US20180052152A1 - Macromolecular conjugates for visualization and separation of proteins and cells - Google Patents

Macromolecular conjugates for visualization and separation of proteins and cells Download PDF

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US20180052152A1
US20180052152A1 US15/541,824 US201615541824A US2018052152A1 US 20180052152 A1 US20180052152 A1 US 20180052152A1 US 201615541824 A US201615541824 A US 201615541824A US 2018052152 A1 US2018052152 A1 US 2018052152A1
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conjugate
tag
gcpii
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cells
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Pavel SACHA
Jan Konvalinka
Jiri SCHIMER
Tomas KNEDLIK
Vaclav NAVRATIL
Jan TYKVART
Frantisek SEDLAK
Pavel Majer
Petr Cigler
Vladimir Subr
Karel Ulbrich
Jiri Strohalm
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USTAV MAKROMOLEKULARNI CHEMIE AV CR VVI
Karlova Univerzita v Praze
Institute of Organic Chemistry and Biochemistry CAS
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Karlova Univerzita v Praze
USTAV MAKROMOLEKULARNI CHEMIE AV CR VVI
Institute of Organic Chemistry and Biochemistry CAS
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Assigned to UNIVERZITA KARLOVA V PRAZE, PRIRODOVEDECKA FAKULTA, USTAV MAKROMOLEKULARNI CHEMIE AV CR, V.V.I., USTAV ORGANICKE CHEMIE A BIOCHEMIE AV CR, V.V.I. reassignment UNIVERZITA KARLOVA V PRAZE, PRIRODOVEDECKA FAKULTA ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: CIGLER, PETR, KNEDLIK, Tomas, KONVALINKA, JAN, MAJER, PAVEL, SACHA, Pavel, SCHIMER, Jiri, SEDLAK, Frantisek, TYKVART, Jan, STROHALM, JIRI, NAVRATIL, Vaclav, SUBR, VLADIMIR, ULBRICH, KAREL
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    • 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
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • G01N33/534—Production of labelled immunochemicals with radioactive label
    • 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
    • A61K47/58—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 obtained by reactions only involving carbon-to-carbon unsaturated bonds, e.g. poly[meth]acrylate, polyacrylamide, polystyrene, polyvinylpyrrolidone, polyvinylalcohol or polystyrene sulfonic acid resin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0013—Luminescence
    • A61K49/0017—Fluorescence in vivo
    • A61K49/005—Fluorescence in vivo characterised by the carrier molecule carrying the fluorescent agent
    • A61K49/0054—Macromolecular compounds, i.e. oligomers, polymers, dendrimers
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K17/00—Carrier-bound or immobilised peptides; Preparation thereof
    • C07K17/02—Peptides being immobilised on, or in, an organic carrier
    • C07K17/08—Peptides being immobilised on, or in, an organic carrier the carrier being a synthetic polymer
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
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    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
    • C08F220/00—Copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and only one being terminated by only one carboxyl radical or a salt, anhydride ester, amide, imide or nitrile thereof
    • C08F220/02—Monocarboxylic acids having less than ten carbon atoms; Derivatives thereof
    • C08F220/10—Esters
    • C08F220/12—Esters of monohydric alcohols or phenols
    • C08F220/16—Esters of monohydric alcohols or phenols of phenols or of alcohols containing two or more carbon atoms
    • C08F220/18—Esters of monohydric alcohols or phenols of phenols or of alcohols containing two or more carbon atoms with acrylic or methacrylic acids
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
    • C08F220/00—Copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and only one being terminated by only one carboxyl radical or a salt, anhydride ester, amide, imide or nitrile thereof
    • C08F220/02—Monocarboxylic acids having less than ten carbon atoms; Derivatives thereof
    • C08F220/52—Amides or imides
    • C08F220/54—Amides, e.g. N,N-dimethylacrylamide or N-isopropylacrylamide
    • C08F220/58—Amides, e.g. N,N-dimethylacrylamide or N-isopropylacrylamide containing oxygen in addition to the carbonamido oxygen, e.g. N-methylolacrylamide, N-(meth)acryloylmorpholine
    • 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
    • G01N33/531—Production of immunochemical test materials
    • 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
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • 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
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • G01N33/533—Production of labelled immunochemicals with fluorescent label
    • 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
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/60—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances involving radioactive labelled substances
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
    • C08F2500/00—Characteristics or properties of obtained polyolefins; Use thereof
    • C08F2500/01—High molecular weight, e.g. >800,000 Da.

Definitions

  • the invention describes synthetically prepared macromolecules having properties of monoclonal antibodies, said macromolecules being capable of replacing the use of antibodies in scientific research, in diagnostics, in biochemical investigations and for the preparation of targeted drugs.
  • These synthetic macromolecules, targeted and binding specifically to certain proteins, are suitable for the visualization, identification and isolation of biomolecules and/or cells in biochemistry, molecular biology and medicine and as targeting ligands in the pharmacy and diagnostics.
  • HPMA copolymers are multivalent macromolecules enabling the covalent attachment of multiple types of low molecular weight compounds such as drugs, radionuclides or fluorescent probes. Likewise, they also permit the binding of various macromolecules, e.g. (glyco)proteins, oligonucleotides and polynucleotides. Multivalency of these copolymers allows to connect both to only one type of molecules and to combinations of different molecules [5-7].
  • the present invention combines the advantages of a specific targeting of proteins by means of their specific ligands with the versatility and stability of the polymer chain.
  • the present invention provides a macromolecular conjugate of a synthetic copolymer with three types of low molecular weight functional compounds (hereinafter also referred to as “functional groups”, i.e., “affinity tag”, “imaging probe” and “targeting ligand”; this designation refers to their function in the final conjugate and has nothing to do with the so-called chemical functional groups).
  • Functional groups i.e., “affinity tag”, “imaging probe” and “targeting ligand”; this designation refers to their function in the final conjugate and has nothing to do with the so-called chemical functional groups.
  • Synthetic copolymer forms the backbone of macromolecular conjugate to which molecules of functional groups are linked via a covalent bond: (a) affinity tag, (b) imaging probe, which may be for example a fluorescent compound, a radionuclide or a metal complex, (b) targeting ligand allowing specific targeting of this conjugate to a given protein.
  • Targeting ligand is attached to the polymer chain
  • the synthetic copolymer is preferably water-soluble.
  • R 1 is selected from H, CH 3 ;
  • R 2 is selected from NH 2 , NH—CH 2 —CH(OH)—CH 3 , NH—CH 3 , NH—CH 2 CH 3 , NH—CH 2 CH 2 —OH, NH—CH 2 CH 2 CH 2 —OH, NHC(CH 2 OH) 3 , NH—CH 2 CH 2 —N + (CH 3 ) 3 Cl ⁇ , O—CH 2 CH 2 —OH, O—(CH 2 CH 2 O) 2 —H O—(CH 2 CH 2 O) 3 —H, O—CH 2 CH 2 —N + (CH 3 ) 3 Cl ⁇ , NH—(CH 2 ) 3 N + (CH 3 ) 2 —(CH 2 ) 2 —COO ⁇ ; and at least one type of monomer of Formula 2:
  • R 1 is selected from H, CH 3
  • X is selected from NH—(CH 2 ) 2 —CO, NH—(CH 2 ) 3 —CO, NH—(CH 2 ) 4 —CO, NH—(CH 2 ) 5 —CO, Gly, GlyGly, GlyPheLeuGly, and R 3 is selected from
  • Content of the reactive groups (i.e. content of the monomer of Formula 2) in the copolymer is preferably in the range of 0.5 to 30 mol. %, more preferably 2 to 20 mol. %.
  • At least one reactive group R 3 is replaced by a targeting ligand, at least one reactive group R 3 is replaced by an affinity tag, and at least one reactive group R 3 is replaced by an imaging probe.
  • more than one reactive group R 3 is replaced by said groups. More preferably, more than 50% of the reactive groups R 3 are replaced by the said groups, even more preferably, 100% of the reactive groups R 3 are replaced by the said groups.
  • Reactive groups remaining in the polymer chain after conjugation are always replaced by 1-amino-propan-2-ol group.
  • HPMA copolymer i.e. poly(HPMA-co-Ma- ⁇ -Ala-TT); copolymer prepared by conventional radical solution copolymerization, or controlled radical copolymerization (e.g. RAFT-copolymerization, reversible addition-fragmentation chain-transfer) of N-(2-hydroxypropyl)methacrylamide (HPMA) and 3-(3-methakrylamidopropanoyl)thiazolidin-2-thione (Ma- ⁇ -Ala-TT), can be preferably used as the basic copolymer.
  • HPMA content is preferably in the range from 70 to 98 mol %, the content of reactive thiazolidine-2-thione groups is preferably 2 to 30 mol %.
  • the functional compounds are attached to the polymer chain via an amide bond, which is formed in the reaction of the amino group present in the molecule of the functional compound, i.e. the affinity tag, the imaging probe and the targeting ligand, with the reactive group (preferably thiazolidine-2-thione) present on the polymer chain.
  • the reactive group preferably thiazolidine-2-thione
  • the molecular weight of the conjugate is preferably in the range of 1000 to 500000 g/mol, preferably in the range of 20000 to 150000 g/mol.
  • the affinity tag can be for example biotin.
  • biotin-avidin/streptavidin/neutravidin Using the very strong interaction biotin-avidin/streptavidin/neutravidin, the conjugate can be easily and specifically immobilized on various types of resins based on Streptravidin Sepharose, whereby it is possible to separate the conjugate from the mixture either by centrifugation or magnetic interaction (depending on the type of resin). Since the interaction of biotin with avidin/streptavidin/neutravidin is very strong (K D ⁇ 10 15 ), there is practically no risk of dissociation of the conjugate from the resin. Biotin can also be used for binding other proteins which are conjugated with streptavidin (either chemically or by genetic fusion)—e.g. neutravidin conjugated to horseradish peroxidase, which can be used for example in ELISA.
  • biotin also for example His tag (polyhistidine sequence, frequently six histidines in succession, bound with complex of chelating agent and nickel), FLAG tag (DYKDDDDK sequence recognized by an antibody), hemagglutinin tag (YPYDVPDYA amino acid sequence derived from the surface glycoprotein of the influenza virus, hemagglutinin, recognized by an antibody), Strep-tag (WSHPQFEK octapeptide sequence bound by modified streptavidin—Strep-Tactin), Avi-tag (peptide sequence recognized by biotin ligase; biotinylation enables subsequent isolation by streptavidin), GST (glutathione-S-transferase, the glutathione binding enzyme), c-myc-tag (EQKLISEEDL peptide sequence recognized by an antibody), V5-tag (GKPIPNPLLGLDST peptide sequence recognized by an antibody), E-tag (GAPVPYPDPLEPR peptide sequence recognized by an antibody), S-tag (
  • the imaging probe may be a fluorophore, preferably the ATTO488 fluorophore, enabling visualization of the polymer and the particles or cells to which the conjugate is bound.
  • This makes it possible to use the conjugate in methods such as e.g. flow cytometry (and a derived FACS technique, fluorescence-activated cell sorting, separating cells based on their fluorescence at a given wavelength), or immunocytochemistry and immunohistochemistry.
  • fluorophores with emission of radiation in the far red region of the spectrum (“far-red” fluorescence), e.g. DY676, can be advantageously used, as radiation with a longer wavelength passes through the tissue better than radiation of shorter wavelength.
  • the imaging probe may be a metal complex, e.g. lanthanide (particularly Gd, Mn, or. Dy, Eu).
  • the imaging probe may be a complex of a radionuclide, e.g. selected from the group consisting of 64 Cu, 68 Ga, 18 F.
  • the imaging probe may be a complex of a radionuclide selected from the group 99m Tc, 123 I, 125 I, 131 I, 57 Co, 51 Cr, 67 Ga, 64 Cu, 111 In, 90 Y.
  • Ligands suitable for complexation of metals referred to are well known in the field, such as macrocyclic ligands, derivatives of cyclopentadienyl, phosphine and azine ligands.
  • the imaging probe may be a ruthenium complex [Ru(Bpy) 3 ] 2+ .
  • Targeting ligand a low-molecular substance, provides specific targeting of the whole conjugate to a given (desired) protein.
  • Targeting ligand may be an inhibitor or substrate of an enzyme, receptor agonists or antagonists, a ligand of a protein carrier or another substance or compound capable of selectively binding to a particular protein or peptide sequence.
  • the targeting specificity of the resulting conjugate is given mostly by the properties of the low molecular weight compound. Since the targeting ligand is usually bound to a site performing certain biological functions, this binding requires a biologically active protein, i.e. in its native conformation. This allows, in contrast to large amounts of antibodies binding an epitope sequence, to distinguish between biologically active and inactive form of the enzyme.
  • Targeting ligand may be attached to the synthetic copolymer via a flexible linker, based on e.g. (oligo)polyethylene glycol, peptide, nucleic acid or oligosaccharide.
  • the linker allows inhibitor binding to the active site of the enzyme so as to avoid steric hindrance of the binding by the polymer and time such linker allows targeting enzymes with active site hidden in the binding cavity of the enzyme.
  • the linker is selected from the group consisting of linkers based on polyethylene glycol, peptide, preferably a peptide having a molecular weight from 100 to 5000 g/mol, or nucleic acid, preferably a nucleic acids comprising 1 to 40 nucleotides, or oligosaccharide, preferably an oligosaccharide containing 1 to 40 monosaccharides.
  • polymeric conjugates are inexpensive, and in comparison to antibodies, if there is an inhibitor of the enzyme, conjugates are also relatively easily prepared.
  • Polymeric conjugates are chemically substantially more stable and their solutions can be repeatedly frozen and thawed without significant influence on their ability to bind the enzyme.
  • One of the biggest advantages of these conjugates is that due to the present inhibitor they bind to the active site of the enzyme and thus bind only to enzymatically active form of the enzyme, i.e. always to a native protein. Antibodies lack this ability.
  • Another advantage is the “non-biological” origin of the polymeric backbone—in many methods with complex matrices (e.g.
  • Polymeric conjugates being synthetic molecules based on an entirely different structural pattern, do not cause these problems and can be used without side effects. Equally important is the fact that the active site of the enzyme is usually the most conserved point of the whole enzyme; this makes it possible to use one inhibitor (and therefore one conjugate) for a whole group of enzymes.
  • This group may be relatively small (e.g. homologous proteins; two paralogs in the same organism, or orthologs in two different organisms), but it can also be e.g. an entire type of enzymes (aspartate proteases, etc.). We can never achieve this with antibodies, since they bind only the surface of enzymes, i.e. a highly variable part.
  • the principal advantage of the polymer conjugate system is its modularity. Since the individual functional compounds are connected to the polymer backbone via an amide bond formed by reaction of an amino group present on the functional compound with the reactive group (e.g. thiazolidine-2-thione) present on the polymer, the polymer chain can be substituted as needed. For example fluorophores may be replaced with others (if they contain an amino group) according to the desired wavelength. It is also possible to have several types of fluorophores on one polymer chain. The advantage is then rather the presence of several different inhibitors on one polymer than more fluorophores. This will ensure the specificity of the conjugate against two (or more) enzymes using one conjugate.
  • the reactive group e.g. thiazolidine-2-thione
  • ELISA Enzyme-Linked Immunosorbent Assay
  • a primary antibody against the protein is adsorbed to the surface of the plate and unoccupied surface of the plate is blocked with a solution of casein.
  • Sample of the protein to be determined is then added, and after its binding to the antibody, the polymer conjugate capable of binding to this protein is added.
  • the amount of the bound conjugate may be determined using Neutravidin (horseradish peroxidase-conjugated) binding biotin present on the conjugate.
  • the concentration can also be determined by fluorescence using fluorophores present on the conjugate.
  • the polymer conjugate can also be immobilized by binding to neutravidin/streptavidin adsorbed on the surface of the plate (through biotin-streptavidin bond). After binding of the protein to be determined, the primary antibody against the protein is added, and its amount is then determined using a secondary antibody conjugated to horseradish peroxidase. Biotin present on the conjugate can thus be used both for immobilization and for detection, while a fluorophore only for detection.
  • Immunoprecipitation involves a polymeric conjugate binding to a solid phase, e.g. streptavidin sepharose. After washing away the unbound conjugate, the resin with bound conjugate is incubated with a sample containing the protein recognized by the conjugate. After incubation, the resin is washed and the protein is released from the resin (by heating in the presence of SDS, changing the pH, changing the ionic strength, etc.). Alternatively, the polymeric conjugate can be added directly to the sample and the resulting protein-conjugate complexes are separated from the sample by addition of streptavidin sepharose.
  • a solid phase e.g. streptavidin sepharose.
  • Immunocytochemistry involves visualizing the proteins, the cell structures and cells by (confocal) fluorescence microscopy.
  • the cells grown on a matrix suitable for microscopy are first incubated in the presence of a polymeric conjugate, and after washing away and eventual fixation of cells (formaldehyde) or cell nuclei staining (using DAPI or Hoechst) the coupled polymeric conjugate is visualized using fluorescence microscopy, preferably confocal microscopy.
  • Flow Cytometry allows the detection cell surface proteins; subsequently counting, sorting and separating the cells.
  • Cells are first incubated in the presence of the polymeric conjugate and then the cells are suspended in a solution.
  • the cell suspension is then passed through a capillary, which involves detection of the fluorescently labeled conjugates bound to the surface antigen.
  • Based on the presence or absence of fluorescence on the cell surface i.e. the presence or absence of surface antigen
  • cells can be separated from each other (i.e. FACS—fluorescence-activated cell sorting).
  • Measurement of surface plasmon resonance (SPR) is a biophysical technique to analyze the binding process (and consequently the strength of this bond) of two interacting substances.
  • SPR surface plasmon resonance
  • the polymeric conjugate can be used to immobilize the protein to the biosensor surface, and then analyze the bond between the given protein and another substance.
  • the protein is bound to the antibody immobilized on a gold biosensor chip and then the bond of the conjugate to the protein is analyzed.
  • the polymeric conjugate is first attached to neutravidin immobilized on the gold biosensor chip, then a particular protein bound to it and thereafter binding of the test substance to the protein is analyzed.
  • conjugates can be provided that enable for example the targeting of glutamate carboxypeptidase II (GCPII), glutamate carboxypeptidase III (GCPIII), HIV-1 protease, aspartic proteases, carbonic anhydrase II (CA-II), carbonic anhydrase VII (CA-VII), carbonic anhydrase IX (CA-IX).
  • GCPII glutamate carboxypeptidase II
  • GCPIII glutamate carboxypeptidase III
  • HIV-1 protease aspartic proteases
  • CA-II carbonic anhydrase II
  • CA-VII carbonic anhydrase VII
  • CA-IX carbonic anhydrase IX
  • Glutamate carboxypeptidase II is a membrane metallopeptidase, expressed most of all in the central nervous system (involved there in degradation of the N-acetyl-L-aspartyl-glutamate neurotransmitter; cleaved free glutamate then causes glutamate excitotoxicity) and in prostate. Due to the increased expression in prostate cancer and neovasculatures of most solid tumors, GCPII has for several years been considered as target for therapeutic intervention (both for the visualization of tumors and for targeted drug delivery).
  • Field of application of the present invention is not only in scientific research, particularly in biochemistry and molecular biology, and methods employing antibodies, but also in diagnostics, in biochemical laboratories, in biochemical investigations and in specific separation of biologically active substances.
  • FIG. 1 shows a schematic structure of the polymeric conjugates.
  • FIG. 2 shows the structure of the inhibitor intended for targeting of GCPII.
  • FIG. 3 shows the structure of the inhibitor intended for targeting of CA-IX.
  • FIG. 4 shows the structure of the inhibitor intended for targeting of HIV-1 protease.
  • FIG. 5 shows the structure of the inhibitor intended for targeting of aspartic proteases.
  • FIG. 6 shows the structure of Conjugate 1 intended for targeting of GCPII.
  • FIG. 7 shows the structure of comparative Conjugate 2 without inhibitor serving as a negative control.
  • FIG. 8 shows the structure of Conjugate 3 intended for targeting of CA-IX.
  • FIG. 9 shows the structure of Conjugate 4 intended for targeting of HIV-1 protease.
  • FIG. 10 shows the structure of Conjugate 5 intended for targeting of aspartic proteases.
  • FIG. 11A shows the silver-stained gel demonstrating the affinity isolation of GCPII (“pull-down”) from a lysate of LNCaP cells with Conjugate 1.
  • Lane 1 All Blue Marker (0.5 ⁇ l); 2: rhGCPII standard (50 ng); 3: Lysate of LNCaP cells; 4: FT: Conjugate 2 (negative control); 5: FT: Conjugate 1; 6: FT: 2G7 antibody; 7: FT: negative control for 2G7 antibody; 8: Elution: Conjugate 2 (negative control); 9: Elution: Conjugate 1; 10: Elution: 2G7 antibody; 11: Elution: negative control for the 2G7 antibody. All lanes were loaded with 8 ⁇ l of the sample.
  • FIG. 11B shows a Western blot demonstrating the affinity isolation of GCPII (“pull-down”) from a lysate of LNCaP cells with Conjugate 1.
  • GCP-04 antibody [9] was used to visualize the GCPII.
  • Lane 1 All Blue Marker (0.5 ⁇ l); 2: rhGCPII standard (5 ng); 3: lysate of LNCaP cells; 4: FT: Conjugate 2 (negative control); 5: FT: Conjugate 1; 6: FT: 2G7 antibody; 7: FT: negative control for 2G7 antibody; 8: Elution: Conjugate 2 (negative control); 9: Elution: Conjugate 1; 10: Elution: 2G7 antibody; 11: negative control for the 2G7 antibody. All lanes were loaded with 6 ⁇ l of the sample.
  • FIG. 12 shows a typical course of Conjugate 1 binding Avi-GCPII analyzed by SPR (surface plasmon resonance).
  • Extracellular recombinant GCPII (Avi-GCPII) was immobilized on a gold chip coated with D2B antibody against native GCPII.
  • Four different concentrations of Conjugate 1 were then applied to the prepared layer (a) 8 nM; b) 4 nM; c) 2 nM; d) 1 nM) and the association and dissociation phases of binding were monitored. Acquired curves were processed and then fitted in the TraceDrawer program v.1.5 (Ridgeview Instruments AB, Sweden).
  • FIG. 13 shows flow cytometry of cells expressing GCPII (LNCaP) and not expressing GCPII (PC-3). Cells were incubated in the presence of 10 nM Conjugate 1 or Conjugate 2 and then analyzed on a FortessaTM BD LSR cytometer.
  • FIG. 14 shows immunocytochemistry using Conjugate 1 and Conjugate 2.
  • LNCaP cells expressing GCPII
  • PC-3 cells not expressing GCPII
  • FIG. 14 shows immunocytochemistry using Conjugate 1 and Conjugate 2.
  • LNCaP cells expressing GCPII
  • PC-3 cells not expressing GCPII
  • 10 nM Conjugate 1 or Conjugate 2 were incubated in the presence of 10 nM Conjugate 1 or Conjugate 2; to verify the selectivity of binding, cells were incubated also in the presence of 10 nM Conjugate 1 or Conjugate 2 and 500 nM 2-PMPA inhibitor.
  • Cell nuclei were stained with Hoechst 33258 and the cells were observed using a Zeiss LSM 780 confocal microscope.
  • FIG. 15 shows immunocytochemistry using Conjugate 3 and Conjugate 2.
  • HT-29 cells expressing CA-IX were incubated in the presence of 1 ⁇ M Conjugate 3 or Conjugate 2.
  • Cell nuclei were stained with Hoechst 33258 and the cells were observed using a Zeiss LSM 780 confocal microscope.
  • FIG. 16 shows a typical course of binding CA-IX to Conjugate 3 analyzed by SPR (surface plasmon resonance).
  • Conjugate 3 was bound to streptavidin immobilized on a gold chip surface.
  • Four different concentrations of recombinant CA-IX in TBS were then applied to the prepared layer (a) 510 nM; b) 255 nM; c) 128 nM; d) 64 nM) association phase was monitored and then dissociation phase (only TBS application).
  • Acquired curves were processed and then fitted in the TraceDrawer program v.1.5 (Ridgeview Instruments AB, Sweden).
  • FIG. 17 shows a Western blot demonstrating the affinity isolation of CA-IX (“pull-down”) from the lysate of HT-29 cells using Conjugate 3.
  • CA-IX protein was visualized on the membrane using an M75 antibody.
  • Lane 1 All Blue Marker (2 ⁇ l); 2: lysate of HT-29 cells (Load); 3: free lane; 4: Elution: Conjugate 3; 5: Elution: Conjugate 2; 6: Elution: M75 antibody; 7: Elution: negative control for M75 antibody; 8: free lane; 9: FT: Elution: Conjugate 3; 10: FT: Conjugate 2; 11: FT: M75 antibody; 12: FT: negative control for M75 antibody. All lanes were loaded with 10 ⁇ l of the sample.
  • FIG. 18 shows the silver-stained gel demonstrating the affinity isolation of HIV-1 protease (“pull-down”) from a LNCaP cell lysate spiked with HIV-1 protease using Conjugate 4 and Conjugate 5.
  • Lane 1 All Blue Marker (0.5 ⁇ l); 2: HIV-1 protease standard (600 ng); 3: Load (LNCaP cell lysate spiked with HIV-1 protease); 4: Elution: Conjugate 4; 5: Elution: Conjugate 5; 6: Elution: Conjugate 2 (negative control). Lanes 3-6 were loaded with 10 ⁇ l of the sample.
  • FIG. 19 shows the silver-stained gel demonstrating the affinity isolation of pepsin (the representant of aspartic proteases) from a LNCaP cell lysate spiked with pepsin using Conjugate 5.
  • Lane 1 All Blue Marker (0.5 ⁇ l); 2: pepsin standard (2 ⁇ g); 3: Load (LNCaP cell lysate spiked with pepsin); 4: Elution: Conjugate 5; 5: Elution: Conjugate 2 (negative control).
  • Lanes 3-5 were loaded with 10 ⁇ l of the sample.
  • RTV ritonavir
  • the structure of the inhibitor is based on pepsatin A, an inhibitor of aspartic proteases.
  • the pepstatin inhibitor was synthesized by standard amino-Fmoc synthesis on solid phase, using 2-chlortrityl chloride resin (Iris-Biotech).
  • the first amino acid (Fmoc-Sta-OH) was attached to the solid phase according to the manufacturer's instructions: the resin was left to react with Fmoc-Sta-OH (0.6 eq to resin substitution) in presence of 4 equivalents of DIEA for 2 hours in DCM.
  • the remaining reactive residues were quenched with mixture of DCM/MeOH/DIEA (17:2:1) for 15 minutes. All other amino acids and the linker Boc-O2Oc-O2Oc-OH (Iris-Biotech, #BAA1485) were added using HOBt/DIC method.
  • Example 5 Synthesis of a Conjugate of HPMA Copolymer with a GCPII Inhibitor (Compound A), an ATTO488 Fluorophore and Biotin (Conjugate 1)
  • Monomeric compounds N-(2-hydroxypropyl)methacrylamide (HPMA) and 3-(3-methacrylamido-propanoyl)thiazolidine-2-thione (Ma- ⁇ -Ala-TT) were prepared according to published procedure [3, 7].
  • the polymeric precursor poly(HPMA-co-MA- ⁇ -Ala-TT) was prepared using RAFT-copolymerization (reversible addition-fragmentation chain-transfer).
  • ATTO488-NH 2 (2.5 mg) was dissolved in 0.1 ml of DMSO and added to the solution of polymeric precursor.
  • N,N-diisopropylethylamine (2.5 ⁇ l) was then added and the reaction mixture was stirred for 4 hours at room temperature, then 1-amino-propan-2-ol (5 ⁇ l) was added to the solution and the reaction mixture was stirred for 10 min.
  • the polymeric conjugate poly(HPMA-co-MA-3-Ala-CompoundA-co-MA- ⁇ -Ala-ATTO488-co-MA- ⁇ -Ala-NH-biotin) was then isolated by precipitation into acetone:diethyl ether (3:1), filtered, washed with acetone and diethyl ether and dried in vacuum.
  • Polymeric conjugate was purified from low-molecular impurities by column chromatography on Sephadex LH-20 in methanol, precipitated in diethyl ether, filtered and dried in vacuum.
  • the yield of the poly(HPMA-co-Ma- ⁇ -Ala-CompoundA-co-Ma- ⁇ -Ala-ATTO488-co-Ma- ⁇ -Ala-NH-biotin) conjugate was 33 mg, the content of inhibitor (Compound A) was 9.8%, the content of ATTO488 was 3.9% and biotin content was 9.8%.
  • ATTO488-NH 2 (2.5 mg) was dissolved in 0.1 ml of DMSO and added to the solution of polymeric precursor.
  • DIPEA N,N-diisopropylethylamine
  • the polymeric conjugate poly(HPMA-co-Ma- ⁇ -Ala-ATTO488-co-Ma- ⁇ -Ala-NH-biotin) was then isolated by precipitation into acetone:diethyl ether (3:1), filtered, washed with acetone and diethyl ether and dried in vacuum. Polymeric conjugate was purified by column chromatography on Sephadex LH-20 in methanol, precipitated in diethyl ether, filtered and dried in vacuum. The yield of the poly(HPMA-co-Ma- ⁇ -Ala-ATTO488-co-Ma- ⁇ -Ala-NH-biotin) conjugate was 32 mg, ATTO488 content was 5.1% and biotin content 10.8%.
  • Example 7 Synthesis of a Conjugate of HPMA Copolymer with a CA-IX Inhibitor (Compound B), the ATTO488 Fluorophore and Biotin (Conjugate 3)
  • N,N-diisopropylethylamine (DIPEA) (8 ⁇ l) was added and the reaction mixture was stirred for 4 hours at room temperature; subsequently, 1-amino-propan-2-ol (5 ⁇ l) was added to the solution of and the reaction mixture was stirred for 10 min.
  • the polymeric conjugate poly (HPMA-co-MA- ⁇ -Ala-CompoundB-co-MA- ⁇ -Ala-ATTO488-co-MA- ⁇ -Ala-NH-biotin) was then isolated by precipitation into acetone:diethyl ether (3:1), filtered, washed with acetone and diethyl ether and dried in vacuum.
  • Polymeric conjugate was purified from low-molecular impurities by column chromatography on Sephadex LH-20 in methanol, precipitated in diethyl ether, filtered and dried in vacuum.
  • the yield of the poly(HPMA-co-MA- ⁇ -Ala-CompoundB-co-MA- ⁇ -Ala-ATTO488-co-MA- ⁇ -Ala-NH-biotin) conjugate was 39 mg, the content of inhibitor (Compound B) was 10.5%, content of ATTO488 3.7% and content of biotin 8.6%.
  • Example 8 Inhibition of the GCPII Activity by Inhibitors and Conjugate 1
  • Inhibitory potency of the inhibitors and polymeric conjugates on the hydrolytic activity of GCPII were tested by HPLC (described in [12]) using a recombinant extracellular GCPII (Avi-GCPII; prepared according to [13]). 210 pg of GCPII was mixed with a solution of 25 mM Bis-Tris propane, 150 mM NaCl, pH 7.4, 0.001% monododecyl(oktaethylenglycol)ether (Affymetrix, Octaethylene glycol monododecyl ether) and inhibitor solution to a total volume of 180 ⁇ l in a 96-well plate. Ten different inhibitor concentrations covering the whole inhibition curve were used.
  • Reactions were first incubated 5 min at 37° C., then initiated by addition of 20 ⁇ l of pteroyl-bis(L-glutamate) to a final concentration of 400 nM and incubated at 37° C. for 20 min. Reactions were stopped with 20 ⁇ l of 25 ⁇ M 2-(phosphonomethyl)pentanedioic acid (2-PMPA). Subsequently, 100 ⁇ l of the reaction mixture was analyzed in Agilent 1200 Infinity (Agilent Technologies, Inc.) on an RP-HPLC column Waters Acquity UPLC HSS T3 1.8 ⁇ m, 2.1 ⁇ 100 mm (Waters).
  • HPLC analysis was performed isocratically in 2.7% acetonitrile and 97.3% 20 mM phosphate, pH 6.0. Substrate and product absorbance was measured at 281 nm. IC 50 values were obtained from GraFit v.5.0.11 (Erithacus Software Ltd.).
  • TAB Prepared inhibitors and polymeric conjugates and their inhibition constants (K i ) for GCPII Number Compound M r Targeted of designation g/mol towards inhibitors K i [pM] Modifications 2-PMPA 226 GCPII — 370 ⁇ 30 — Compound A 780 GCPII — 2,033 ⁇ 426 — Conjugate 1 107,000 GCPII 13.7 3.1 ⁇ 0.5 Compound A, ATTO488, biotin Conjugate 2 96,000 — 0 N/A ATTO488, biotin
  • Example 9 Affinity Isolation (“Pull-Down”) of GCPII Using Polymeric Conjugates and Subsequent Detection of GCPII by Western Blot
  • LNCaP cells (cultured in 100 mm Petri dish) derived from cells of metastatic prostate adenocarcinoma and endogenously expressing GCPII were lysed by sonication in a water bath (3 min/0° C.) in 450 ⁇ l of 50 mM Tris-HCl, 150 mM NaCl, pH 7.4, 1% Tween 20. The resulting cell lysate was further diluted in 20 mM Tris-HCl, 150 mM NaCl, 0.1% Tween 20, pH 7.4 (TBST) to a final protein concentration of 200 ⁇ g/ml (concentration of GCPII was approximately 100 ng/ml).
  • Conjugate 1 and Comparative Conjugate 2 (negative control showing nonspecific binding) were pre-bound to 20 ⁇ l Streptavidin Sepharose (5 ⁇ M solution in 200 ⁇ l TBST, 1 hour, 6° C.), and after washing twice with 200 ⁇ l TBST, the resin was mixed with 200 ⁇ l of LNCaP cell lysate and incubated at 6° C. for 12 h. The resin was then washed with 2 ⁇ 200 ⁇ l of TBST and subsequently, proteins were eluted by addition of 50 ⁇ l of sampled buffer for SDS-PAGE and by heating to 98° C. for 10 min.
  • GCPII was isolated at the same time using 2G7 antibody [15] (ie. immunoprecipitation).
  • the experiment was performed analogously to the experiment with polymeric conjugates: 5 ⁇ g of the antibody was pre-bound to 20 ⁇ l of Protein G Sepharose and the procedure followed as described above. Resin Protein G Sepharose without the antibody was used as negative control.
  • Conjugate 1 was able to affinity isolate GCPII from lysate of LNCaP cells endogenously expressing GCPII.
  • the quantity of GCPII isolated with Conjugate 1 and with 2G7 antibody designed against native GCPII was practically the same ( FIG. 11A , B).
  • the advantage of polymeric conjugates against antibodies lies in the possibility of their use in cases where the use of antibodies is impossible or difficult, e.g. in immunoprecipitation of proteins from blood plasma, where large quantities of endogenous antibodies in blood compete with protein G for binding sites on the resin. Biotinylation of these antibodies may be a solution, which, however, may damage the antibody.
  • biotinylated polymeric conjugates and a resin with streptavidin solves this problem, because endogenous antibodies are not biotinylated.
  • the chip is attached to an SPR chip prism; all measurements were performed at 25° C.
  • Activation of the terminal carboxyl groups on the sensor surface was carried out in situ by addition of a mixture (1:1) 11.51 mg/ml N-hydroxysuccinimide (NHS, Biacore), and 76.68 mg/ml 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC, Biacore) in deionized water for 5 min at a flow rate 20 ⁇ l/min. Following parts of the experiment were then conducted at a flow rate of 30 ⁇ l/min.
  • NHS N-hydroxysuccinimide
  • EDC 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride
  • Curves describing the bond were exported and analyzed in TraceDrawer v.1.5 (Ridgeview Instruments AB) to obtain the k on and k off parameters ( FIG. 12 ).
  • Sandwich ELISA normally implemented with two antibodies, has been modified for the use of polymeric conjugates either for immobilisation or in the role of the second specific detection antibody. All steps of the experiment were performed at room temperature.
  • streptavidin 500 ng/well in 100 mM borate buffer, pH 9.5, was sorbed (1 hour) to 96-well Maxisorb plates (Nunc). After washing with 3 ⁇ 200 ⁇ l of TBS, the uncovered surface of the well was blocked with 0.55% (w/v) solution of casein in TBS (Casein Buffer 20 ⁇ -4 ⁇ Concentrate, SDT, 24 hod). After further washing with 3 ⁇ 200 ⁇ l TBST, Conjugate 1 (100 nM in TBST) was bound to streptavidin (2 hours).
  • the plate was first coated with 2G7 antibody in borate buffer (500 ng/well). After blocking the surface with casein and washing (see above) incubation with recombinant extracellular GCPII followed (rhGCPII; prepared according to [18]) in TBST (in amounts 1 ng-1 pg/well, 20 min). After washing with 3 ⁇ 200 ⁇ l of TBST, the solution of Conjugate 1 was added (at concentrations 0.5-1000 nM in TBST, 1 hour), subsequently washed away with 3 ⁇ 200 ⁇ l of TBST and NeutrAvidin conjugated to horseradish peroxidase (100 ng/well, Thermo Scientific) was added to the well.
  • the detection limit was approximately 30 pg.
  • the detection limit decreased to 0.5 pg of GCPII, i.e. lower value, as in the case of using the best sandwiches for GCPII quantification.
  • An important advantage of polymers is their insensitivity to the presence of interfering antibodies, i.e. a frequent and serious cause of false positivity in the case of using two antibodies. Interfering antibodies recognize epitopes on antibodies and can couple the antibodies used in the sandwich without the presence of the determined antigen itself. Polymeric conjugates, as molecules of completely different chemical nature, do not cause such problems.
  • the K D value should correspond to the K i determined by measuring the inhibition of GCPII activity (see Tab. 1), or to the K D value determined by measuring this interaction using SPR.
  • the K D value determined with the ELISA method was approximately 40 times higher (for Conjugate 1, the K D value was 115 pM; the K i was 3.1 pM, and the K D (SPR) ⁇ 20 pM). The difference is probably caused by using different methods.
  • Example 12 Modified ELISA Method for Testing GCPII Inhibitors
  • ELISA method was also used to test the GCPII inhibitors; the procedure was analogous to Example 11. This method is based on the competition of Conjugate 1 and the tested potential GCPII inhibitor for binding into the active site of GCPII. The amount of bound Conjugate 1 is then determined by chemiluminescence and subsequently related to the sample without test inhibitor (see below).
  • the Maxisorp plate (Nunc) was first coated with 2G7 antibody in borate buffer (500 ng/well). After blocking the surface with casein and washing it away (see above), incubation with recombinant extracellular GCPII followed (rhGCPII; prepared according to [18]) in TBST (10 ng/well, 1 hr). After washing with 3 ⁇ 200 ⁇ l of TBST, either a solution containing either conjugate 1 alone was added (5 mM in TBST for 1 hour; reference sample), or a mixture of Conjugate 1 (5 mM in TBST) and the test substance in the selected concentration (typically 0.1-100 ⁇ M in TBST).
  • NCaP prostate cancer cells
  • PC3 Complete RPMI-1640 medium (Sigma-Aldrich)
  • PC-3 cells in complete DMEM-High Glucose medium (GE Healthcare), containing L-glutamine (final concentration 4 mM) and FBS (final concentration 10%).
  • the medium was removed, cells rinsed with PBS and incubated for 3 min in 1.5 ml of 0.25% trypsin and 0.01% EDTA. Cells were resuspended in this solution and transferred to 8 ml of DMEM or RPMI complete medium, centrifuged 250 ⁇ g/2 min and washed with 5 ml PBS. Subsequently, 500 ⁇ l of 10% fetal bovine serum in PBS was added to block the cell surface (1 hr/37° C.). The amount of cells was counted using Countess® Automated Cell Counter (Invitrogen).
  • GCPII Fluorescence visualization of GCPII on cell surface (immunocytochemistry) using polymeric conjugates was performed on two types of cell lines derived from prostate cancer: LNCaP cells (endogenously expressing GCPII) and PC-3 (non-expressing endogenous GCPII).
  • LNCaP cells endogenously expressing GCPII
  • PC-3 non-expressing endogenous GCPII
  • Cells were cultured overnight in complete RPMI-1640 medium (LNCaP) or DMEM-High glucose medium (PC-3).
  • a solution of Conjugate 1 or Conjugate 2 was added to the medium to a final concentration of 10 nM and the cells were incubated in their presence for 2 hours at 37° C.
  • ELISA sandwich arrangement can detect and quantify the order of picograms and fractions of picograms of GCPII. Thanks to the combination of antibody-conjugate, sandwich ELISA does not suffer from false positive results caused by binding of endogenous interfering antibodies. This can be used for very sensitive and specific quantification of GCPII in biologically relevant samples, e.g. blood, blood plasma, blood serum, cerebrospinal fluid, urine, synovial fluid, amniotic fluid, ascites, pleural fluid, pericardial fluid, saliva, sweat or seminal plasma.
  • biologically relevant samples e.g. blood, blood plasma, blood serum, cerebrospinal fluid, urine, synovial fluid, amniotic fluid, ascites, pleural fluid, pericardial fluid, saliva, sweat or seminal plasma.
  • the same conjugate with the same inhibitor selectively binds also into the active site of glutamate carboxypeptidase III (GCPIII), a close homolog of GCPII.
  • GCPIII glutamate carboxypeptidase III
  • ELISA was selective for GCPIII, and GCPII presence did not interfere with the determination.
  • affinity tag With immobilization through affinity tag and using the same conjugate we managed to detect and quantify the amount of recombinantly prepared proteins GCPII and GCPIII with great sensitivity.
  • Sandwich ELISA for quantification of CA-IX was carried out analogously to the ELISA method for GCPII quantification (see Example 11); all steps of the experiment were performed at room temperature.
  • the plate was first coated with antibody against CA-IX M75 in TBS (500 ng/well, 2 hours). After blocking the surface with casein (18 hr), and its washing away incubation followed with a lysate of HT-29 cells, diluted in 20 mM Tris-HCl, 200 mM NaCl, 0.1% Tween 20, pH 7.4 (TBST′) (in amounts of 32 ⁇ g-32 ng of total protein/well, 4 hours).
  • CA-IX with PG carbonic anhydrase IX
  • Conjugate 3 was used with an inhibitor selectively binding to the active site of human carbonic anhydrases, especially carbonic anhydrase IX (CA-IX).
  • CA-IX carbonic anhydrase IX
  • a monoclonal antibody in ELISA sandwich arrangement we achieved a highly sensitive determination of CA-IX in solution and in various biological matrices, particularly in tissue and cell lysates, as well as blood plasma and serum.
  • ELISA for the CA-IX quantification with Conjugate 3 allowed detecting picogram quantities of CA-IX; using a combination of M75 antibody (binding CA-IX) and Conjugate and 3, it was possible to detect 1 pg of CA-IX in the HT-29 cell lysate. Thanks to several inhibitors of CA-IX present on one conjugate, it was possible to develop highly sensitive ELISA method using relatively weak (submicromolar) inhibitor of CA-IX. Incubating polymeric conjugate with CA-IX in the presence of test compounds allowed to determine the bond strength (ie. the inhibition constant) of these test substances with great precision.
  • the chip was attached to a SPR chip prism; all measurements were performed at 25° C.
  • Activation of the terminal carboxyl groups on the sensor surface was carried out in situ by addition of a mixture (1:1) 11.51 mg/ml N-hydroxysuccinimide (NHS, Biacore), and 76.68 mg/ml 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC, Biacore) in deionized water for 5 min at a flowrate of 20 ⁇ l/min. Following parts of the experiment were then performed at a flow rate of 30 ⁇ l/min.
  • NHS N-hydroxysuccinimide
  • EDC 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride
  • Curves describing the binding were exported and analyzed in TraceDrawer v.1.5 (Ridgeview Instruments AB) to obtain the parameters k on and k off .
  • HT29 cells (cultured in 100 mm Petri dish) endogenously expressing CA-IX were lysed by sonication in a water bath (3 min/0° C.) in 450 ⁇ l of 50 mM Tris-HCl, 150 mM NaCl, pH 7.4, 1% Tween 20. Resulting cell lysate was further diluted in 20 mM Tris-HCl, 150 mM NaCl, 0.1% Tween 20, pH 7.4 (TBST) to a final protein concentration of 360 ⁇ g/ml.
  • Conjugate 3 and Conjugate 2 (negative control showing nonspecific binding) were pre-bound to 25 ⁇ l of Streptavidin Sepharose (200 nM solution in 200 ⁇ l of TBST, 1 hour, 6° C.), and after washing with 3 ⁇ 200 ⁇ l TBST, the resin was mixed with 200 ⁇ l of HT-29 cell lysate and incubated at 25° C. for 3 hours. The resin was then washed with 3 ⁇ 200 ⁇ l of TBST and subsequently, proteins were eluted with 25 ⁇ l of sample buffer for SDS-PAGE and with heating to 98° C. for 10 min.
  • CA-IX was also isolated with M75 antibody.
  • the experiment was performed analogously to experiment with polymeric conjugates: 1 ⁇ g of antibody was pre-bound to 20 ⁇ l of Protein G Sepharose the procedure followed as described above. Protein G Sepharose resin without the antibody was used as negative control.
  • Conjugate 3 it was possible to affinity isolate CA-IX from lysate of HT-29 cells, endogenously expressing CA-IX. Quantities of CA-IX isolated with Conjugate 3 and with M75 antibody prepared against native CA-IX were practically the same ( FIG. 17 , lanes 4 and 6). Comparative Conjugate 3 serving as a negative control (without CA-IX inhibitor) showed no binding of CA-IX, which shows selective binding of CA-IX to polymeric conjugate through the inhibitor present on Conjugate 3.
  • Example 19 Modified ELISA Method for Testing CA-IX Inhibitors
  • the Maxisorp plate (Nunc) was first coated with M75 antibody in borate buffer (500 ng/well). After blocking the surface with casein and washing it away (see above), incubation followed with recombinant CA-IX (prepared according to [20]) in TBST; 10 ng/well, 1 hr). After washing with 3 ⁇ 200 ⁇ l of TBST a solution was added containing either Conjugate 3 alone (5 nM in TBST for 1 hour; Reference sample) or a mixture of Conjugate 3 (5 nM in TBST) and the test substance in the selected concentration (typically 0.1-100 ⁇ M in TBST).
  • the inhibition analyses were performed by spectrophotometric assay using the chromogenic peptide substrate KARVNle*NphEANle-NH 2 as previously described.
  • the 1 ml reaction mixture contained 100 mM sodium acetate, 300 mM NaCl, pH 4.7, 6.8 ⁇ mol of HIV-1 protease and inhibitor in concentrations ranging between 2 and 130 nM. Substrate was added to a final concentration of 16 ⁇ M. Afterwards, the hydrolysis of substrate was followed as a decrease in absorbance at 305 nm using a UNICAM UV500 UV-VIS spectrophotometer (Thermo, Cambridge, Mass.). The data were analyzed using the equation for competitive inhibition according to Williams and Morrison. The mechanism of inhibition was determined by analysis of Lineweaver-Burk plots.
  • Pepstatin A is a potent inhibitor of aspartic proteases, such as HIV-1 protease, pepsin, cathepsin D and cathepsin E.
  • TAB. 2 Prepared inhibitors and polymer conjugates and their inhibition constants towards HIV-1 protease No. of inhibitor Compound M r Targeting moieties K i [nM] Modification ritonavir 721 HIV-1 protease — 0.015 ⁇ 0.002 — pepstatin A 686 aspartic proteases — 110 ⁇ 12 — compound C 815 HIV-1 protease — 0.012 ⁇ 0.001 — compound D 892 aspartic proteases — 590 ⁇ 2 — Conjugate 4 37,000 HIV-1 protease 5.3 7.2 ⁇ 0.5 compound C, biotin Conjugate 5 71,200 aspartic proteases 12.2 30.3 ⁇ 0.2 compound D, biotin
  • Example 21 Affinity Isolation (“Pull-Down”) of HIV-1 Protease from Spiked LNCaP Lysate Using Conjugates 4 and 5
  • Conjugate 4 (containing ritonavir-based inhibitor) and Conjugate 5 (containing pepstatin A-based inhibitor) were used.
  • Conjugate 2 which lacks the targeting ligand, was used as a negative control.
  • the resin was incubated with 1 ml of 2 mM biotin, 20 mM Tris-HCl, 150 mM NaCl, pH 7.4. Then, the resin was washed three times with 1 ml of 100 mM sodium acetate, 300 mM NaCl, 0.1% Tween 20, pH 4.7.
  • the washed resin was mixed with 200 ⁇ l of LNCaP cell lysate spiked with HIV-1 protease (12 ng/ ⁇ l, total protein concentration 1 mg/ml) in 100 mM sodium acetate, 300 mM NaCl, 0.1% Tween 20, pH 4.7, and incubated for 30 min at room temperature.
  • the resin was washed four times with 1 ml of 100 mM sodium acetate, 300 mM NaCl, 0.1% Tween 20, pH 4.7.
  • bound HIV-1 protease was eluted from Streptavidin Agarose by adding 30 ⁇ l reducing SDS sample buffer and heating to 98° C. for 10 min. Ten microliters of the samples was loaded onto the gel.
  • HIV-1 protease is a homodimeric aspartic protease, with an active site located among the monomers.
  • Conjugate 4 containing ritonavir-based inhibitor, i.e. specific HIV-1 protease inhibitor
  • Conjugate 5 containing pepstatin A-based inhibitor, i.e. class specific inhibitor of aspartic proteases
  • conjugate 4 and conjugate 5 specifically bind HIV-1 protease ( FIG. 18 , lane 4 a 5) and contrastingly, negative control conjugate does not bind HIV-1 protease at all ( FIG. 18 , lane 6).
  • Synthetic macromolecular conjugates that are the subject of the present invention can be used in any laboratory and diagnostic applications, where polyclonal or monoclonal antibodies are commonly used, their fragments or derivatives. These can be a cheap and stable substitute of antibodies used in the ELISA diagnostic method (Enzyme-Linked Immunosorbent Assay), as well as in isolation and quantification of biomolecules in complex mixtures (substitute of antibodies in immunoprecipitation), in visualization of tumor markers and other surface molecules (substitute of antibodies in immunohistochemical analysis), and finally substituting antibodies in fluorescent cytometry.
  • MRI diagnostic method for example polymeric conjugate with gadolinium atom intended for in vivo detection can be used.
  • the invention was developed under the project “Management of the structure and function of biomolecules at the molecular level: the interplay between theory and experiment,” Center of Excellence GACR, P208/12/016.

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US11306170B2 (en) 2016-12-15 2022-04-19 Clariant International Ltd. Water-soluble and/or water-swellable hybrid polymer
US11311473B2 (en) 2016-12-12 2022-04-26 Clariant International Ltd Use of a bio-based polymer in a cosmetic, dermatological or pharmaceutical composition
US11339241B2 (en) 2016-12-15 2022-05-24 Clariant International Ltd. Water-soluble and/or water-swellable hybrid polymer
US11384186B2 (en) 2016-12-12 2022-07-12 Clariant International Ltd Polymer comprising certain level of bio-based carbon
US11401362B2 (en) 2016-12-15 2022-08-02 Clariant International Ltd Water-soluble and/or water-swellable hybrid polymer
US11447682B2 (en) 2015-06-17 2022-09-20 Clariant International Ltd Water-soluble or water-swellable polymers as water loss reducers in cement slurries
US11542343B2 (en) 2016-12-15 2023-01-03 Clariant International Ltd Water-soluble and/or water-swellable hybrid polymer

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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11447682B2 (en) 2015-06-17 2022-09-20 Clariant International Ltd Water-soluble or water-swellable polymers as water loss reducers in cement slurries
US11311473B2 (en) 2016-12-12 2022-04-26 Clariant International Ltd Use of a bio-based polymer in a cosmetic, dermatological or pharmaceutical composition
US11384186B2 (en) 2016-12-12 2022-07-12 Clariant International Ltd Polymer comprising certain level of bio-based carbon
US11306170B2 (en) 2016-12-15 2022-04-19 Clariant International Ltd. Water-soluble and/or water-swellable hybrid polymer
US11339241B2 (en) 2016-12-15 2022-05-24 Clariant International Ltd. Water-soluble and/or water-swellable hybrid polymer
US11401362B2 (en) 2016-12-15 2022-08-02 Clariant International Ltd Water-soluble and/or water-swellable hybrid polymer
US11542343B2 (en) 2016-12-15 2023-01-03 Clariant International Ltd Water-soluble and/or water-swellable hybrid polymer

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CZ201520A3 (cs) 2016-07-27
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US20190033300A1 (en) 2019-01-31
WO2016112883A3 (en) 2016-08-25

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