EP1856528A2 - Compositions et procede de purification et de cristallisation de molecules d'interet - Google Patents

Compositions et procede de purification et de cristallisation de molecules d'interet

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Publication number
EP1856528A2
EP1856528A2 EP06711155A EP06711155A EP1856528A2 EP 1856528 A2 EP1856528 A2 EP 1856528A2 EP 06711155 A EP06711155 A EP 06711155A EP 06711155 A EP06711155 A EP 06711155A EP 1856528 A2 EP1856528 A2 EP 1856528A2
Authority
EP
European Patent Office
Prior art keywords
cell
molecule
composition
virus
ligand
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP06711155A
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German (de)
English (en)
Inventor
Guy Patchornik
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Affisink Biotechnology Ltd
Original Assignee
Affisink Biotechnology Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from IL16680005A external-priority patent/IL166800A0/xx
Priority claimed from US11/330,112 external-priority patent/US20060121519A1/en
Application filed by Affisink Biotechnology Ltd filed Critical Affisink Biotechnology Ltd
Publication of EP1856528A2 publication Critical patent/EP1856528A2/fr
Withdrawn legal-status Critical Current

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
    • G01N33/537—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with separation of immune complex from unbound antigen or antibody
    • G01N33/539—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with separation of immune complex from unbound antigen or antibody involving precipitating reagent, e.g. ammonium sulfate
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/66—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid the modifying agent being a pre-targeting system involving a peptide or protein for targeting specific cells
    • A61K47/665—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid the modifying agent being a pre-targeting system involving a peptide or protein for targeting specific cells the pre-targeting system, clearing therapy or rescue therapy involving biotin-(strept) avidin systems
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6891—Pre-targeting systems involving an antibody for targeting specific cells
    • A61K47/6897—Pre-targeting systems with two or three steps using antibody conjugates; Ligand-antiligand therapies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6891—Pre-targeting systems involving an antibody for targeting specific cells
    • A61K47/6897—Pre-targeting systems with two or three steps using antibody conjugates; Ligand-antiligand therapies
    • A61K47/6898—Pre-targeting systems with two or three steps using antibody conjugates; Ligand-antiligand therapies using avidin- or biotin-conjugated antibodies
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery

Definitions

  • the present invention relates to compositions, which can be used for purifying and crystallizing molecules of interest.
  • Proteins and other macromolecules are increasingly used in research, diagnostics and therapeutics. Proteins are typically produced by recombinant techniques on a large scale with purification constituting the major cost (up to 60 % of the total cost) of the production processes. Thus, large-scale use of recombinant protein products is hindered because of the high cost associated with purification.
  • Affinity precipitation is the most effective and advanced approach for protein precipitation [Mattiasson (1998); Hilbrig and Freitag (2003) J Chromatogr B Analyt Technol Biomed Life Sci. 790(l-2):79-90].
  • Current state of the art AP employs ligand coupled "smart polymers”.
  • Smart polymers or stimuli-responsive "intelligent” polymers or Affinity Macro Ligands (AML)] are polymers that respond with large property changes to small physical or chemical stimuli, such as changes in pH, temperature, radiation and the like.
  • polymers can take many forms; they may be dissolved in an aqueous solution, adsorbed or grafted on aqueous-solid interfaces, or cross-linked to form hydrogels [Hoffman J Controlled Release (1987) 6:297-305; Hoffman Intelligent polymers. In: Park K, ed. Controlled drug delivery. Washington: ACS Publications, (1997) 485-98; Hoffman Intelligent polymers in medicine and biotechnology. Artif Organs (1995) 19:458-467].
  • the smart polymer in solution will show a sudden onset of turbidity as it phase-separates; the surface-adsorbed or grafted smart polymer will collapse, converting the interface from hydrophilic to hydrophobic; and the smart polymer (cross-linked in the form of a hydrogel) will exhibit a sharp collapse and release much of its swelling solution.
  • Smart polymers may be physically mixed with, or chemically conjugated to, biomolecules to yield a large family of polymer-biomolecule systems that can respond to biological as well as to physical and chemical stimuli.
  • Biomolecules that may be polymer-conjugated include proteins and oligopeptides, sugars and polysaccharides, single- and double-stranded oligonucleotides and DNA plasmids, simple lipids and phospholipids, and a wide spectrum of recognition ligands and synthetic drug molecules.
  • a number of structural parameters control the ability of smart polymers to specifically precipitate proteins of interest; smart polymers should contain reactive groups for ligand coupling; not interact strongly with the impurities; make the ligand available for interaction with the target protein; give complete phase separation of the polymer upon a change of medium property; form compact precipitates; exclude trapping of impurities into the gel structure and be easily solubilized after the precipitate is formed.
  • Membrane proteins present the most challenging group of proteins for crystallization.
  • the number of 3D structures available for membrane proteins is still around 20 while the number of membrane proteins is expected to constitute a third of the proteome.
  • Numerous obstacles need to be traversed when wishing to crystallize a membrane protein. These include, low abundance of proteins from natural sources, the need to solubilize hydrophobic membrane proteins from their native environment (i.e., the lipid bilayer) and their tendency to denaturate, aggregate and/or degrade in the detergent solution.
  • the choice of the solubilizing detergent presents another problem as some detergents may interfere with binding of a stabilizing partner to the target protein.
  • composition of matter comprising at least one antibody binding moiety capable of binding an antibody-labeled target molecule, cell or virus of interest, said at least one antibody binding moiety being attached to at least one coordinating moiety selected capable of directing the composition of matter to form a non-covalent complex when co-incubated with a coordinator ion or molecule.
  • said target cell is a prokaryotic cell. According to still further features in the described preferred embodiments said target cell is a eukaryotic cell.
  • said eukaryotic cell is a stem cell or a cancer cell.
  • said antibody-labeled molecule, target cell or virus comprises at least two distinct antibody labels.
  • said antibody binding moiety is selected from the group consisting of a protein A, a protein G. protein L and an antibody.
  • said antibody comprises an antibody fragment.
  • said complex is a polymeric complex.
  • composition further comprising said coordinator ion or molecule.
  • at least one antibody binding moiety is attached to said at least one coordinating moiety via a linker.
  • said coordinating moiety is selected from the group consisting of a biotin, a nucleic acid sequence, an epitope tag, an electron poor molecule and an electron-rich molecule.
  • said coordinating moiety is a chelator.
  • said coordinator ion or molecule is selected from the group consisting of an avidin, a nucleic acid sequence, an electron poor molecule and an electron-rich molecule.
  • said coordinator ion or molecule is a metal ion.
  • said molecule is a toxin or a prion.
  • said toxin is an endotoxin.
  • a method of purifying a target molecule, cell or a virus of interest comprising: (a) labeling the target molecule, cell or the virus with at least one antibody, so as to obtain antibody labeled target molecule, cell or the virus; (b) contacting the antibody labeled target molecule, cell or the virus with the composition of claim 1; and (b) collecting a precipitate including said complex bound to the target molecule cell or the virus, thereby purifying the molecule, target molecule or cell of interest.
  • step a and step b are effected sequentially.
  • step a and step b are effected concomitantly.
  • the method further comprising recovering the target molecule, cell or the virus from said precipitate.
  • a method of detecting predisposition to, or presence of a disease associated with a molecule, a cell or virus of interest in a subject comprising contacting an immunolabeled biological sample obtained from the subject with the composition of claim 1, wherein formation of said complex including the molecule, cell or virus of interest is indicative of predisposition to, or presence of the disease associated with the molecule, cell or virus of interest in the subject.
  • a target molecule, cell or virus of interest from a sample, the method comprising:
  • kits for isolating a target molecule cell or a virus of interest from a biological sample comprising a packaging material which comprises the composition of matter of claim 1.
  • kit further comprising an antibody for specifically labeling the target molecule, cell or the virus.
  • the present invention successfully addresses the shortcomings of the presently known configurations by providing compositions and methods for the purification of molecules.
  • FIGs. la-f schematically illustrate several configurations of the compositions of the present invention.
  • Figures la-c show ligands bound to two coordinating moieties.
  • Figures ld-f show ligands bound to multiple coordinating moieties.
  • Z denotes the coordinating moiety;
  • FIGs. 2a-b schematically illustrate precipitation of a target molecule using the compositions of the present invention.
  • a ligand covalently attached to a bis-chelator is incubated in the presence of a target molecule ( Figure 2a).
  • Addition of a metal (M + , M 2+ , M 3+ , M 4+ ) binds the chelator and forms a matrix including the target molecule non-covalently bound to the metal ion ( Figure 2b);
  • FIGs. 3a-e schematically illustrate stepwise recovery of the target molecule from the precipitate.
  • Figure 3 a shows the addition of a free chelator, which competes with the binding of the ligand-bound chelator to the metal.
  • Figure 3b shows gravity- based separation of the ligand-bound target molecule from the free competing chelator and the complexed metal (Figure 3c).
  • Figure 3d shows loading of the ligand- bound target molecule on an immobilized metal column to allow binding of the complex. Under proper elution conditions the target molecule is eluted while the ligand-coordinating moiety molecule is not. A desalting stage may be added for further purification of the target molecule. Regeneration of the ligand-chelator molecule is achieved by addition of a competing chelator to the column, followed by dialysis or ultrafiltration (Figure 3e);
  • FIG. 4 schematically illustrates direct elution of the target molecule from the precipitate, wherein the chelator-metal complex is maintained, while binding between the target molecule and the ligand decreases;
  • FIG. 5 schematically illustrates regeneration of the precipitating unit (i.e., ligand-coordinating moiety) following elution of the target molecule.
  • the precipitating unit i.e., ligand-coordinating moiety
  • recovery is achieved by the addition of a competing chelator and application of an appropriate separation procedure, such as, dialysis and ultrafiltration;
  • FIGs. 6a-c schematically illustrate precipitation of a target molecule using nucleic acid sequences as the coordinating moiety.
  • a ligand with a covalently bound bis-nucleotide sequence (coordinating moiety) is incubated in the presence of a target molecule ( Figure 6a).
  • Addition of a complementary sequence results in the formation of matrix including ligand-coordinating moiety:target molecule:the complementary sequence (coordinator molecule, Figure 6b).
  • Non-symmetrical coordinating sequences are shown as well ( Figure 6c);
  • FIGs. 7a-b schematically illustrate precipitation of a target molecule using biotin as the coordinating moiety.
  • a ligand with a covalently bound bis-biotin or biotin derivative such as: DSB-X Biotin is incubated in the presence of a target molecule ( Figure 7a).
  • Introduction of avidin (or its derivatives) creates a network comprising ligand-coordinating moiety (biotin): target molecule: avidin ( Figure 7b);
  • FIGs. 8a-c schematically illustrate precipitation of a target molecule using electron rich molecules as the coordinating moiety.
  • a ligand with a covalently bound bis-electron rich entity is incubated in the presence of a target molecule ( Figure 8a).
  • FIG. 9 schematically illustrates precipitation of a target antibody with protein A (ProA) bound used as a ligand. Addition of an appropriate coordinator results in a network of: Protein A-coordinating moiety : coordinator : target molecule;
  • FIGs. 10a-b schematically illustrate the use of the complexes of the present invention for crystallization of membrane proteins.
  • the general formation of 2D (or 3D) structures in the presence of crystallizing composition is presented, where the coordinators are not interconnected between themselves ( Figure 10a).
  • Figure 10b A more detailed example utilizing a specific ligand modified with two antigens, and a monoclonal antibody (niAb) directed at the specific antigen, serving as the coordinator, is illustrated in Figure 10b;
  • FIGs. l la-b schematically illustrate the use of metallo complexes (Figure Ha) and nucleo-complexes (Figure lib) for the formation of crystals of membrane proteins;
  • FIG. lie schematically illustrates a three-dimensional membrane complex using the compositions of the present invention.
  • the hydrophobic domain of the protein is surrounded by detergent micelles.
  • Z denotes a multi valent coordinator (i.e., at least bi-valent coordinator);
  • FIG. 12 schematically illustrates the formation of a non-covalent composition consisting of three ligands bound to a single metal coordinator, through suitable chelators which are bound to the ligands through covalent linkers;
  • FIGs. 13a-b schematically illustrate the modification of three ligands of interest to include the hydroxamate derivatives (Figure 13a), such that a tri-non- co valent ligand complex is formed in the presence of Fe 3+ ions ( Figure 13b);
  • FIG. 14 schematically illustrates a two-step synthesis procedure for the generation of ligand-chelator molecules
  • FIGs. 15a-b schematically illustrate the formation of di ( Figure 15a) and tri
  • FIGs. 16a-c schematically illustrate the compositions of the present invention coordinated by electron poor / rich relations.
  • FIG. 17 schematically illustrates a two step synthesis process for the preparation of ligand-electron rich or ligand-electron poor derivatives
  • FIG. 18 schematically illustrates the use of peptides for the formation of ligand complexes utilizing electron rich and electron poor moieties
  • FIG. 19 schematically illustrates the formation of ligand complexes which utilize a chelator-metal as well as electron rich and poor relationships
  • FIG. 20 schematically illustrates a single step synthesis procedure for the preparation of a chelator-electron poor derivative
  • FIGs. 21a-b schematically illustrate formation of di and tri non-covalent electron poor moieties by utilizing the same chelator-electron poor (catechol-TNB) derivative and changing only the cation in the medium;
  • FIGs. 22a-b schematically illustrate the addition of a peptide containing an electron rich moiety to form a dimer and a trimer
  • FIGs. 23a-b schematically illustrate the formation of a polymer complex by the addition of a composition including ligand attached to two chelators which are coordinated through electron rich/poor relations;
  • FIG. 24 schematically illustrates one possibility of limiting the freedom of motion of non-covalent protein dimers.
  • a covalent electron poor moiety e.g. trinitrobenzene-trinitrobenzene (TNB-TNB)
  • Trp accessible electron rich residues
  • FIG. 25 schematically illustrates chelators and metals, which can be used as the coordinating moiety and coordinator ion, respectively, in the compositions of the present invention
  • FIG. 26 schematically illustrates electron rich and electron poor moieties which can be used as the coordinating moiety in the compositions of the present invention
  • FIGs. 27a-b illustrate purification of rabbit IgG from normal rat kidney (NRK) cell lysate ( Figure 27a) or from mouse myoblasts (C2) cell lysate ( Figure 27b), utilizing Desthiobiotinylated protein A (DB-ProA) and free avidin.
  • Figure 27a - lane 1 rabbit IgG; lane 2 DB-ProA; lane 3 NRK cell lysate; lane 4 mixture of rabbit IgG, DB- ProA and NRK cell lysate; lane 5 recovered IgG (yield: ⁇ 90% by densitometry); lane 6 content of supernatant after specific precipitation of the IgG from the cell lysate.
  • Figure 27b lane 1 rabbit IgG; lane 2 DB-ProA; lane 3 C2 cell lysate; lane 4 mixture of rabbit IgG, DB-ProA and C2 cell lysate; lane 5 recovered IgG (yield: ⁇ 90% by densitometry); lane 6 content of supernatant after specific precipitation of the IgG from the cell lysate;
  • FIG. 28 illustrates purification of rabbit IgG from E. coli cell lysate, utilizing desthiobiotinylated protein A (DB-ProA) and free avidin.
  • Lane 1 rabbit IgG; lane 2 DB-ProA; lane 3 E. coli cell lysate; lane 4 mixture of rabbit IgG, DB-Pro A and E. coli cell lysate; lane 5 Biorad prestained protein markers; lane 6 recovered IgG (yield: 85% by densitometry); lane 7 content of supernatant after specific precipitation of the IgG from the cell lysate;
  • FIG. 29a illustrates the effect of increase background contamination (BSA) on the precipitation process.
  • FIG. 29b illustrates the effect of increase background contamination (E. coli lysate) on the precipitation process.
  • FIG. 30a illustrates purification of rabbit IgG from E. coli cell lysate utilizing Protein A modified with the strong chelator catechol (ProA-CAT) and Fe 3+ ions.
  • Lane 1 rabbit IgG; lane 2 native Protein A; lane 3 ProA-CAT; lane 4 E. coli cell lysate; lane 5 rabbit IgG, ProA-CAT and E. coli cell lysate; lane 6 recovered rabbit IgG; lane 7 content of supernatant after addition of Fe 3+ ions to the mixture in lane 5;
  • FIG. 30b illustrates the effect of increased background contamination on the precipitation process.
  • FIGs. 31a-d illustrate antibody purification utilizing a modified Protein A (ProA-CAT) and Fe 3+ ions.
  • Figure 31a specific binding of ProA-CAT to the target IgG leads to the formation of the: [ProA-CAT : target IgG] soluble complex.
  • Figure 31b addition of Fe 3+ ions to the complex shown in Figure 31a generates insoluble macro-complexes containing the target IgG. Impurities, left in the supernatant are discarded via centrifugation.
  • Figure 31c - target IgG is eluted under acidic conditions without dissociating the [ProA-CAT : Fe 3+ ] macro-complex of the insoluble pellet.
  • the complexed Fe 3+ ions and free chelators are excluded by dialysis while the free ProA-CAT can be reused;
  • FIGs. 32a-c illustrate a comparison of the basic chemical architecture of affinity chromatography (AC), affinity precipitation (AP) and affinity sinking (AS).
  • Figure 32a - Ligands in AC are immobilized to non-soluble polymeric matrixes.
  • Figure 32b - Ligands in AP are immobilized to water soluble polymers which would change reversibly to water in-soluble upon a physiochemical change such as low pH.
  • Figure 32c - Ligands in AS are not immobilized but modified with a complexing entity enabling their precipitation upon addition of an appropriate Mediator. Thus, no polymeric entity is present within the precipitation process and ligands are free in the medium;
  • FIGs. 33a-b schematically illustrate positive or negative cell selection ( Figure 33a) and virus depletion ( Figure 33b), utilizing a core complex comprised of [DB- ProA- avidinj;
  • FIG. 34 illustrates simultaneous depletion of several impurities upon addition of different biotinylated ligands and free avidin.
  • the resulting supernatant in stage C. contains enriched mixture of target proteins whereas impurities are left insoluble in the pellet;
  • FIG. 35 illustrates purification of fusion proteins with a modified human IgG
  • FIG. 36 illustrates covalent modification of a protein (e.g. Ovalbumin) with a small ligand (e.g. peptide) and a complexing entity (e.g. desthiobiotin) would lead to a modified protein (b) possessing multi-complexing features. Its incubation in a medium containing a Target would lead to specific binding of the Target (c) and precipitation of the latter complex upon addition of free Avidin (d). Thus, the Target is specifically precipitated whereas impurities are left soluble in the supernatant and are excluded.
  • a protein e.g. Ovalbumin
  • a small ligand e.g. peptide
  • a complexing entity e.g. desthiobiotin
  • Elution of the Target is obtained by incubating the above macro- complex under conditions favoring dissociation of the [Ovalbumin-Ligand : Target] complex while maintaining the: [Ovalbumin-Desthiobiotin : avidin] complex, intact;
  • FIG. 37 illustrates purification of an Anti-FITC niAb utilizing modified ovalbumin and free avidin.
  • Lane 1 native ovalbumin; lane 2 - modified ovalbumin; lane 3 - niAb Anti-FITC; lane 4 - mixture the mAb and the modified ovalbumin; lane 5 - content of supernatant after addition of avidin to lane 4 in the absence of free Fluorescein; lane 6 - content of supernatant after addition of avidin to lane 4 in the presence of Fluorescein; lane 7 - recovered mAb from the pellet generated in the absence of free Fluorescein; lane 8 - recovered mAb from the pellet generated in the presence of free Fluorescein;
  • FIG. 38 illustrates Purification of His-Tag-Target utilizing non-immobilized
  • Ovalbumin-NTA-Desthiobioitin multi-ligand Modification of a protein (e.g. Ovalbumin) with a metal chelator (e.g. NTA) and desthiobiotin generates the non- immobilized modified ligand (b). Incubation of the above under proper conditions (e.g. low imidazole concentration); an appropriate metal (e.g. Ni2+, Co2+) and a medium containing the His-Tag-Target will lead to specific binding (c). Addition of free avidin will generate insoluble macro-complexes that will precipitate together with the His-Tag-Target (d). Elution of the His-Tag-Target could then be performed leaving the: [modified ovalbumin: avidin] macro-complex in the pellet; and
  • FIG. 39 illustrates gel chromatography of a precipitate obtained from a regular network and defective network.
  • FIGs. 40a-b are pictures showing precipitation of immuno-labeled antigen using desthiobiotinylated-Protein-G.
  • Figure 40a shows immunoprecipitation of HA- LacZ from normal rat kidney (NRK) lysate. Time of incubation: 10 minutes.
  • the present invention is of compositions, which can be used for purifying and crystallizing molecules of interest.
  • the principles and operation of the present invention may be better understood with reference to the drawings and accompanying descriptions.
  • Affinity Precipitation which is based on the use of "smart" polymers coupled to a recognition unit, which binds the protein of interest.
  • These smart polymers respond to small changes in environmental stimuli with large, sometimes discontinuous changes in their physical state or properties, resulting in phase separation from aqueous solution or order-of- magnitude changes in hydrogel size and precipitation of the molecule of interest.
  • compositions of the present invention specifically bind target molecules to form non- covalent complexes which can be precipitated and collected under mild conditions.
  • the compositions of the present invention are not immobilized (such as to a smart polymer) which reduces affinity of the ligand towards the target molecule, limits the amount of ligand used, necessitates the use of sophisticated laboratory equipment (HPLC) requiring high maintenance, leads to column fouling and limits column usage to a single covalently bound ligand.
  • HPLC sophisticated laboratory equipment
  • composition-of-matter which is suitable for purification of a target molecule or cell of interest.
  • the target molecule can be a macromolecule such as a protein (e.g., a prion), a carbohydrate, a glycoprotein, a lipid or a nucleic acid sequence (e.g. DNA such as plasmids, RNA) or a small molecule such as a chemical or a combination of same (e.g., toxins such as endotoxins).
  • a protein e.g., a prion
  • a carbohydrate e.g., a glycoprotein, a lipid or a nucleic acid sequence
  • DNA such as plasmids, RNA
  • a small molecule such as a chemical or a combination of same
  • toxins such as endotoxins
  • the target cell can be a eukaryotic cell, a prokaryotic cell or a viral cell.
  • the composition-of-matter of the present invention includes at least one ligand capable of binding the molecule or cell of interest and at least one coordinating moiety which is selected capable of directing the composition of matter to form a non- covalent complex when co-incubated with a coordinator ion or molecule.
  • ligand refers to a synthetic or a naturally occurring molecule preferably exhibiting high affinity (e.g. K D ⁇ 10 "5 ) binding to the target molecule of interest and as such the two are capable of specifically interacting.
  • the ligand is selected capable of binding a protein, a carbohydrate or chemical, which is expressed on the surface of the cell (e.g. cellular marker).
  • ligand binding to the molecule or cell of interest is a non- covalent binding.
  • the ligand according to this aspect of the present invention may be mono, bi (antibody, growth factor) or multi-valent ligand and may exhibit affinity to one or more molecules or cells of interest (e.g. bi-specific antibodies).
  • Examples of ligands which may be used in accordance with the present invention include, but are not limited to, antibodies, mimetics (e.g. Affibodies® see: U.S. Pat. Nos.
  • calmodulin protein A, protein G and protein L or mimetics thereof (e.g. PAM, see Fassina (1996) J. MoI. Recognit. 9:564-9], chemicals (e.g. cibacron Blue which bind enzymes and serum albumin; amino acids e.g. lysine and arginine which bind serine proteases) and magnetic molecules such as high spin organic molecules and polymers (see http://www.chem.unl.edu/rajca/highspin.html).
  • PAM see Fassina (1996) J. MoI. Recognit. 9:564-9
  • chemicals e.g. cibacron Blue which bind enzymes and serum albumin; amino acids e.g. lysine and arginine which bind serine proteases
  • magnetic molecules such as high spin organic molecules and polymers (see http://www.chem.unl.edu/rajca/highspin.html).
  • the ligand is a an antibody binding moiety.
  • an antibody binding moiety can be any molecule which is capable of binding an immunoglobulin region of an antibody. Examples include but are not limited to protein A/G/L as well as antibodies (e.g., secondary antibodies) or antibody fragments. Methods of generating antibodies or fragments of same are well known in the art.
  • coordinating moiety refers to any molecule having sufficient affinity (e.g. K D ⁇ 10 "5 ) to a coordinator ion or molecule.
  • the coordinating moiety can direct the composition of matter of this aspect of the present invention to form a non-covalent complex when co-incubated with a coordinator ion or molecule.
  • Examples of coordinating moieties which can be used in accordance with the present invention include but are not limited to, epitopes (antigenic determinants antigens to which the paratope of an antibody binds), antibodies, chelators (e.g.
  • coordinating moieties can be attached to the ligand such as a chelator and an electron rich/poor molecule to form a complex such as is shown in Figure 19.
  • a combination of binding moieties may mediate the formation of polymers or ordered sheets (i.e., networks) containing the molecule of interest as is illustrated in Figures 23a-b and 24, respectively.
  • the coordinating moiety is selected so as to negate the possibility of coordinating moiety-ligand interaction or coordinating moiety-target molecule interaction.
  • the ligand is an antigen having an affinity towards an immunoglobulin of interest than the coordinating moiety is preferably not an epitope tag or an antibody capable of binding the antigen.
  • coordinator ion or molecule refers to a soluble entity (i.e., molecule or ion), which exhibits sufficient affinity (i.e., K D ⁇ 10 "5 ) to the coordinating moiety and as such is capable of directing the composition of matter of this aspect of the present invention to form a non-covalent complex.
  • coordinator molecules which can be used in accordance with the present invention include but are not limited to, avidin and derivatives thereof, antibodies, electron rich molecules, electron poor molecules and the like.
  • coordinator ions which can be used in accordance with the present invention include but are not limited to, mono, bis or tri valent metals.
  • Figure 25 illustrates examples of chelators and metals which can be used as a coordinator ion by the present invention.
  • Figure 26 lists examples of electron rich molecules and electron poor molecules which can be used by the present invention.
  • Methods of generating antibodies and antibody fragments as well as single chain antibodies are described in Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York, 1988, incorporated herein by reference; Goldenberg, U.S. Pat. Nos. 4,036,945 and 4,331,647, and references contained therein; See also Porter, R. R. [Biochem. J. 73: 119-126 (1959);
  • the composition of this aspect of the present invention includes the coordinator ion or molecule.
  • the ligand of this aspect of the present invention may be bound directly to the coordinating moiety, depending on the chemistry of the two. Measures are taken, though, to maintain recognition (e.g. affinity) of the ligand to the molecule of interest. When needed (e.g. steric hindrance), the ligand may be bound to the coordinating moiety via a linker.
  • a general synthetic pathway for modification of representative chelators with a general ligand is shown in Figure 14. Margherita et al. (1993) J. Biochem. Biophys. Methods 38:17-28 provides synthetic procedures which may be used to attach the ligand to the coordinating moiety of the present invention.
  • fusion protein When the ligand and coordinating moiety bound thereto are both proteins (e.g. growth factor and epitope tag, respectively), synthesis of a fusion protein can be effected by molecular biology methods (e.g. PCR) or biochemical methods (solid phase peptide synthesis).
  • molecular biology methods e.g. PCR
  • biochemical methods solid phase peptide synthesis
  • Complexes of the present invention be of various complexity levels, such as, monomers (see Figures 12 and 13a-b depicting a three ligand complex), dimers, polymers (see Figures 23a-b depicting formation of a polymer via a combined linker as described in Example 3 of the Examples section), sheets (see Figure 24 in which sheets are formed when a single surface exposed Trp residue of a target molecule forms electron rich/poor relations with a TNB — TNB entity) and lattices which may form three dimensional (3D) structures (such as when more than one surface exposed Trp residues form electron rich/poor relations).
  • monomers see Figures 12 and 13a-b depicting a three ligand complex
  • dimers see Figures 23a-b depicting formation of a polymer via a combined linker as described in Example 3 of the Examples section
  • sheets see Figure 24 in which sheets are formed when a single surface exposed Trp residue of a target molecule forms electron rich/poor relations with a TNB — TNB entity
  • the ligand is selected such that the target molecule/cell is uniformly bound to the complex.
  • the ligand can be selected such that the target molecule/cell bound by the complex is only associated with a single ligand molecule of the complex or with a predetermined number of ligand molecules.
  • such uniform association between ligand and target molecule/cell ensures that purification of the target from the complex is uniform, i.e. that a single elution step releases substantially all of the complex-bound target.
  • ligand configuration which enable such uniform binding of the target molecule/cell, include: peptides (i.e., cyclic or linear), Protein A or G or L, antibodies, lectines (e.g., concanavalin A from Jack bean, Jacalin from Jack fruit), various dyes (e.g., Cibacron Blue 3GA) and aptamers.
  • peptides i.e., cyclic or linear
  • Protein A or G or L antibodies
  • lectines e.g., concanavalin A from Jack bean, Jacalin from Jack fruit
  • various dyes e.g., Cibacron Blue 3GA
  • compositions of the present invention can be packed in a purification kit which may include additional buffers and additives, as described hereinbelow. It will be appreciated that such kits may include a number of ligands for purifying a number of molecules from a single sample. However, to simplify precipitation (e.g. using the same reaction buffer, temperature conditions, pH and the like) and further purification steps, the coordinating moieties and coordinator ions or molecules are selected the same.
  • compositions of the present invention may be used to purify a molecule or cell of interest from a sample.
  • a method of purifying a molecule of interest refers to at least separating the molecule of interest from the sample by changing its solubility upon binding to the composition of the present invention and precipitation thereof (i.e., phase separation).
  • the method of this aspect of the present invention is effected by contacting a sample including the molecule of interest with a composition of the present invention and collecting a precipitate which includes a complex formed from the composition- of-matter of the present invention and the molecule of interest, thereby purifying the molecule of interest.
  • sample refers to a solution including the molecule of interest and possibly one or more contaminants (i.e., substances that are different from the desired molecule of interest).
  • the sample can be the conditioned medium, which may include in addition to the recombinant polypeptide, serum proteins as well as metabolites and other polypeptides, which are secreted from the cells.
  • purifying refers to concentrating.
  • the composition-of-matter of the present invention is first contacted with the sample. This is preferably effected by adding the ligand attached to the coordinating moiety to the sample allowing binding of the molecule of interest to the ligand and then adding the coordinator ion or molecule to allow complex formation and precipitation of the molecule of interest. In order to avoid rapid formation of complexes (which may result in the entrapment of contaminants) slow addition of the coordinator to the sample while stirring is preferred.
  • Controllable rate of precipitation can also be achieved by adding free coordinating entity (i.e., not bound to the ligand), which may also lead to the formation of smaller complexes which may be beneficial in a variety of applications such as for the formation of immunogens, further described hereinbelow.
  • precipitation of the complex may be facilitated by centrifugation (e.g. ultra-centrifugation), although in some cases (for example, in the case of large complexes) centrifugation is not necessary.
  • centrifugation e.g. ultra-centrifugation
  • the precipitate may be subjected to further purification steps in order to recover the molecule of interest from the complex.
  • This may be effected by using a number of biochemical methods which are well known in the art. Examples include, but are not limited to, fractionation on a hydrophobic interaction chromatography (e.g.
  • reaction solution e.g. buffer
  • simple addition of clean reaction solution may be added to the precipitate to elute low affinity bound impurities which were precipitated during complex formation.
  • any of the above-described purification procedures may be repetitively applied on the sample (i.e., precipitate) to increase the yield and or purity of the target molecule.
  • the composition of matter and coordinator ion or molecule are selected so as to enable rapid and easy isolation of the target molecule from the complex formed.
  • the molecule of interest may be eluted directly from the complex, provided that the elution conditions employed do not disturb binding of the coordinating moiety to the coordinator (see Figures 4-5).
  • the coordinating moiety used in the complex is a chelator, high ionic strength may be applied to elute the molecule of interest, since it is well established that it does not effect metal-chelator interactions.
  • elution with chaotropic salt may be used, since it has been shown that metal-chelator interactions are resistant to high salt conditions enabling elution of the target molecule at such conditions [Porath (1983) Biochemistry 22:1621-1630].
  • the complex can be re-solubilized by the addition of free (unmodified) chelator (i.e., coordinating moiety), which competes with the coordinator metal ( Figure 3). Ultrafiltration or dialysis may be used, thereafter, to remove most of the chelated metal and the competing chelator.
  • the solubilized complex i.e., molecule of interest: ligand-coordinating moiety
  • an immobilized metal affinity column e.g. iminodiacetic acid (IDA) and nitrilotriacetic acid (NTA)].
  • IDA iminodiacetic acid
  • NTA nitrilotriacetic acid
  • Regeneration of the ligand-coordinating moiety is of high economical value, since synthesis of such a fusion molecule may contribute most of the cost and labor involved in the methodology described herein.
  • regeneration of the ligand-coordinating moiety can be achieved by loading the above-described column with a competing chelator or changing column pH followed by ultrafiltration that may separate between the free chelator and the desired ligand-coordinating moiety.
  • the Examples section which follows provides specific examples of binding/elution protocols which can be used with the present invention. It will be appreciated however, that the described parameters can be varied according to the immobilized target and purity needs.
  • binding/washing/elution/regeneration parameters can be utilized by the present invention, including:
  • GIy at various concentrations and pH values; (v) presence or absence of radical scavengers; (vi) addition of divalent, trivalent or tetravalent metals (Ca2+, Mg2+,
  • various incubation times e.g. the binding of the modified ligand to the target may change when that target is at low concentration or binding between the two is relatively weak, ligand will be required when the target is at low concentration or when the affinity of the ligand toward the target is low;
  • different sequences of additions for example, addition of salt, then ligand then free chelator, then metal, or, addition of salt, then ligand, then metal, then free chelator;
  • compositions of the present invention may also be used to isolate particular populations of cells, antigens, viruses, plasmids and the like. The following section exemplifies use of the present invention in such applications.
  • the present invention can be utilized to isolate cancer cells or stem cells which possess unique surface markers.
  • cells displaying CD34 and CD105 [see Pierelli (2001) Leuk. Lymphoma 42(6): 1195-206]) can be isolated by incubation of a cell suspension with a niAb directed at an epitope on the target cell (imrnuno labeled), followed by addition of desthiobiotinylated protein A (which could be added together with the mAb itself).
  • the target cell-mAb-modified protein A (or G or L) complex also referred to herein as the Precipitating complex
  • the supernatant will be discarded while the pellet containing the target cell would be either directly used; agitated to free bound cells from the precipitate; incubated in the presence of a competing molecule (e.g. peptide) which would release the target cell by competing with the epitope of the cell on binding to the mAb; or incubated in the presence biotin (or its analogues) for partial or total dissolution of the pellet thereby, enabling an effective cell release (for further detail see Figure 33).
  • Negative selection of cells the precipitating complex described above can be used along with a single mAb or several mAbs targeted at non-relevant cells in order to precipitate non-target cells and form a supernatant containing enriched medium of target cells.
  • Specific antigen precipitation the precipitating complex described above can be utilized with a target antigen known to bind to an niAb/s forming a part of the complex.
  • the precipitating complex described above can be used with virus or viruses containing an epitope known to bind to an mAb/s forming a part of the complex.
  • Precipitation of DNA/RNA-protein complexes the precipitating complex described above can utilize an mAb/s which can bind DNA/RNA-protein.
  • Plastnid purification the Precipitating complex described above can utilize an antibody which binds directly to a plasmid.
  • an antibody or mAb utilized by the precipitating complex could be used as "modification platform", into which ligands or nucleotide sequences are covalently attached.
  • the modified antibody could then be utilized for all the above described applications. Such an approach will circumvent the need for antibodies specific to target biomolecules.
  • compositions can also be utilized for reducing contamination or background.
  • several ligands may be modified with the same coordinating entity (e.g. biotin) and incubated in a medium containing impurities known to bind to the modified ligands. Removal of impurities will be initiated by addition of free avidin (for example), and the enriched supernatant could be used for further applications (see Figure 34 for further detail).
  • a coordinating entity e.g. biotin
  • impurities will be initiated by addition of free avidin (for example), and the enriched supernatant could be used for further applications (see Figure 34 for further detail).
  • Purification of recombinant proteins possessing fusion partners such as the Z (or
  • ZZ domain of Protein A could be purified in the presence of a modified human IgG (hlgG) to which the Z domain binds specifically, followed by addition of an appropriate transition metal which would generate insoluble macro-complexes containing the fusion protein (see Figure 35 for further detail). These macro-complexes would precipitate while impurities left soluble in the supernatant will be excluded.
  • hlgG modified human IgG
  • Recombinant protein - ABP Albumin Binding Protein of Protein G
  • HSA Human Serum Albumin
  • Recombinant protein - MBP E. coli Maltose Binding Protein
  • the present invention can also utilize non-immobilized multivalent ligands (NML) which can be generated via covalent linking of a protein (e.g. ovalbumin) with any ligand (e.g. Fluorescein) and a complexing entity (e.g. desthiobiotin).
  • NML non-immobilized multivalent ligands
  • the modified protein serves as the MNL since it is capable of interacting specifically with a Target molecule ( Figure 36 step b) and be further precipitated upon addition of an appropriate mediator entity (e.g. free avidin) (Figure 36 step c) which will interconnect modified ovalbumins ( Figure 36 step d).
  • an appropriate mediator entity e.g. free avidin
  • the Target is then eluted from the precipitate (i.e. pellet) under conditions favoring dissociation of the Target rather than dissociation of the [ovalbumin-desthiobiotin : avidin] multi-complex ( Figure 36 step d)
  • An efficient elution may be accomplished by using networks with lower degree of complexity (e.g. a network which includes larger holes). These could be generated by an avidin solution containing also bis, tris or multi avidin complexes that were cross-linked prior to their incubation with bis, tris or multi biotin moieties, (or their derivatives), via modification of the ligand with a complexing (coordinating) entity having extended spacer arms or by using avidin molecules that were incubated with free biotin prior to their use as a coordinator molecule. Similarly, free biotin may be present before the addition of avidin (see Example 7).
  • stem cells which are capable of differentiating to any desired cell lineage must be isolated.
  • a number of ligands may be employed which bind to surface markers which are unique to this cell population, such as CD34 and CD105 [see Pierelli (2001) Leuk. Lymphoma 42(6): 1195-206].
  • Another example is the isolation of erythrocytes using lectin ligands, such as concanavalin A [Sharon (1972) Science 177:949; Goldstein (1965) Biochemistry 4:876].
  • Viral cell isolation may be effected using various ligands which are specific for viral cells of interest [see www.bdbiosciences.com/clontech/ archive/JAN04UPD/Adeno-X.shtml].
  • retroviruses may be isolated by the compositions of the present invention which are designed to include a heparin ligand [Kohleisen (1996) J Virol Methods 60(l):89-101].
  • Cell isolation using the above-described methodology may be effected with preceding steps of sample de-bulking which is effected to isolate cells based on cell density or size (e.g. centrifugation) and further steps of selective cell-enrichment (e.g. FACS).
  • compositions of the present invention may also be used to deplete a sample from undesired molecules or cells.
  • This is effected by contacting the sample including the undesired target molecule or cell of interest with the composition of the present invention such that a complex is formed (described above) and removing the precipitate.
  • the clarified sample is the supernatant.
  • This method have various uses such as in depleting tumor cells from bone marrow samples, depleting B cells and monocytes for the isolation and enrichment of T cells and CD8 + cells or CD 4 + cells from peripheral blood, spleen, thymus, lymph or bone marrow samples, depleting pathogens and unwanted substances (e.g. prions, toxins) from biological samples, protein purification (e.g. depleting high molecular weight proteins such as BSA) and the like.
  • BSA high molecular weight proteins
  • multiple ligands may be employed for the depletion of a number of targets from a given sample such as for the removal of highly abundant proteins from biological fluids (e.g. albumin, IgG, anti-trypsin, IgA, transferrin and haptoglobin, see http://www.chem.agilent.eom/cag/prod/ca/51882709small.pdf).
  • biological fluids e.g. albumin, IgG, anti-trypsin, IgA, transferrin and haptoglobin, see http://www.chem.agilent.eom/cag/prod/ca/51882709small.pdf.
  • the unique properties of the novel compositions of the present invention provide numerous advantages over prior art precipitation compositions (e.g. smart polymers), some of these advantages are summerized infra. (i) Low cost purification; the present methodology does not rely upon sophisticated laboratory equipment such as HPLC, thereby circumventing machine maintenance and operating costs.
  • precipitation may be governed by, slow addition of an appropriate coordinator ion or molecule to the precipitation mixture; use of mono and/or multi-valent coordinators; use of coordinator ions or molecules with different affinities towards the coordinating moiety; addition of the non- immobilized free coordinating moieties to avoid non-specific binding and entrapment of impurities prior to, during or following formation of a non-covalent polymer, sheet or lattice [Mattiasson et al, (1998) J. MoI. Recognit. 11:211-216; Hilbrig and Freitag (2003) J. Chromatogr. B 790:79-90]; as well as by varrying temperature conditions.
  • compositions of the present invention to arrange molecules of interest in ordered complexes such as in dimers, trimers, polymers, sheets or lattices also enables use thereof in facilitating crystallization of macromolecules such as proteins, in particular membraneous proteins.
  • a crystal structure represents ordered arrangement of a molecule in a three dimensional space.
  • Such ordered arrangement can be egenerated by reducing the number of free molecules in a given space (see Figures 10a-b and 1 la-c).
  • composition for crystallizing a molecule of interest there is provided a composition for crystallizing a molecule of interest.
  • crystallizing refers to the solidification of the molecule of interest so as to form a regularly repeating internal arrangement of its atoms and often external plane faces.
  • the composition of this aspect of the present invention includes at least one ligand capable of binding the molecule of interest, wherein the ligand is attached to at least one coordinating moiety; and a coordinator capable of non-covalently binding the at least one coordinating moiety, wherein the at least one coordinating moiety and the coordinator are capable of forming a complex when co-incubated and whereas the composition is selected so as to define the relative spatial positioning and orientation of the molecule of interest when bound thereto, thereby facilitating formation of a crystal therefrom under inducing crystallization conditions.
  • the present invention circumvents these, by synthesizing only the basic unit in the non-covalent multi-ligand, (having the general structure of: Ligand — coordinating moiety) which is far easier to achieve, faster and cheaper.
  • This basic unit would form non-covalent tri-ligand only by adding the multi valent coordinator ion or molecule.
  • a single synthesis step is used to form di, tri, terra or higher multi ligands that may be used for crystallization experiments.
  • compositions of the preset invention are contacted with a sample, which includes the molecule of interest preferably provided at a predetermined purity and concentration.
  • the crystallization sample is a liquid sample.
  • the crystallization sample is a membrane preparation. Methods of generating membrane preparations are described in Strategies for Protein Purification and Characterization - A Laboratory Course Manual" CSHL Press (1996).
  • the sample is subjected to suitable crystallization conditions.
  • crystallization approaches which are known in the art can be applied to the sample in order to facilitate crystalization of the molecule of interest. Examples of crystallization approaches include, but are not limited to, the free interface diffusion method [Salemme, F. R. (1972) Arch. Biochem. Biophys. 151:533-539], vapor diffusion in the hanging or sitting drop method (McPherson, A. (1982) Preparation and Analysis of Protein Crystals, John Wiley and Son, New York, pp 82-127), and liquid dialysis (Bailey, K. (1940) Nature 145:934-935).
  • the hanging drop method is the most commonly used method for growing macromolecular crystals from solution; this approach is especially suitable for generating protein crystals.
  • a droplet containing a protein solution is spotted on a cover slip and suspended in a sealed chamber that contains a reservoir with a higher concentration of precipitating agent.
  • the solution in the droplet equilibrates with the reservoir by diffusing water vapor from the droplet, thereby slowly increasing the concentration of the protein and precipitating agent within the droplet, which in turn results in precipitation or crystallization of the protein.
  • Crystals obtained using the above-described methodology have a resolution of preferably less than 3 A, more preferably less than 2.5 A, even more preferably less than 2 A.
  • compositions of the present invention may have evident utility in assaying analytes from complex mixtures such as serum samples, which may have obvious diagnostic advantages.
  • the present invention envisages a method of detecting predisposition to, or presence of a disease associated with a molecule of interest in a subject.
  • prostate cancer which may be detected by the presence of prostate specific antigen [PSA, e.g. >0.4 ng/ml, Boccon-Gibod Int J Clin Pract. (2004) 58(4): 382-90].
  • PSA prostate specific antigen
  • compositions of the present invention are contacted with a biological sample obtained from the subject whereby the level of complex formation including the molecule of interest is indicative of predisposition to, or presence of the disease associated with the molecule of interest in the subject.
  • biological sample refers to a sample of tissue or fluid isolated from a subject, including but not limited to, for example, plasma, serum, spinal fluid, lymph fluid, the external sections of the skin, respiratory, intestinal, and genitourinary tracts, tears, saliva, milk, blood cells, tumors, neuronal tissue, organs, and also samples of in vivo cell culture constituents.
  • the biological sample or the composition is preferably labeled (e.g. fluorescent, radioactive labeling).
  • compositions of the present invention may also be utilized to qualify and quantify substances present in a liquid or gaseous samples which may be of great importance in clinical, environmental, health and safety, remote sensing, military, food/beverage and chemical processing applications.
  • Abnormal protein interaction governs the development of many pathogenic disorders. For example, abnormal interactions and misfolding of synaptic proteins in the nervous system are important pathogenic events resulting in neurodegeneration in various neurological disorders. These include Alzheimer's disease (AD), Parkinson's disease (PD), and dementia with Lewy bodies (DLB).
  • AD Alzheimer's disease
  • PD Parkinson's disease
  • DLB dementia with Lewy bodies
  • misfolded amyloid beta peptide 1-42 (Abeta), a proteolytic product of amyloid precursor protein metabolism, accumulates in the neuronal endoplasmic reticulum and extracellularly as aggregates (i.e., plaques).
  • the compositions of the present invention can be used to disturb such macromolecular complexes to thereby treat such disorders.
  • compositions of the present invention can be included in a diagnostic or therapeutic kits.
  • compositions of a specific disease can be packaged in a one or more containers with appropriate buffers and preservatives and used for diagnosis or for directing therapeutic treatment.
  • the ligand and coordinating moiety can be placed in one container and the coordinator molecule or ion can be placed in a second container.
  • the containers include a label.
  • Suitable containers include, for example, bottles, vials, syringes, and test tubes.
  • the containers may be formed from a variety of materials such as glass or plastic.
  • compositions of the present invention may be used to mediate the same.
  • the present invention also envisages a method of enhancing immunogenicity of a molecule of interest using the compositions of the present invention.
  • immunogenicity refers to the ability of a molecule to evoke an immune response (e.g. antibody response) within an organism.
  • the method is effected by contacting the molecule of interest with the composition of the present invention whereby the complex thus formed serves as an immunogen.
  • Such a complex can be injected to an animal host to generate an immune response.
  • the above-described immunogenic composition is subcutaneously injected into the animal host (e.g. rabbit or mouse). Following 1-4 injections (i.e., boosts), serum is collected (about 14 weeks of first injection) and antibody titer is determined such as by using the above-described methods of analyte detection in samples, where the ligand is protein A for example. Alternatively or additionally, affinity chromatography or ELISA is effected. It will be appreciated that the compositions of the present invention may have numerous other utilities, which are not distinctly described herein such as those utilities, which are attributed to affinity chromatography [see e.g. Wen-Chien and Kelvin (2004) Analytical Biochemistry 324:1-10].
  • a hydroxamate (which is a known Fe 3+ chelator) derivative is synthesized (Figure 13 a) such that in the presence Of Fe 3+ ions, a non-covalent multi- ligand complex is formed ( Figure 13b).
  • Figure 14 a A general synthetic pathway for modification of representative chelators with a general ligand is shown in Figure 14. Such a synthesis can be similar to the one presented by Margherita et al, 1999 supra.
  • chelators for the preparation of a non-covalent multi-ligand complex, may have an additional advantage which arises from the ability of some chelators to bind different metals with different stochiometries, as in the case of [1,10- phenanthroline] 2 -Cu 2+ , or [l,10-phenanthroline] 3 -Ru 3+ [Onfelt et al., (2000) Proc. Natl. Acad. Sci. USA 97:5708-5713].
  • Electron acceptors form molecular complexes readily with the " ⁇ excessive" heterocyclic indole ring system.
  • Indole picric acid was the first complex of this type to be described nearly 130 years ago [Baeyer, and Caro, (1877) Ber. 10:1262] and the same electron acceptor was used a few years later to isolate indole from jasmine flower oil.
  • Picric acid had since been used frequently for isolating and identifying indoles as complexes from reaction mixtures. Later, 1,3,5-trinitro benzene was introduced as a complexing agent and often used for the same purpose [Merchant, and Salagar, (1963) Current Sci. 32:18].
  • Figure 16a illustrates one example of a ligand — linker — electron poor (E. poor) derivative
  • Figure 16b presents an example of an electron rich covalent trimer that could be used. It is expected, that by mixing together the trinitrobenzene (Figure 16a) and the indole ( Figure 16b) derivatives, a multi-ligand complex will be formed ( Figure 16c). It will be appreciated that the reverse complex could be synthesized as well, i.e., a ligand derivative with an electron rich moiety, and an electron poor covalent trimer.] A possible synthetic pathway for the preparation of the above ligand derivatives is shown in Figure 17.
  • Synthetic peptides (or any peptide) containing Trp residues (or any other electron rich or poor moieties) may also be of use for the preparation of non-covalent multi ligand complexes.
  • Figure 18 shows an example of a synthetic peptide with four Trp residues (four electron rich moieties) that can be formed, a tetra-non-covalent- ligand in the presence of a ligand derivative modified with an electron poor moiety (trinitrobenzene).
  • a chelator e.g. catechol
  • M 2+ M 2+
  • M 3+ metals is capable in the presence of M 2+ and M 3+ metals, to form a non-covalent- di-ligand, ( Figure 21a), or a non-covalent-tri-ligand ( Figure 21b).
  • a dimer, trimer, tetramer etc. is formed, (by a ligand — chelator derivative for example) it may be desired to limit the freedom of motion of the above, in order to achieve more order. If the protein of interest has an electron rich moiety
  • Recombinant Protein A was modified with desthiobiotin N- Hydroxysuccinimidyl ester and yielded the modified Protein A derivative (DB-ProA) utilized in all purification experiments shown in Figures 27-29.
  • Precipitation and elution of rabbit IgG were carried out at 4 0 C in a medium containing: 50 mM sodium phosphate at pH 8; 0.23 mg/niL of DB-ProA; 0.6 mg/mL rabbit IgG and cell lysate (either NRK, C2 or E. col ⁇ ) in a total volume of 50 ⁇ L.
  • a freshly prepared avidin solution (1.5 mg/mL final concentration) was added and a precipitate was formed. This was followed by a short spin at 14,000 RPM and removal of the supernatant. The pellet was resuspended once with 200 ⁇ L of 50 mM sodium phosphate buffer pH 8 and the supernatant discarded.
  • the pellet was further resuspended with 0.1M sodium citrate pH 2.5 or 3, with or without 0.9 M urea at 4°C for 3-10 minutes in a total volume of 50 ⁇ L with or without gentle agitation. After an additional spin, the supernatant was neutralized with IN NaOH or 3M Tris pH 9 and applied to the gel. Regeneration ofDB-ProA. Recovery of DB-ProA was achieved by incubating the pellets in 0.1M sodium citrate pH 3 and 5 mM of biotin at 4 0 C for 10 minutes. Centrifugation at 14,000 RPM was performed and the supernatant was neutralized with IN NaOH and loaded onto an acrylamide gel.
  • rabbit IgG was purified from bacterial cell lysates ( Figures 27-28) by preparing a medium containing whole cell lysate, DB-ProA and rabbit IgG. Upon addition of avidin, a precipitate was generated and the resulting pellet was washed once with 200 ⁇ L of fresh buffer. The washed pellet was further incubated under eluting conditions (0.1M sodium citrate at pH 2.5-3, 4 0 C, for 5 minutes) and the supernatant of the resuspended pellet was applied to the gel after being neutralized to pH 7.
  • eluting conditions 0.1M sodium citrate at pH 2.5-3, 4 0 C, for 5 minutes
  • the recovery yield of the IgG was 85% ( Figure 27a, lane 5; Figure 27b, lane 5; Figure 28, lane 6). Since no DB-ProA was observed by Coomassie staining in the eluted IgG (lane 6), the degree of leached DB-ProA was assessed by silver staining and was determined to be less than 1% (data not shown).
  • the modified ligands used in this study were desthiobiotinylated protein A (DB-ProA) and desthiobiotinylated concanavalin A (DB-ConA).
  • DB-ProA desthiobiotinylated protein A
  • DB-ConA desthiobiotinylated concanavalin A
  • Incubation of the modified ligand with the target protein and addition of the interconnecting entity (free avidin) generated a precipitate, composed primarily of the [modified ligand - target protein - avidin] multi-complex ( Figure 32c).
  • the target protein is then eluted from the generated percipitate (i.e. pellet) under conditions that essentially do not dissociate the [modified ligand - avidin] multi-complex.
  • Thyroglobulin Concanavalin A 70-75 % 95 % 85-89 %
  • ligands utilized by the present approach are modified with a complexing entity (e.g. desthiobiotin, metal chelator)
  • a complexing entity e.g. desthiobiotin, metal chelator
  • removal of minute amounts ( ⁇ 1%) of leached ligand can be accomplished by passing the sample containing primarily the eluted protein through an appropriate affinity column that would remove traces of leached modified ligand rather than the target protein.
  • a desthiobiotinylated-ligand could be removed from a solution containing the target protein by an avidin column.
  • greater volumes of buffer are needed to remove impurities that bind non-specifically to the polymeric matrix.
  • regeneration of the modified ligand could be accomplished by a simple dialysis procedure. Since desthiobiotin has a lower association constant for biotin binding proteins (K a ⁇ 5xl0 13 M '1 for streptavidin) than biotin (K 3 ⁇ IxIO 15 M "1 ), the pellet will dissociate upon addition of biotin (28). Dialysis will remove excess of unbound biotin, leaving the modified ligand (DB-ProA or DB-ConA) and the [avidin-(biotin) 4 ] complex in the dialysis container. This mixture (devoid of free biotin) could be used directly in the next batch, since the free [avidin-(biotin) 4 ] complex is blocked
  • the non-immobilized state of the modified ligand might posses additional theoretical advantages which include higher yields of purified product due to faster and more efficient binding to the target protein in homogenous solutions where no additional steric hindrances are imposed by the polymeric matrix.
  • the non- immobilized ligand is expected to be more available for binding, while in its immobilized state may also interact with the polymeric matrix making itself less available for binding.
  • the measured affinity of the modified ligand should represent its affinity upon use, enabling easier judgment as to the most appropriate modified Hgand derivative to be utilized in a particular purification process. It has been argued that once a ligand is immobilized its affinity may be reduced by up to a factor of 1000 (30).
  • the approach does not introduce a new chemical principle but rather a different chemical architecture which could utilize any ligand, provided that specificity and affinity as well as uniformity are preserved following ligand modification.
  • modified ligands e.g. ligand-chelator, ligand-antigen, ligand-nucleotide sequence, ( Figure 32c) emphasizes the wide applicability of the present approach.
  • the [DB-ProA - avidin] complex may serve as a "core complex" for additional applications such as positive/negative cell selection - target cells could be purified (or depleted) with the above "core complex” and an antibody targeted at an epitope on the target cell ( Figure 33a) or depletion of viruses via use of an antibody specific to the virus ( Figure 33b).
  • the supernatant primarily contained impurities with no evidence of ProA-CAT and the IgG ( Figure 30a, lane 7).
  • the pellet (containing the complexed IgG) was then washed once with 100 ⁇ l of fresh buffer containing 20 mM NaPi pH 7, to remove traces of impurities.
  • the present inventors presented a novel purification approach, utilizing free nonimmobilized desthiobiotinylated ligands (e.g., protein A; concanavalin A) and free avidin.
  • the nonimmobilized state of the ligand circumvents the need for immobilizing ligands to polymeric supports hence, polymers are excluded from the process and purification is accomplished without chromatographic columns.
  • This study further demonstrated the implementation of the present approach on a novel, more challenging platform, the Metal: Chelator platform.
  • Protein A a 42 kDa factor produced by several stains of Staphylococcus aureus, which binds specifically to the Fc region of different classes of immunoglobulins (35), was modified with an active ester derivative of the strong metal chelator catechol, catechol-NHS according to Bayer et al. (36).
  • the modified protein A serves as the nonimmobilized ligand and is used for purification of rabbit and bovine IgGs from E. coli cell lysate.
  • Catechol was chosen as the preferred chelator since it: (a) exhibits high affinity toward diverse transition metals (37), therefore enabling the use of a variety of transition metals; (b) requires three independent catechol moieties to chelate a single Fe 3+ ion, thereby increasing the possibility of interconnecting adjacent [ProA-CAT : IgG] soluble complexes; (c) was expected to retain its chelating ability even at acidic conditions (pH 3) due to the absence of basic atoms (e.g. nitrogen) required for complex formation. A nitrogen atom (if existed) would be protonated at low pH and not be available for chelating Fe 3+ ions.
  • basic atoms e.g. nitrogen
  • HPLC HPLC
  • Targets are not diluted within the process (unlike column chromatography) and are eluted into small volumes of elution buffer, resulting in concentrated preparations which may be used directly for crystallization trials.
  • the present approach may be applicable to positive or negative cell selection, virus depletion and immunoprecipitation via epitope capture by a free antibody.
  • FIG. 32 illustrates the differences in chemical architecture between well established approaches (e.g. affinity chromatography, affinity precipitation) and the present approach (labeled as "affinity sinking"), in which, precipitation of the target protein requires two water soluble entities: a modified ligand and an interconnecting entity.
  • ovalbumin (Sigma A5503) was modified with desthiobiotin N- Hydroxysuccinimidyl ester and 6-[Fluorescein-5(6)-carboxamido]hexanoic acid JV- hydroxysuccinimide ester (Sigma - Fl 756) in the following stoichiometric ratio: Ovalbumin : Desthiobiotin : Fluorescein, 1 : 22 : 12. Modification was carried out in 0.1M NaHCO3 pH 8.5 for 4 hours at room temperature followed by extensive dialysis to remove excess of free desthiobiotin and fluorescein. The modified ovalbumin serves as the multi-nonimmobilized ligand of the present invention. Purification of anti-Fluorescein niAb
  • the pellet was further resuspended with 20 mM sodium phosphate buffer pH 7 and 5 mM of free Fluorescein at 4 0 C for 3-10 minutes in a total volume of 50 ⁇ L with or without gentle agitation. After an additional spin, the supernatant containing the recovered (i.e. eluted) mAb was neutralized and applied on the gel (lane 7, Figure 37). Similar recovery the anti-Flourescein mAb was obtained under acidic conditions (0.1M sodium citrate) data not shown. An identical elution procedure was performed on the pellet generated in the presence of free Flourescein.
  • networks/matrices which have "larger holes".
  • One approach for generation of such networks can be effected by initiating a precipitation process in the presence of free biotin which would occupy some of the binding sites of avidin and avoid maximum interconnections between modified ligands. (e.g. desthiobiotinylated ligand). Similarly, prior incubation of avidin with biotin would be applicable as well.
  • Desthiobiotinylated Protein G was synthesized according to DB- ProA in Example 4 and incubated at indicated times at 4 0 C in a medium containing: normal rat kidney (NRK) cell lysate, 0.0135 mg/ml HA-LacZ (i.e. Target antigen), 0.008 mg/ml anti-HA mAb (Sigma H9658), 0.019 mg/ml DB-ProG; 20 mM NaPi at pH 7, in a total volume of 600 ⁇ L.
  • NRK normal rat kidney
  • HA-LacZ i.e. Target antigen
  • 0.008 mg/ml anti-HA mAb Sigma H9658
  • DB-ProG Desthiobiotinylated Protein G
  • a freshly prepared avidin solution was then added to the medium (0.125 mg/ml final concentration) and a precipitate was formed.
  • the pellet was separated from the supernatant (containing most of the impurities) by a short centrifugation at 14K and removal of the supernatant.
  • the pellet could then be resusupended with fresh buffer (e.g. 20 mM NaPi pH 7) to remove traces of impurities.
  • the Target (HA- LacZ) was eluted from the washed (or unwashed) pellet by further resuspending it in 0.1M Glycine pH 2.5 at 4 0 C for 3-10 minutes in a total volume of 50 ⁇ L with or without gentle agitation. After an additional spin, the supernatant was neutralized with IN NaOH or 3M Tris pH 9 and applied to the gel (see the gel below).

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Abstract

L'invention concerne une composition de matière. Ladite composition contient au moins un fragment de liaison à l'anticorps pouvant se lier à une molécule, une cellule ou un virus cible anticorps d'intérêt, ledit fragment de liaison à l'anticorps étant rattaché à au moins un fragment de coordination sélectionné pouvant diriger la composition de matière de manière à former un complexe non covalent lors de son incubation avec un ion ou une molécule de coordination.
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US10030224B2 (en) 2015-11-01 2018-07-24 Ariel-University Research And Development Company Ltd. Methods of analyzing cell membranes
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