wavelength was set at 580 run for TRX-DBA and 510 run for TRX-DBB. The slit widths were both 10 nm and concentrations of the proteins were 0.1 mg/mL and .about.0.9 mg/mL, respectively. Fluorescence excitation spectra were recorded from 400-560 nm for TRX-DBA and 350-460 nm for TRX-DBB.
"FIG. 4 is a graphical representation showing fluorescence emission spectra of the refolded TRX Demibodies. The excitation wavelength was set at 510 nm for TRX-DBA and 400 nm for TRX-DBB. The slit widths were both 10 nm and concentrations of the proteins were 0.1 mg/mL and -0.9 mg/mL, respectively. Fluorescence emission spectra were recorded from 550-650 nm for TRX-DBA and 450-600 nm for TRX-DBB 5.
"FIG. 5 is a graphical representation showing relative fluorescence properties of Demibodies. An eGFP fusion protein (1 .mu.M) was excited at 430 nM; Refolded Trx-DBA (.about.1.5 .mu.M) was excited at 510 nm; refolded Trx-DBB(.about.15 .mu.M) was excited at 400 nm. Excitation and emission slit widths were both 5 nm in all cases.
"FIG. 6 is a graphical representation showing fluorescence properties of a mixture of TRX-DBA and TRX-DBB. The excitation wavelength was set at 400 nm for TRX-DBB; the slit widths were both 10 nm and concentrations of the proteins were 0.05 mg/mL and .about.0.45 mg/mL, respectively for TRX-DBA and TRX-DBB. This corresponds to estimated active concentrations of .ltoreq..about.130 nM and .ltoreq..about.75 nM. A fluorescence emission spectrum was recorded from 450-600 nm for a mixture of TRX-DBA and TRX-DBB (ABMIX) and is compared with that of TRX-DBB alone.
"FIG. 7 is a photographic representation of the identification of pET32a-30103 S1 in E. coli BL21. M=Marker; 1=Uninduced colony; 2=Colony 1; 3=Colony 2; 4=Colony 3.
"FIG. 8 is a photographic representation showing the solubility detection of 30103 S1. 1=supernatant of 30103 S1 induced at 15.degree. C. overnight; 2=supernatant of 30103 S2 induced at 25.degree. C. for 6 hours; 3=precipitation of 30103 S1 induced at 15.degree. C. for overnight; 4=precipitation of 30103 S1 induced at 25.degree. C. for 6 hours; M=low weight marker.
"FIG. 9 is a diagrammatic representation showing SDS-PAGE of Refolded 30103 S1. M=marker; 1=refolded 30103 S1.
"FIG. 10 is a photographic representation of the identification of the identification of pET32a-30103s2 in E. coli BL21. 1=marker; 2=colony #1 uninduced; 3-7=colony #2-#6 induced.
"FIG. 11 is a photographic representation showing the solubility detection of 30103s2. 1=precipitate of 30103s2 induced at 15.degree. C. overnight; 2=supernatant of 30103s2 induced at 15.degree. C. overnight; 3=precipitate of 30103s2 induced at 25.degree. C. for 6 hours; 4=supernatant of 30103s2 induced at 25.degree. C. for 6 hours.
"FIG. 12 is a photographic representation of the SDS-PAGE of Refolded 30103S2.1=marker; 2=refolded 30103 S2.
"FIG. 13 is a schematic representation showing the polypeptide domains of proposed chimeric antibody (demibody).
"FIG. 14 is a diagrammatic representation showing the structure of protein components of the hlgG1 Fc fragment-Fc RIII complex. hlgG1 Fc fragment is shown as ribbon diagram with chain A (light) and chain B (dark). The N and C termini are indicated. sFc III is shown as a wire representation (coordinates taken from RCSB Protein Databank Accession code: 1e4k; Sondermann et al, Nature 406:267-273, 2000).
"FIG. 15 is a diagrammatic representation showing leucine zipper heterodimerization. Top: helical wheel representation (looking down long axis of helices) of heterodimerization. Bottom: schematic showing that complementary electrostatic charges mediate heterodimerization not homodimerization.
"FIG. 16 (A) a diagrammatic representation of enhanced FACS with one color for two markers using demibodies. (B) Cell type-specific detection.
"FIG. 17 is a diagrammatic representation showing enhanced immunoassays using demibodies where A=.alpha.-phospho-tyrosine, B=.alpha.-protein-epitope, and C=e.g. detection of multimeric modified proteins in one step.
"FIG. 18 is a diagrammatic representation showing enhanced immunoaffinity chromatography using demibodies where A=.alpha.-phospho-tyrosine, B=.alpha.-Fc, e.g. protein A, and C=.alpha.-protein-epitope.
"FIG. 19 is a diagrammatic representation showing antibody-dependent cellular cytotoxicity (ADCC) mediated against CD5.sup.+ CD19.sup.+ chronic lymphocytic leukemia (CLL) cells by a pair of demibodies."
For more information, see this patent: Christopherson,
Keywords for this news article include: Antibodies, Antigens, Therapy, Peptides, Treatment, Immunology, Amino Acids, Blood Proteins, Immunoproteins, Immunoglobulins, Serum Globulins, Biological Factors, Drug Delivery Systems, The
Our reports deliver fact-based news of research and discoveries from around the world. Copyright 2014, NewsRx LLC
Most Popular Stories
- Obama Administration Releases Proposal to Regulate For-Profit Colleges
- Apple, HP, Intel May Take a Hit from Slowdown in Smartphone Sales Growth
- Elizabeth Vargas' Husband Marc Cohn Addresses Rumors
- Keurig Adds Peet's coffee, Alters Starbucks deal
- FDIC Files Lawsuit on Behalf of Banks Allegedly Hurt by Libor Scandal
- Motley Crue's Nikki Sixx Marries Model Courtney Bingham
- U.S. to Relinquish Gov't Control Over Internet
- Chinese e-Commerce Giant Alibaba Gears for IPO in U.S.
- Some California Cities Seeking Water Independence
- Will Missing Malaysian Jet Prompt Aviation System Change?