Patent Description:
The use of fluorescent probes in immunology, haematology and microbiology has grown enormously in the last two decades. These are basic methods used daily in workplaces around the world, from basic research to hospital laboratories and analytical departments. Commercially available, fluorescently labelled antibodies are used in clinical diagnostics for analysing the state of the immune system of patients, in haemato-oncology for the diagnosis of malignant disease, in pathology for the detection of changes and disorders and changes in tissues. They are also used for hundreds of applications in science and research from physiological and immunological to evolutionary, anatomical and developmental analyses.

Hundreds of different antibodies against various biological targets are currently available on the market, either labelled with fluorophores or in the form of so-called purifiers - i.e. without covalently bound fluorescent labels, which must be subsequently detected using so-called labelled secondary antibodies.

Etrych et al. , HPMA copolymer conjugates with reduced anti-CD20 antibody for cell-specific drug targeting. Synthesis and in vitro evaluation of binding efficacy and cytostatic activity; <NPL>, describes semitelechelic polymers having a pHPMA backbone to which an anticancer drug doxorubicine is bound via an oligopeptide linker degradable by lysosomal enzymes or via a pH-labile hydrazone linkage. The polymer backbone has a reactive thiazolidine-<NUM>-thione amide or maleimide end functionality, which enables to target the polymers to anti-CD20 monoclonal antibody forming a star-shaped structure with central antibody surrounded by hydrophilic polymer chains. The star-shaped structure targets cancer cells wherein degradation of the pH-labile or enzyme-labile linker hydrolyses releasing doxorubicin at the site of action.

<CIT> discloses fluorescent polymers for theranostic applications, designed to generate a fluorescent signal in response to a chemical event (upon contacting an analyte - an enzyme - over-expressed in a diseased tissue). The enzyme degrades the peptidic binding of the fluorophore inside the polymer, the fluorophore is thus released from the polymer and the fluorescence in a diseased tissue is enhanced. The peptidic binding of fluorophore is designed to undergo enzymatic degradation.

The growth of bioinformatics and technological progress in the design of detection systems (whether flow cytometers, imaging systems or chip and membrane readers) led to the massive development of multiplex analysis, allowing to significantly multiply the number of features detected simultaneously in individual experiments. This development was also reflected in need for new fluorophores to supplement the spectrum of fluorescent labels used. Hundreds of different fluorophores with different absorption and emission characteristics and fluorescence intensity derived from various modifications of other starting chemical structures are currently available on the market.

In the multiplex analysis, a panel of labelled antibodies should be used, each carrying its fluorophore distinguishable by its spectral profile (absorption and emission maxima) from all other antibodies in the panel. When setting up an antibody diagnostic/analytical panel in the case of more extensive analyses (<NUM> antibodies and more), users encounter the problem of unavailability of specific labelled commercial antibodies against the minor, little experimentally used or newly defined markers which they require to analyse.

Many products are available on the market, allowing users to conjugate a particular fluorophore to its unlabelled antibody. The problem with these kits, however, is the small choice of fluorophores - most of them spectrally overlap with other commonly available fluorophores, and especially the low level of labelling. The results are fluorescently labelled antibodies, but their use is limited, and low-expression markers cannot be detected with these products at all. Spectrally 'interesting' fluorophores emitting in regions where they can be easily distinguished, then usually have a low fluorescence quantum yield and directly labelled conjugates are difficult to distinguish. Polymer fluorescent probes amplify the number of fluorescent molecules and thus significantly amplify the detectable signal. It is, therefore, possible to use non-traditional, spectrally and functionally unique fluorophores and combinations of fluorophores.

The present invention relates to a fluorescent polymer, a fluorescent probe and a conjugation kit for rapid labelling of antibodies, proteins and other structures with a suitably modified surface. The fluorescent polymer and conjugation kit of the present invention allows the labelling of own appropriately modified structures by introducing on their surface semitelechelic copolymer chains having fluorescent label molecules (fluorophores) attached to them. The reactive end groups of the semitelechelic polymer chain allow a single point binding of the fluorescent polymer to the protein structure and thus the fluorescent labelling of the antibody.

Thanks to this approach, up to several times higher amounts of fluorophore can be introduced into a suitable protein structure than with conventional commercially available kits. The resulting fluorescent probe allows the amplification of a signal which reaches and exceeds the intensity of labelling using commercially available biomolecules and nanoparticles. A suitable protein structure can be labelled using the conjugation kit of the present invention in units of minutes and with a minimum number of steps by a fluorescently labelled hydrophilic copolymer, e.g. based on N-(<NUM>-hydroxypropyl)methacrylamide (HPMA), to prepare a polymeric fluorescent probe. Along the chain of this copolymer, functional groups are introduced to which suitable fluorophore derivatives can be attached; at one of its ends, they contain a reactive group for single-point grafting onto the protein structure. Another field of application of such systems according to the present invention is the possibility of creating own combination of several fluorophores bound in several different desired ratios to the structure of interest to create a precisely defined fluorescence pattern that is unique for each particular structure. The resolution of the intensity ratios gives rise to a unique fluorescent signature, where it is then possible to distinguish up to <NUM> different spectral combinations in two channels, and thus to distinguish up to eight types of cell subtypes (subsets). These 'barcoding' spectral technologies can be used to distinguish between different samples, which will first be fluorescently 'coded' and then mixed and labelled all at once, which eliminates differences in labelling techniques and allows direct comparison of the expressions of the monitored markers. The 'barcoding' marker occupies only two spectral bands but allows up to eight different samples to be distinguished. The remaining part of the detection spectrum can be used for other evaluated (diagnostic) markers. In addition, two or more fluorophores reacting differently to physicochemical or biological parameters of the microenvironment of labelled cells, e.g. pH, temperature, ion concentration, enzyme activity, etc., can be combined. One of the used markers is stable in the environment and allows normalising the signal from different cells, and other signals can be variable. The ratio of the normalised and sensitive signal can then be used to determine the parameters of the environment directly. The whole system according to the present invention thus makes it possible to increase the detectability of the monitored markers in one cell and at the same time significantly accelerate the preparation of fluorescently labelled structures with a suitably modified surface.

The object of the present invention is a fluorescent polymer for the rapid labelling of antibodies, proteins and other structures with a suitably modified surface, comprising a linear semitelechelic statistical copolymer to which at least one fluorescent label (fluorophore) is attached in an amount from <NUM> to <NUM> mol %, preferably from <NUM> to <NUM> mol %, based on the number of monomer units, wherein the linear semitelechelic statistical polymer is based on polyacrylamide copolymers, polymethacrylamide copolymers, polyacrylate copolymers or polymethacrylate copolymers, preferably comprises copolymers of poly(N-(<NUM>-hydroxypropyl)methacrylamide, wherein in the linear semitelechelic statistical copolymer from <NUM> to <NUM> mol % of monomer units is statistically replaced by monomer units of general formula (I)
<CHM>
wherein.

By natural amino acids are meant histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, valine, arginine, cysteine, glutamine, glycine, proline, tyrosine, alanine, aspartic acid, asparagine, glutamic acid, serine, selenocysteine. The side chains are chains attached to the alpha-carbon of the amino acid.

In one preferred embodiment, the natural amino acid side chain is selected from the group comprising methyl, isopropyl, isobutyl, -CH(CH<NUM>)(CH<NUM>CH<NUM>), -CH<NUM>OH, -CH(OH)(CH<NUM>), -CH<NUM>-(C<NUM>H<NUM>)OH, -(CH<NUM>)<NUM>-S-CH<NUM>, -CH<NUM>SH, -(CH<NUM>)<NUM>-NH<NUM>, -CH<NUM>COOH, -CH<NUM>C(O)NH<NUM>, -(CH<NUM>)<NUM>COOH, -(CH<NUM>)<NUM>C(O)NH<NUM>, -(CH<NUM>)<NUM>NH-C(=NH)(NH<NUM>), benzyl.

The fluorescent polymer may further contain up to <NUM> mol % of monomer units of general formula (II), based on the number of monomer units,
<CHM>
wherein R is as defined above and Z is selected from the group comprising -C(=O)-NH-(CH<NUM>)a-CH<NUM>(OH); -C(=O)-NH-(CH<NUM>)b-CH(OH)-CH<NUM>; -C(=O)-NH-(CH<NUM>)b-CH(OH)-(CH<NUM>)c-CH<NUM>; wherein a is an integer from <NUM> to <NUM>, b is an integer from <NUM> to <NUM> and c is from <NUM> to <NUM>; and -NH-C(=O)-CH<NUM>,
wherein in total the monomer units of general formula (I) and (II) in the fluorescent polymer are at most <NUM> mol %, based on the number of monomer units.

In one preferred embodiment, R is selected from the group comprising ethan-<NUM>,<NUM>-diyl (-CH<NUM>-CH<NUM>-); propane-<NUM>,<NUM>-diyl (-CH<NUM>-CH<NUM>-CH<NUM>-); hexane-<NUM>,<NUM>-diyl (-(CH<NUM>)<NUM>-).

A linear semitelechelic copolymer is a statistical copolymer formed by radical polymerisation. Thus, the end groups of the resulting linear copolymer contain portions of polymerisation initiator molecules (the initiator may be, for example, <NUM>,<NUM>'-azobis[N-(<NUM>-carboxyethyl)-<NUM>-methylpropionamidine] (V-<NUM>)) and a transfer agent (the transfer agent may be, for example, N-(<NUM>-azidopropyl)-<NUM>-ethylsulphanylcarbothioylsulphanyl-<NUM>-methylpentanamide (CTA-N<NUM>)), or their derivatives formed by reaction with, for example, <NUM>,<NUM>'-azobisisobutyronitrile (AIBN). Thus, the end groups of the copolymer contain an azide group, which can be subsequently modified by reaction with dibenzocyclooctyne-maleimide (DBCO-MI) to another maleimide reactive functional group, by reaction with dibenzocyclooctyne-carboxylic acid (DBCO-Cbx), which is subsequently modified with thiazolidine-<NUM>-thione to a thiazolidine-<NUM>-thione reactive functional group, by reaction with the dibenzocyclooctyne-N-hydroxysuccinimide ester (DBCO-NHS) to an N-hydroxysuccinimide ester reactive functional group. Unlike conventional radical polymerisation with an initiator, which in principle does not allow the formation of a semitelechelic copolymer (does not allow the introduction of reactive end groups at the end of the chain, because no transfer agent is used in the polymerisation), to enable this polymer to be grafted onto the protein in a single point, the presence of a so-called transfer agent is necessary, which introduces a suitable reactive group into the polymer structure. An appropriate reaction is, for example, the RAFT radical polymerisation.

In one preferred embodiment, the linear semitelechelic copolymer is poly(N-(<NUM>-hydroxypropyl) methacrylamide), the resulting fluorescent polymer structure in this embodiment comprises the general formula (III),
<CHM>
wherein R, Y and Z are as defined above, n is an integer in the range of from <NUM> to <NUM>; the proportion of units of general formula (I) in this linear statistical copolymer is from <NUM> to <NUM> mol %, based on the number of monomer units, and the proportion of units of general formula (II) in this linear statistical copolymer is from <NUM> to <NUM> mol %, based on the number of monomer units, the total monomer units of the general formulas (I) and (II) being at most <NUM> mol %, based on the number of monomer units. The end groups of the resulting linear copolymer of general formula (III) contain portions of the polymerisation initiator and transfer agent molecules, and a functional group for binding to a protein structure, preferably N-hydroxysuccinimide ester, thiozolidine-<NUM>-thione amide, maleimide, azide, or propargyl.

In one embodiment, the semitelechelic copolymers that are subsequently part of the fluorescent polymer of the present invention contain -N<NUM> end groups introduced during the polymerisation reaction which can be used to introduce maleimide for conjugation to a protein structure with a suitably modified surface.

The fluorescent label (fluorophore) is selected according to excitation and emission wavelengths, wherein the excitation wavelengths are in the range of from <NUM> to <NUM>, and the emission wavelengths are in the range of from <NUM> to <NUM>,<NUM>. By low-molecular-weight fluorophore is meant a fluorophore having a molecular weight in the range of from <NUM> to <NUM>,<NUM>/mol. Preferably, the fluorophore or derivative thereof contains a functional group, selected from an amino group, an isothiocyanate group and an N-hydroxysuccinimide group, for attachment to the amino or TT group of the linear copolymer side chain. Preferably, the fluorescent label is selected from the group comprising fluorescein, fluorescein isothiocyanate (FITC), derivatives of ruthenium-bipyridine complexes Ru(bpy)<NUM>, in particular bis(<NUM>,<NUM>'-bipyridine)-[<NUM>-(<NUM>'-methyl-<NUM>,<NUM>'-bipyridin-<NUM>-yl)butan-<NUM>-aminium ruthenium tris(perchlorate) (RUB-C4), cyanines, especially Cyanine3 or Sulfo-Cyanine7. <NUM>, also <NUM>,<NUM>-diamino-<NUM>-[<NUM>-(<NUM>-aminoethylcarbamoyl)-<NUM>-carboxyfenyl]-<NUM>-(<NUM>-sulphonatopropylsulphamoyl)xanthene-<NUM>-ium-<NUM>-sulphonate (Dy-<NUM>), (2E)-<NUM>-[<NUM>-(<NUM>,<NUM>-dioxopyrro-lidin-<NUM>-yl)oxy-<NUM>-oxohexyl]-<NUM>-[(2E,4E)-<NUM>-[<NUM>-(<NUM>-methoxyethyl)-<NUM>,<NUM>-dimethyl-<NUM>-sulphonato-indol-<NUM>-ium-<NUM>-yl]penta-<NUM>,<NUM>-dienylidene]-<NUM>,<NUM>-dimethyl-indoline-<NUM>-sulphonate (Dy-647P1), <NUM>-[<NUM>-[(E)-<NUM>-[<NUM>-[<NUM>-(<NUM>-aminoethylamino)-<NUM>-oxohexoxy]-<NUM>,<NUM>-benzoxazol-<NUM>-yl]vinyl]-<NUM>-pyridyl]propane-<NUM>-sulphonate (Dy-396XL), <NUM>-[<NUM>-[<NUM>-[[<NUM>-(<NUM>,<NUM>-dioxopyrrolidin-<NUM>-yl)oxy-<NUM>-oxohexyl]carbamoylamino]-<NUM>,<NUM>-benzoxazol-<NUM>-yl]pyridin-<NUM>-ium-<NUM>-yl]propane-<NUM>-sulphonate (Dy-395XL), <NUM>-[[<NUM>-(<NUM>-azaniumyl-ethylamino)-<NUM>-oxohexyl]carbamoyl]-<NUM>-chloro-<NUM>-hydroxy-<NUM>-oxo-chromene-<NUM>-sulphonate (Dy-<NUM>), <NUM>-[<NUM>-[<NUM>-[[<NUM>-(<NUM>-aminoethylamino)-<NUM>-oxohexyl]-ethyl-amino]-<NUM>-oxo-chromen-<NUM>-yl]pyridin-<NUM>-ium-<NUM>-yl]propane-<NUM>-sulphonate (Dy-485XL), <NUM>-[<NUM>-(<NUM>-aminoethylamino)-<NUM>-oxo-hexyl]-<NUM>-[(E)-<NUM>-[<NUM>-(diethylamino)-<NUM>-oxo-chromen-<NUM>-yl]vinyl]pyridin-<NUM>-ium-<NUM>-sulphonate (Dy-<NUM>), <NUM>-[<NUM>-(<NUM>-aminoethylamino)-<NUM>-oxo-hexyl]-<NUM>-[(E)-<NUM>-[<NUM>-(diethylamino)-<NUM>-oxo-chromen-<NUM>-yl]vinyl]pyridin-<NUM>-ium-<NUM>-sulphonate (Dy-520XL), (2E)-<NUM>-[(E)-<NUM>-(<NUM>-amino-<NUM>-terc-butyl-chromenylium-<NUM>-yl) prop-<NUM>-enylidene]-<NUM>-[<NUM>-(<NUM>-aminoethylamino)-<NUM>-oxohexyl]-<NUM>,<NUM>-dimethyl-indoline-<NUM>-sulphonate (Dy-<NUM>), (2Z)-<NUM>-[<NUM>-(<NUM>-aminoethylamino)-<NUM>-oxobutyl]-<NUM>-[(E)-<NUM>-[<NUM>-terc-butyl-<NUM>-[ethyl(<NUM>-sulpho-natopropyl)amino]chromenylium-<NUM>-yl]prop-<NUM>-enylidene]-<NUM>-methyl-<NUM>-(<NUM>-sulphonatopropyl)indo-line-<NUM>-sulphonate (Dy-<NUM>) and (2Z)-<NUM>-[<NUM>-(<NUM>-aminoethylamino)-<NUM>-oxo-butyl]-<NUM>-[(E)-<NUM>-[<NUM>-(diethylamino)-<NUM>-methyl-<NUM>-phenyl-chromenylium-<NUM>-yl]prop-<NUM>-enylidene]-<NUM>-methyl-<NUM>-(<NUM>-sulpho-natopropyl)indoline-<NUM>-sulphonate (Dy-<NUM>), or their derivatives containing a functional group selected from an amino group, an isothiocyanate group and an N-hydroxysuccinimide group.

In one preferred embodiment, the fluorescent polymer of the present invention comprises at least two different fluorescent labels, for example, combinations Dy-<NUM> and Dy-647P1, Dy-396XL and Dy-647P1, Dy-<NUM> and Dy-647P1, Dy-396XL and Dy-<NUM>, Dy-<NUM> and Dy-480XL, Dy-396XL and Dy-480XL and a variety of other, spectrally non-overlapping fluorophores. These different fluorescent labels can be bound to one fluorescent polymer in different molar ratios, preferably from <NUM>:<NUM> to <NUM>:<NUM>, resulting in a different ratio of fluorescence intensities. More preferably, fluorophores with different excitation and emission wavelengths are combined in one polymer.

Another object of the present invention is a fluorescent probe comprising at least one fluorescent polymer according to the present invention, and further comprising a protein structure selected from the group of a peptide having an amino acid number ranging from <NUM> to <NUM>, a protein, a lipoprotein, an antibody, an enzyme, a hormone, a receptor, a structural protein, a transport protein, a cell signalling pathway protein, a synthetic protein, wherein the protein structure may be modified before conjugation with the fluorescent polymer to contain at least one group selected from -NH<NUM>, -SH, sDBCO (sulphodibenzocyclooctyne group), DBCO (dibenzocyclooctyne group), azide, to which the fluorescent polymer is covalently bound via a functional group, preferably selected from N-hydroxysuccinimide ester, thiozolidine-<NUM>-thione amide, maleimide, azide, or propargyl, contained at the end of the linear polymer chain of the fluorescent polymer. This functional group is covalently bound to an -NH<NUM> group (e.g. lysine residues), -SH group (introduced, e.g. by reduction of disulphide bonds), or to sDBCO, DBCO, and azide groups introduced onto the protein structure to form a covalent bond.

In one preferred embodiment, the fluorescent probe comprises a monoclonal antibody to which at least one fluorescent polymer according to the present invention is covalently bound. The monoclonal antibody may be, for example, anti-CD20, anti-CD3, anti-CD4, anti-CD8, anti-CD71, anti-MHCII, anti-CD45, anti-JNK. Monoclonal antibodies have a monovalent affinity and bind specifically to a single epitope of an antigen.

The monoclonal antibody contains a heavy chain which is type-specific and two light chains which have an antigen-binding site. The fluorescent polymer is covalently bound either to the light chain of the monoclonal antibody at sites where the monoclonal antibody contains disulphide bonds which are reduced before binding to the fluorescent polymer, or along the entire antibody structure at sites containing free amine groups (e.g. from lysine side chains). The reduction of disulphide bonds can be carried out, for example, by treatment with dithiothreitol (DTT), tris(<NUM>-carboxyethyl)phosphine (TCEP), β-mercaptoethanol or glutathione. The monoclonal antibody thus prepared can be immediately conjugated to the fluorescent polymer of the present invention, wherein the SH groups formed by reducing the disulphide bonds of the monoclonal antibody react with a linear polymer chain end group of the fluorescent polymer, e.g., a maleimide group, or the SH groups of the antibody can be further converted by reaction with sDBCO-MI (sulphodibenzocyclooctyne maleimide) to sDBCO groups, or the free NH<NUM> groups of the antibody can be converted to DBCO groups by reaction with DBCO-NHS (dibenzocyclooctyne-N-hydroxysuccinimide ester). Alternatively, NH<NUM> groups of the antibody can be converted to azide groups by reaction with NHS-Azide. The resulting DBCO, sDBCO or azide groups of the antibody are then subsequently reacted with a linear polymer chain end group of the fluorescent polymer, preferably selected from the group comprising azide, or propargyl, to form a covalent bond between the fluorescent polymer and the antibody.

The method of preparing the fluorescent probe (the fluorescent polymer with a bound protein structure) comprises the following steps:.

Step a) of providing the monomers of a linear semitelechelic copolymer includes providing N-(<NUM>-hydroxypropyl)methacrylamide (HPMA), acrylamide or derivatives thereof, methacrylamide or derivatives thereof, acrylate or derivatives thereof or methacrylate or derivatives thereof, and providing monomers of the general formula (IV)
<CHM>.

The amino group may be protected with a protecting group, for example, a tert-butoxycarbonyl (Boc) protecting group. Carbonyl means a -C(= O)- group. N-(<NUM>-hydroxypropyl)methacrylamide (HPMA) and other acrylamide, methacrylamide, acrylate and methacrylate type monomers are commercially available.

Compounds of formula (IV) were either obtained from commercially available sources, for example, methacryolyl-<NUM>-aminoropylamine protected on amine groups by tert-butyloxycarbonyl groups (MA-Pr-NH-Boc), or were prepared according to procedures mentioned in the literature (Pola R. , Parnica J. , Böhmová E. , Filipová M. , Pechar M. , Pankrác J. , Mucksová J. , Kalina J. , Trefil P. , Větvička D. , Poučková P. , Bouček J. , Janoušková O. , Etrych T. Multifunct. <NUM>, <NUM>: <NUM>).

Step b) of polymerisation of the monomers of the linear semitelechelic copolymer is carried out by controlled RAFT radical polymerisation of the monomers from step a) with a content of <NUM> to <NUM> mol % of the monomer of general formula (IV), and at least <NUM> mol % (<NUM> to <NUM> mol %) of monomer units selected from the group comprising N-(<NUM>-hydroxypropyl) methacrylamide (HPMA) and other monomers based on acrylamides, methacrylamides, acrylates and methacrylates, preferably selected from the group comprising HPMA, acrylamide, methacrylamide. The reaction takes place at a temperature in the range of from <NUM> to <NUM>, preferably from <NUM> to <NUM>, and a solvent preferably selected from the group comprising dimethylsulphoxide, dimethylacetamide, dimethylformamide, methanol, ethanol, dioxane, tetrahydrofuran, propanol, tert-butanol or mixtures thereof.

The reaction is initiated by an initiator, preferably selected from the group comprising V-<NUM> and ABIK-N<NUM>, wherein V-<NUM> is <NUM>,<NUM>'-azobis[N-(<NUM>-carboxyethyl)-<NUM>-methylpropionamidine] and ABIK-N<NUM> is N-(<NUM>-azidopropyl)-<NUM>-[(Z)-[<NUM>-(<NUM>-azidopropylamino)-<NUM>-cyano-<NUM>-methyl-<NUM>-oxobutyl] azo]-<NUM>-cyano-pentanamide, optionally in the presence of a transfer agent, preferably CTA-N<NUM>, wherein CTA-N<NUM> is N-(<NUM>-azidopropyl)-<NUM>-ethylsulphanylcarbothioylsulphanyl-<NUM>-methyl-pentanamide. The polymeric carrier is then terminated by a residue from the radical formed by the decomposition of the initiator used.

The resulting linear semitelechelic copolymer preferably contains an -N<NUM> end group; wherein the - N<NUM> end group of this resulting semitelechelic copolymer may be further reacted with suitable maleimide derivatives, preferably selected from the group of dibenzocyclooctyne-amine (DBCO-NH<NUM>) or propargyl derivatives.

The prepared linear semitelechelic copolymer from step b) may be further subjected to removal of protecting groups protecting the amine groups of the side chains (e.g. Boc groups). Deprotection can be accomplished by established procedures known to those skilled in the art, for example, removal of the Boc group with trifluoroacetic acid or by heating the copolymer in water. The resulting copolymer/product can be stored without the risk of decomposition. The types of reactions which are preferably used to prepare the semitelechelic copolymers of the present invention are the RAFT polymerisation (using a pre-prepared transfer agent, e.g., CTA-N<NUM>) and the 'click reaction' (e.g. when using dibenzocyclooctyne, propargyl, or azide end groups for semitelechelic copolymers).

Step c) of binding a fluorescent label to the linear semitelechelic copolymer to form a fluorescent polymer is accomplished by conjugating the free amine groups or thiazolidine-<NUM>-thione (TT) groups of the monomer units of general formula (IV) of the linear semitelechelic copolymer described above to a low-molecular-weight fluorescent label (fluorophore), which contains suitable reactive groups (e.g. amine or SCN) and which may be used in free form or the form of a salt with an acid, e.g. HCl; wherein the low-molecular-weight fluorophore is a fluorophore having a molecular weight in the range of from <NUM> to <NUM>,<NUM>/mol. The fluorophore is selected according to excitation and emission wavelengths, wherein the excitation wavelengths range from <NUM> to <NUM>, and the emission wavelengths range from <NUM> to <NUM>,<NUM>; wherein the molecule of the low-molecular-weight fluorophore is bound to the linear semitelechelic copolymer by an amide or thioamide bond;.

Alternatively, the fluorescent polymer can then be subjected to further reactions leading to a change of the reactive group at the end of the semitelechelic chain. An example is the click reaction of an azide group with dibenzocyclooctyne maleimide to form a fluorescent polymer with a maleimide functional group at one end of the linear polymer chain;.

The optional step d) is the modification of a protein structure selected from the group of peptide, protein, lipoprotein, antibody, enzyme, hormone, receptor, structural protein, transport protein, cell signalling pathway protein, synthetic protein, preferably such protein structure that contains disulphide bonds; wherein the modification consists in reducing the disulphide bonds with a reducing agent selected from the group of dithiothreitol (DTT), tris(<NUM>-carboxyethyl)phosphine (TCEP), β-mercaptoethanol, glutathione, preferably DTT, in an aqueous buffer (pH <NUM> to <NUM>) at room temperature to form SH-groups in the protein structure. These SH groups can then be further modified with sDBCO-MI (sulphodibenzocyclooctyne-maleimide) to sDBCO groups, or free NH<NUM> groups of the antibody (e.g. amino groups of lysine residues) can be modified to DBCO or azide groups by reaction with DBCO-NHS (dibenzocyclooctyne-N-hydroxysuccinimide ester) or NHS-azide. The resulting -SH, azide, DBCO and sDBCO groups or the original -NH<NUM> groups of the antibody are subsequently reacted in step e) with an end group of the linear polymer chain of the fluorescent polymer, preferably selected from the group consisting of N-hydroxysuccinimide ester, thiozolidine-<NUM>-thione amide, maleimide propargyl or azide, to form a covalent bond between the fluorescent polymer and the antibody.

The step e) of binding the protein structure to the fluorescent polymer - the step of conjugating of at least one fluorescent polymer according to the present invention and the protein structure or protein structure with introduced sulphhydryl (-SH), azide, sulphodibenzocyclooctyne (sDBCO) or dibenzocyclooctyne (DBCO) groups prepared in the previous step, wherein the reaction takes place at room temperature in aqueous buffer (pH <NUM> to <NUM>); the end group of the fluorescent polymer reacts with -NH<NUM>, -SH, propargyl, sDBCO or DBCO groups present on the protein structure in the order of minutes and in high yield, and the resulting conjugate retains its unaffected biological effect;.

In one preferred embodiment, at least two different fluorescent polymers are bound to the protein structure, preferably with different fluorophores.

The intermediate product for preparing the fluorescent polymer according to the present invention is, for example, a linear semitelechelic copolymer containing poly(N-(<NUM>-hydroxypropyl) methacrylamide) in which from <NUM> to <NUM> mol % of monomer units are replaced by a monomer unit of general formula (I),
wherein.

Further object of the present invention is a conjugation kit for labelling antibodies comprising the fluorescent polymer according to the present invention, a reaction buffer (<NUM> phosphate buffer, <NUM> EDTA, pH <NUM>), dithiothreitol (DTT) and at least one column having a volume in the range of from <NUM> to <NUM>, preferably with a volume of <NUM>, which contains cross-linked dextran with a molecular weight of from <NUM> to <NUM>,<NUM>/mol, suitable for centrifugal separation of high-molecular-weight substances from low-molecular-weight substances.

The procedure for labelling antibodies using the above conjugation kit consists of the following steps:.

In step A), the antibody is dissolved in the reaction buffer, and DTT in the reaction buffer is added. The reaction mixture reacts at room temperature. The product (reduced antibody in reaction buffer) is purified on a column containing cross-linked dextran having a molecular weight of from <NUM> to <NUM>,<NUM>/mol, and added to the fluorescent polymer in the next step.

In step B), the purified reduced antibody is conjugated to the fluorescent polymer, preferably in a molar ratio of <NUM>: <NUM>.

Using such a conjugation kit, it is also possible to label the protein structures themselves, which have suitable functional groups (disulphide bonds) in their molecule.

Another object of the present invention is the use of the fluorescent polymer, fluorescent probe and/or conjugation kit according to the present invention for fluorescent labelling of protein structures, selected from the group consisting of peptide, protein, lipoprotein, antibody, enzyme, hormone, receptor, structural protein, transport protein, cell signalling pathway protein, synthetic protein.

The fluorescent polymer according to the present invention may carry a combination of several fluorophores bound in several different defined ratios and with an end group for single-point grafting on the protein structure. Various combinations of several fluorophores can then be used to distinguish different spectral combinations, and thus distinguish different types of cell subsets.

In one preferred embodiment, the fluorescent polymer or the fluorescent probe carries a combination of two fluorophores, one of which has fluorescence independent of the environment and thus allows to normalise the signal from different cells or environments. The other fluorophore has fluorescence dependent on various parameters of the external environment, and its fluorescence can react to changes in the external environment. Fluorescent polymers also carry an end group for single-point grafting on the protein structure single point grafting end group on the protein structure according to the present invention for use in determining various physicochemical or biological parameters of cells or environments.

Another object of the present invention is the use of the fluorescent polymer, fluorescent probe and/or conjugation kit according to the present invention in fluorescent imaging techniques, preferably selected from the group comprising flow cytometry for detecting cellular structures and markers, multiplex analysis in flow cytometry to label various antibodies against cellular structures and markers, which is compiled to detect these antibodies simultaneously on individual cells, fluorescence 'barcoding', bead assays, microscopy, western blot, fluorescence flow cytometry for the analysis of cellular events, where one of the fluorophores is responsive to this event. The second fluorophore is stable, thus setting the reference quantification level. The fluorescent polymer and/or fluorescent probe of the present invention can be used, for example, as a secondary reporter probe in sandwich ELISA assays using beads as a carrier for the detection of soluble proteins in suspension; in microscopic techniques based on fluorescence detection for imaging and detection of cellular structures and markers; in microscopic techniques based on fluorescence detection for multiplex analyses and detection of cellular structures and markers; in microscopic techniques based on fluorescence detection for analysis of cellular events, where one of the fluorophores is responsive to this event and the second fluorophore is stable, setting a reference quantification level; in western blot techniques for direct labelling and detection of proteins on the membrane; and direct multiplex labelling and detection of proteins on the membrane.

Other uses are in medical diagnostics, whole-body imaging and/or fluorescence assisted surgery, preferably in diagnosing and monitoring the success of treatment for cancers, hematopoietic diseases (leukaemia, lymphomas, hematopoietic failure), the immune system diseases (primary and secondary immunodeficiency, immune dysregulation, autoimmune diseases), both inflammatory and bacterial. The fluorescent probe and/or fluorescence kit of the present invention can be used, for example, in fluorescence detection-based whole-body imaging techniques to detect organs, single cells, cellular structures and markers; for multiplex detection of organs, single cells, cell structures and markers; for the analysis of physiological and cellular events at the level of organs, single cells, cellular organelles or components as well as soluble proteins, wherein one of the fluorophores is responsive to this event, and the other fluorophore is stable, setting the reference quantification level; in fluorescence-guided surgery to label and display fluorescence in target structures of organs and body tissues.

Thus, the fluorescent probe and fluorescent polymer of the invention can be tailored to a given application for flow cytometry, microscopy, whole-body imaging, western blot, and other techniques using fluorescence as a reporter system. This is made possible by the choice and arbitrary combination of fluorophore and protein as a targeting and specificity-determining structure. The user can therefore create a whole spectrum of their own probes according to their requirements directly in the laboratory, for single- and multi-colour experiments, without the need to purchase different kits from different manufacturers.

The present invention will also allow the preparation of a fluorescent 'barcode' probe for multiplex analyses with the ability to distinguish <NUM> to <NUM> different fluorescent 'barcodes' in a single experiment.

The present invention further allows the determination of parameters of the outside environment or physiological parameters of cells by combining one fluorophore responsive to the respective stimulus with another fluorophore serving as an internal fluorescence standard for normalising the assay result, which represents a significant advance in fluorescence flow cytometry.

Semitelechelic raft-poly(HPMA-co-MA-βAla-TT)-N<NUM> copolymer containing an end azide group (N<NUM>) and thiazolidine-<NUM>-thione (TT) groups along the polymer chain was prepared by controlled RAFT radical polymerisation, where the molar ratio of initiator/RAFT agent/comonomers was <NUM>/<NUM>/<NUM>. The ratio of HPMA/MA-βAla-TT comonomers was <NUM>/<NUM> mol %. <NUM> (<NUM> mmol) of N-(<NUM>-hydroxypropyl)methacrylamide (HPMA) was dissolved in tert-butanol (<NUM>), <NUM> (<NUM> mmol) of <NUM>-(<NUM>-methacryloylamidopropanoyl)thiazolidine-<NUM>-thione (MA-βAla-TT), <NUM> (<NUM>µmol) of initiator, <NUM>,<NUM>'-azobis[N-(<NUM>-carboxyethyl)-<NUM>-methylpropionamidine] (V-<NUM>) and <NUM> (<NUM>µmol) of RAFT agent, N-(<NUM>-azidopropyl)-<NUM>-ethylsulphanyl-carbothioylsulphanyl-<NUM>-methyl-pentanamide (CTA-N<NUM>) was dissolved in dimethylacetamide (DMA; <NUM>). These solutions were mixed in a polymerisation ampoule. The polymerisation mixture was bubbled with argon for <NUM> minutes. The copolymerisation was carried out at <NUM> for <NUM> hours in a sealed ampoule. The product was isolated by precipitation into a <NUM>: <NUM> mixture of acetone/diethyl ether, filtered and dried to constant weight. Reactive ω-end trithiocarbonate groups (TTc) were removed with <NUM>,<NUM>'-azobisisobutyronitrile (AIBN) to prepare a <NUM>% solution of polymer with AIBN (<NUM> wt %) in DMA. The solution in the ampoule was bubbled with argon for <NUM> minutes, sealed and left at <NUM> for <NUM> hours. The product was isolated by precipitation into a <NUM>: <NUM> mixture of acetone/diethyl ether, filtered and dried to constant weight. Characterization of the resulting copolymer: Mn (SEC) <NUM>,<NUM>/mol; Ð ~ <NUM>; content of (TT) ~ <NUM> mol %.

Following the procedure in Example <NUM>, other raft-poly(HPMA-co-MA-βAla-TT)-N<NUM> copolymers with different molar masses Mn in the range from <NUM>,<NUM> to <NUM>,<NUM>/mol were prepared analogously, wherein the polydispersity index Ð was in all cases in the range of <NUM> to <NUM>. The content of TT-groups along the chain was <NUM> to <NUM> mol %. According to this procedure, also other raft-poly(HPMA-co-MA-R1-TT)-N<NUM> copolymers were prepared, which contained in their structure aminoacyl R1 according to formula I. See Table <NUM>.

Semitelechelic raft-poly(HPMA-co-MA-Pr-NH-Boc)-N<NUM> copolymer containing an end azide group (N<NUM>) and protected amine groups (-NH-Boc) along the polymer chain was prepared by controlled RAFT radical polymerisation, where the molar ratio of initiator/RAFT agent/comonomers was <NUM>,<NUM>/<NUM>/<NUM>. The ratio of HPMA/MA-Pr-NH-Boc comonomers was <NUM>/<NUM> mol %. <NUM> (<NUM> mmol) of N-(<NUM>-hydroxypropyl)methacrylamide (HPMA) was dissolved in tert-butanol (<NUM>), <NUM> (<NUM> mmol) of methacryolyl-<NUM>-aminoropylamine protected on amine groups by tert-butyloxycarbonyl groups (MA-Pr-NH-Boc), <NUM> (<NUM>µmol) of initiator, <NUM>,<NUM>'-azobis[N-(<NUM>-carboxyethyl)-<NUM>-methylpropionamidine] (V-<NUM>) and <NUM> (<NUM>µmol) of RAFT agent, N-(<NUM>-azidopropyl)-<NUM>-ethylsulphanylcarbothioylsulphanyl-<NUM>-methyl-pentanamide (CTA-N<NUM>) was dissolve in dimethylacetamide (DMA; <NUM>). These solutions were mixed in a polymerisation ampoule. The polymerisation mixture was bubbled with argon for <NUM> minutes. The copolymerisation was carried out at <NUM> for <NUM> hours in a sealed ampoule. The product was isolated by precipitation into a <NUM>: <NUM> mixture of acetone/diethyl ether, filtered and dried to constant weight. Reactive ω-end trithiocarbonate groups (TTc) were removed with <NUM>,<NUM>'-azobisisobutyronitrile (AIBN) to prepare a <NUM>% solution of polymer with AIBN (<NUM> wt %) in DMA. The solution in the ampoule was bubbled with argon for <NUM> minutes, sealed and left at <NUM> for <NUM> hours. The product was isolated by precipitation into a <NUM>: <NUM> mixture of acetone/diethyl ether, filtered and dried to constant weight. Characterization of the resulting copolymer: Mn (SEC) <NUM>,<NUM>/mol; Ð ~ <NUM>; content of (NH<NUM>) ~ <NUM> mol %.

Following the procedure in Example <NUM>, other raft-poly(HPMA-co-MA-Pr-NH-Boc)-N<NUM> copolymers with different molar masses Mn in the range from <NUM>,<NUM> to <NUM>,<NUM>/mol were prepared analogously, wherein the polydispersity index Ð was in all cases in the range of <NUM> to <NUM>. According to this procedure, also other raft-poly(HPMA-co-MA-R2-NH-Boc)-N<NUM> copolymers were prepared, which contained in their structure aminoacyl R2 according to formula II. The content of NH<NUM> groups along the chain was <NUM> to <NUM> mol %. See Table <NUM>.

The semitelechelic raft-poly(HPMA-co-MA-Pr-NH<NUM>)-N<NUM> copolymer was prepared from the semitelechelic raft-poly(HPMA-co-MA-Pr-NH-Boc)-N<NUM> copolymer of Example <NUM> (<NUM>), which contained amine groups protected by a tert-butyloxycarbonyl group (Boc). The semitelechelic raft-poly(HPMA-co-MA-Pr-NH-Boc)-N<NUM> copolymer was dissolved in <NUM> of distilled water and transferred to a polymerisation ampoule. The reaction mixture was bubbled with argon for <NUM> minutes. Deprotection was performed at <NUM> for <NUM> hour in a sealed polymerisation ampoule. The product was finally lyophilised.

Yield <NUM> (<NUM> %). Characterization: Mw = <NUM>,<NUM>/mol, Mn = <NUM>,<NUM>/mol. Similarly, the protecting groups were removed from other polymers prepared according to procedure <NUM>.

The fluorescent polymer raft-poly(HPMA-co-MA-βAla-Dy-<NUM>)-N<NUM> was prepared by aminolytic reaction of TT groups along the chain of the copolymer (Example <NUM>) and the amino derivative of the fluorophore Dy-<NUM> (ex/em <NUM>/<NUM>), which is commercially available. The starting copolymer raft-poly(HPMA-co-MA-βAla-TT)-N<NUM> (<NUM>, <NUM>µmol) was dissolved in anhydrous dimethyl sulphoxide (DMSO, <NUM>µl) and a solution of the amino derivative Dy-<NUM> (<NUM>, <NUM>µmol, <NUM> mol %) in DMSO was added along with DIPEA (<NUM>µl, <NUM>µmol). The reaction mixture was allowed to react in the dark for <NUM> hours under constant stirring. Unreacted TT groups were removed by the addition of <NUM>-aminopropan-<NUM>-ol (<NUM>µl, <NUM>µmol). The product was purified by column chromatography (Sephadex LH20, methanol). The fraction with the sample was evaporated under vacuum, and the product was lyophilised from aqueous solution. Yield <NUM> % (<NUM>). Dy-<NUM> content ~ <NUM> mol %. Following the procedure in Example <NUM>, other fluorescent polymers derived from the polymers prepared in Example <NUM> were prepared analogously with amino derivatives of various fluorophores whose excitation wavelengths were in the range of from <NUM> to <NUM> and emission wavelengths in the range of from <NUM> to <NUM>,<NUM>. The fluorophore content ranged from <NUM> to <NUM> mol %. See Table <NUM>.

The fluorescent polymer raft-poly(HPMA-co-MA-Pr-FITC)-N<NUM> was prepared by reacting NH<NUM> groups along the chain of a raft-poly(HPMA-co-MA-Pr-NH<NUM>)-N<NUM> copolymer and fluorescein isothiocyanate (FITC, ex/em <NUM>/<NUM>). The starting copolymer (<NUM>, <NUM>µmol) was dissolved in anhydrous dimethyl sulphoxide (DMSO, <NUM>µl) and a solution of FITC (<NUM>, <NUM>µmol, <NUM> mol %) in DMSO was added. The reaction mixture was allowed to react in the dark for <NUM> hours under constant stirring. Unreacted NH<NUM> groups were removed by the addition of acetylthiazolidine-<NUM>-thione (<NUM>, <NUM>µmol). The product was purified by column chromatography (Sephadex LH20, methanol). The fraction with the sample was evaporated under vacuum, and the product was lyophilised from aqueous solution. Yield <NUM> % (<NUM>). FITC content ~ <NUM> mol %.

The polymer precursor raft-poly (HPMA-co-MA-Pr-NH<NUM>)-N<NUM> prepared according to Example <NUM> was also used to prepare a fluorescent polymer using other fluorophore derivatives, e.g. N-hydroxysuccinimide esters according to the procedure described in Example <NUM>. Other polymer precursors prepared according to Example <NUM> were used analogously for the preparation of fluorescent polymers.

Following the procedure in Example <NUM>, other fluorescent polymers raft-poly(HPMA-co-MA-R-NH-FITC)-N<NUM> with different FITC contents ranging from <NUM> to <NUM> mol % were prepared analogously. See Table <NUM> (R is alkylene).

The fluorescent polymer raft-poly(HPMA-co-MA-βAla-Dy-<NUM>)-maleimide was prepared by a click reaction of an azide group present at the end of the polymer chain of raft-poly(HPMA-coMA-βAla-Dy-<NUM>)-N<NUM> (Example <NUM>) and dibenzocyclooctyne-maleimide (DBCO-MI). The starting fluorescent polymer (<NUM>, <NUM>µmol) was dissolved in anhydrous DMSO (<NUM>µl), and a solution of DBCO-MI (<NUM>, <NUM>µmol) in DMSO was added. The reaction mixture was allowed to react in the dark for <NUM> hours under constant stirring. The formation of the product was monitored chromatographically (Shimadzu HPLC system equipped with a Chromolith Performance RP-18e reverse phase column (<NUM> × <NUM>) and a Shimadzu SPD-10AVvp UV-VIS detector (<NUM>); eluent <NUM>% acetonitrile - <NUM>% acetonitrile with gradient <NUM>-<NUM> % by volume <NUM>% acetonitrile, flow rate <NUM>·min-<NUM>) from the decrease of the initial amount of DBCO-MI. The product was purified by column chromatography (Sephadex LH20, methanol). The fraction with the sample was evaporated under vacuum, and the product was lyophilised from aqueous solution. Yield <NUM> % (<NUM>).

In the same manner, maleimide end groups were introduced onto all fluorescent polymers prepared in Examples <NUM> and <NUM>.

The fluorescent polymer raft-poly(HPMA-co-MA-βAla-Dy-<NUM>)-TT was prepared by a click reaction of an azide group present at the end of the polymer chain of raft-poly(HPMA-co-MA-βAla-Dy-<NUM>)-N<NUM> (Example <NUM>) and dibenzocyclooctyne-carboxylic acid (DBCO-Cbx), which was subsequently modified with thiazolidine-<NUM>-thione. The starting fluorescent polymer (<NUM>, <NUM>µmol) was dissolved in anhydrous DMSO (<NUM>µl), and a solution of DBCO-Cbx (<NUM>, <NUM>µmol) in DMSO was added. The reaction mixture was allowed to react in the dark for <NUM> hours under constant stirring. The formation of the product was monitored chromatographically (Shimadzu HPLC system equipped with a Chromolith Performance RP-18e reverse phase column (<NUM> × <NUM>) and a Shimadzu SPD-10AVvp UV-VIS detector (<NUM>); eluent <NUM>% acetonitrile - <NUM>% acetonitrile with a gradient of <NUM>-<NUM>% by volume <NUM>% acetonitrile, flow rate <NUM>·min-<NUM>) from the decrease of the starting amount of DBCO-Cbx. Subsequently, N-(<NUM>-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (<NUM>, <NUM>µmol), thiazolidine-<NUM>-thione (<NUM>, <NUM>µmol) and a catalytic amount of dimethyl aminopyridine were added to the reaction mixture. The reaction mixture was allowed to react in the dark for <NUM> hours under constant stirring. The product was purified by column chromatography (Sephadex LH20, methanol). The fraction with the sample was evaporated under vacuum, and the product was precipitated as described in Examples <NUM> and <NUM>. The TT content was determined spectrophotometrically. Yield <NUM> % (<NUM>), TT content <NUM> % mol. In the same manner, TT end groups were introduced on all fluorescent polymers prepared in Examples <NUM> and <NUM>.

The fluorescent polymer raft-poly(HPMA-co-MA-βAla-Dy-<NUM>)-N- hydroxysuccinimide ester was prepared by a click reaction of an azide group present at the end of the polymer chain of raft-poly(HPMA-co-MA-βAla-Dy-<NUM>)-N<NUM> (Example <NUM>) and dibenzocyclooctyne-N-hydroxysuccinimide ester (DBCO-NHS). The starting fluorescent polymer (<NUM>, <NUM>µmol) was dissolved in anhydrous DMSO (<NUM>µl), and a solution of DBCO-NHS (<NUM>, <NUM>µmol) in DMSO was added. The reaction mixture was allowed to react in the dark for <NUM> hours under constant stirring. The formation of the product was monitored chromatographically (Shimadzu HPLC system equipped with a Chromolith Performance RP-18e reverse phase column (<NUM>× <NUM>) and a Shimadzu SPD-10AVvp UV-VIS detector (<NUM>); eluent <NUM>% acetonitrile - <NUM>% acetonitrile with a gradient of <NUM>-<NUM>% by volume <NUM>% acetonitrile, flow rate <NUM>·min-<NUM>) from the decrease of the starting amount of DBCO-NHS. The product was purified by column chromatography (Sephadex LH20, methanol). The fraction with the sample was evaporated under vacuum, and the product was precipitated as described in Examples <NUM> and <NUM>. In the same manner, NHS end groups were introduced on all fluorescent polymers prepared in Examples <NUM> and <NUM>.

A fluorescent probe PS1 with bound anti-CD20 monoclonal antibody was prepared by aminolytic reaction of the end TT groups present on the fluorescent polymers (Example <NUM>) and the amino groups present on the antibody molecule. A solution of anti-CD20 monoclonal antibody was added to the fluorescent polymer in a molar ratio of anti-CD20: fluorescent polymer = <NUM>:<NUM>. The reaction mixture was allowed to react in the dark for <NUM> minutes under constant stirring; the product was desalted on a PD10 column (Sephadex G25) in water and lyophilised. The presence of the resulting fluorescent probe was confirmed by size exclusion chromatography (SEC) on a Superose6 column in <NUM> acetate buffer with pH <NUM>. Following the same procedure, a fluorescent probe PS2 was prepared with a bound monoclonal antibody and a fluorescent polymer containing end N-hydroxysuccinimide ester groups (Example <NUM>). Similarly, fluorescent probes were prepared from other monoclonal antibodies.

The reduction of disulphide bonds was carried out in the presence of dithiothreitol in reaction phosphate buffer (<NUM> phosphate buffer, <NUM> EDTA; pH <NUM>), which was bubbled with argon for <NUM> minutes before use. To a solution of anti-CD3 monoclonal antibody (<NUM>µmol; OKT-<NUM>), dithiothreitol (<NUM> mmol; DTT) was added, and the mixture was allowed to react for <NUM> minutes under constant stirring. The product was purified on a PD10 column (Sephadex G25) in reaction buffer. The anti-CD3 monoclonal antibody thus prepared was immediately conjugated to the fluorescent polymer prepared according to Example <NUM>. Characterisation: sulphhydryl group content ~ <NUM>.

Following the procedure in Example <NUM>, other reduced monoclonal antibodies were prepared analogously, such as anti-CD-<NUM>, anti-CD-<NUM>, anti-CD-<NUM>, anti-CD45, anti-Thy1,<NUM>, anti-MHC II, anti JNK.

A fluorescent probe PS3 with bound anti-CD3 monoclonal antibody was prepared by a click reaction of the end maleimide groups present on the fluorescent polymers (Example <NUM>) and the sulphhydryl groups present on the reduced antibody (Example <NUM>). A solution of purified reduced anti-CD3 monoclonal antibody was added to the fluorescent polymer in a molar ratio of anti-CD3 : fluorescent polymer = <NUM>: <NUM>. The reaction mixture was allowed to react in the dark for <NUM> minutes under constant stirring; the product was desalted on a PD10 column (Sephadex G25) in water and lyophilised. The presence of the resulting fluorescent probe was confirmed by size exclusion chromatography (SEC) on a Superose6 column in <NUM> acetate buffer with pH <NUM>. The GPC chromatogram of the fluorescent probe is shown in <FIG>. Fluorescent probes PS4 to PS6 were prepared from other monoclonal antibodies according to the same procedure.

DBCO groups for binding azide-terminated fluorescent polymers (Examples <NUM> and <NUM>) were introduced onto the sulphhydryl groups present on the reduced antibody (Example <NUM>) using sulphodibenzocyclooctyne-maleimide (sDBCO-MI). sDBCO-MI binding was carried out in reaction phosphate buffer (<NUM> phosphate buffer, <NUM> EDTA; pH <NUM>), which was bubbled with argon for <NUM> minutes before use. sDBCO-MI (<NUM>µmol) was added to a solution of anti-CD3 monoclonal antibody (<NUM>µmol; OKT-<NUM>), and the mixture was allowed to react for <NUM> minutes under constant stirring. The product was purified on a PD10 column (Sephadex G25) in distilled water and lyophilised. The modified anti-CD3 monoclonal antibody thus prepared was used for conjugation on a fluorescent polymer according to Example <NUM>.

Following this procedure, other monoclonal antibodies with modified sulphhydryl groups were prepared analogously.

The amino groups of the lysine residues present on the antibody were modified to bind the azide-terminated fluorescent polymers (Examples <NUM> and <NUM>) using dibenzocyclooctyne-N-hydroxysuccinimide ester (DBCO-NHS). DBCO-NHS binding was carried out in reaction phosphate buffer (<NUM> phosphate buffer, <NUM> EDTA; pH <NUM>). A solution of DBCO-NHS (<NUM>µmol) in DMSO was added to the solution of anti-CD3 monoclonal antibody (<NUM>µmol; OKT-<NUM>), and the mixture was allowed to react for <NUM> minutes under constant stirring. The product was purified on a PD10 column (Sephadex G25) in distilled water and lyophilised. The modified anti-CD3 monoclonal antibody thus prepared was used for conjugation to a fluorescent polymer prepared according to Examples <NUM> and <NUM>.

Following this procedure, other monoclonal antibodies with modified amino groups were prepared analogously.

Fluorescent probes PS7 and PS8 with bound anti-CD3 monoclonal antibody were prepared by a click reaction of the end azide groups present on the fluorescent polymers (Examples <NUM> and <NUM>) and the DBCO groups introduced on the antibody molecules (Examples <NUM> and <NUM>). A solution of anti-CD3 monoclonal antibody in a buffer (<NUM> phosphate buffer, <NUM> EDTA, pH <NUM>) was added to the fluorescent polymer in a molar ratio of anti-CD3: fluorescent polymer = <NUM>:<NUM>. The reaction mixture was allowed to react in the dark for <NUM> minutes under constant stirring; the product was desalted on a PD10 column (Sephadex G25) in water and lyophilised. The presence of the resulting fluorescent probe was confirmed by size exclusion chromatography (SEC) on a Superose6 column in <NUM> acetate buffer with pH <NUM>. Fluorescent probes with other antibodies and fluorophores were prepared according to the same procedure.

A fluorescent polymer for the preparation of a multi-spectral fluorescent nanoprobe was prepared by aminolytic reaction of the fluorophores Dy-<NUM> and Dy-647P1 with the appropriate polymer precursor. The appropriate Dy-<NUM> and Dy-647P1 derivatives were added to the reaction mixture in a <NUM>:<NUM> molar ratio sequentially after <NUM> minutes. The following procedure was the same as in Examples <NUM>, <NUM> and <NUM>. Characterization: fluorophore content: Dy-<NUM> ~ <NUM> mol %; Dy-647P1 ~ <NUM> mol %. The molar ratio of fluorophores: <NUM>: <NUM>.

Following the procedure in Example <NUM>, other fluorescent polymers were prepared analogously to prepare multi-spectral fluorescent nanoprobes with different fluorophores and their mutual molar ratios. Their excitation wavelengths were in the range of from <NUM> to <NUM> and emission wavelengths in the range of from <NUM> to <NUM>,<NUM>. The content of fluorophores ranged from <NUM> to <NUM> mol %. See Table <NUM>. Other fluorescent polymers containing fluorophore pairs were prepared in the same manner.

A multi-spectral fluorescent nanoprobe was prepared by conjugating a fluorescent polymer (Example <NUM>) and a reduced anti-CD3 monoclonal antibody (Example <NUM>) according to the procedure described in Example <NUM>.

The prepared copolymers, fluorescent polymers and fluorescent probes with monoclonal antibodies were characterised by determining the weight and number average of molecular weights (Mw, Mn) and the corresponding polydispersity index (Ð) using size exclusion chromatography (SEC) on a system equipped with a UV detector (Shimadzu, Japan), RI detector (Optilab REX, Wyatt Technology Corp. , USA) and a multi-angle light scattering detector (DAWN Heleos-II, Wyatt Technology Corp. In the case of SEC, a TSK <NUM> Super SW column was used for characterisation and a mixture of MeOH (<NUM> %) and <NUM> acetate buffer with pH <NUM> (<NUM> %) as the mobile phase; a Superose6 column was used for characterisation of monoclonal antibody probes with <NUM> acetate buffer with pH <NUM> as the mobile phase. The sample concentration was <NUM>/ml in all cases.

The content of TT and NH<NUM> groups, copolymerised statistically along the polymer chain, was determined spectrophotometrically. All measurements were made on a Specord <NUM> UV-VIS spectrophotometer (Analytik Jena, Germany). The content of TT groups was determined according to the literature (Šubr, V. ; Koňák, Č. ; <NPL>) in methanol (e<NUM> = <NUM>,<NUM>·mol-<NUM>· cm-<NUM>). The content of amine groups (after deprotection in distilled water at <NUM> in a sealed polymerisation ampoule) was determined according to the method described in <NPL> (e<NUM> = <NUM>,<NUM>·mol-<NUM>·cm-<NUM>). The content of bound fluorophores was determined spectrophotometrically.

The fluorescent polymer (polymer precursor with FITC fluorescent label according to Examples <NUM> and <NUM>) was used for conjugation to CD20-RTX monoclonal antibody for <NUM> minutes (A), <NUM> minutes (B), <NUM> minutes (C) and <NUM> minutes (D). The fluorescent polymer with FITC and the antibody were incubated for <NUM> minutes with a human peripheral blood sample by a conventional procedure (incubation with the antibody, lysis of erythrocytes with ammonium chloride solution and centrifugation). The prepared cell suspension was measured with a flow cytometer (BD Celesta, <NUM> laser excitation and <NUM>/<NUM> emission detection). All times tested resulted in equally significant fluorescence intensity of CD20-RTX FITC (<FIG>), which was comparable to the commercially available CD20 FITC reagents.

As expected, the blood sample contains a mixture of T-lymphocytes, NK cells and B-lymphocytes, with only B-lymphocytes carrying a CD20 molecule on the surface, which is detected by a polymer probe and represented by a positive peak in the right part of the graph. The negative signal on the left of the figure belongs to T-lymphocytes and NK cells that do not carry the CD20 marker. Thus, the fluorescent probe provides the expected result with an extremely short conjugation time. The resulting polymeric fluorescent probe is useful for detecting human cells carrying an antibody-targeted molecule.

The conjugation kit for labelling antibodies consists of fluorescent polymers which carry various fluorescent labels along the chain (Examples <NUM> and <NUM>) and end maleimide groups. It also contains reaction buffer (<NUM> phosphate buffer, <NUM> EDTA, pH <NUM>), dithiothreitol (DTT) and columns containing cross-linked dextran with a molecular weight of <NUM> to <NUM>,<NUM>/mol (Sephadex G-<NUM>) with a volume of <NUM> (PD SpinTrap G-<NUM>), which is suitable for centrifugal separation of high-molecular-weight substances from low-molecular-weight impurities. The procedure consists of two necessary steps:.

Using such a conjugation kit, it is also possible to label the protein structures themselves, which have suitable functional groups in their molecule (see <FIG>).

Several conjugation kits have been prepared, which consist of a fluorescent polymer with a fluorescent label (fluorophores) with different excitation and emission characteristics (Dy-396XL; Dy395XL; s Dy682; Dy410; Dy480XL). We prepared polymer fluorescent probes by rapid conjugation with CD3 OKT3 or CD20 RTX antibody. These probes were incubated for <NUM> minutes with human peripheral blood samples according to standard procedures. The prepared cell suspension was measured with a BD Celesta flow cytometer, with excitation at <NUM>, <NUM> and/or <NUM>, and emission detection in the bands indicated in the x-axis label in <FIG>. All prepared and tested polymer fluorescent probes led to the expected profile of labelling of the lymphocyte sample, CD3 on T-lymphocytes and CD20 on B-lymphocytes. The separation of positive and negative cells was variable according to the selected fluorophore and the efficiency of its excitation by available lasers. Polymer fluorescent probes allow the detection of cells carrying the target molecule. The variability of the conjugation kit then provides flexibility in the selection of the antibody according to the user's needs, these polymer fluorescent probes can be detected in different channels of the flow cytometer, and thus the optimal composition of antibody conjugates can be tailored to the research or diagnostic task.

Conjugation kits with a multi-colour spectral profile were prepared. The fluorescent polymer was prepared by binding a mixture of two fluorophores Dy-<NUM> and Dy-647P1 in different ratios according to Example <NUM>. At the same time, fluorescent polymers with individual fluorophores Dy-<NUM> and Dy-647P1 were prepared. Fluorescent polymers were conjugated to the CD8 OKT-<NUM> antibody. We tested the principle of the possibility of distinguishing particles labelled with individual types of polymers with different spectrally distinguishable characteristics using UltraComp particles (Invitrogen) stained with antibodies conjugated to polymers containing fluorophores Dy-<NUM> and Dy-647P1 in various ratios and monochromatic polymers with Dy-<NUM> and Dy-647P1. The particles were incubated in six separate aliquots with each type of fluorescent polymer and antibody separately. We performed the measurements using a flow cytometer. As an unlabelled control sample, we added UltraComp particles without incubation with the polymer fluorescent probe. As documented in <FIG>, the different types of spectrally unique polymers can be distinguished as separate groups of particles with the same characteristics (distinguished in <FIG>). In this way, different types of particles can be labelled (the so-called barcoding) for efficient analysis of sample mixtures using multi-coloured antibody panels. By combining only two fluorophores, it makes it possible to distinguish six types of barcode. The successful use of multi-spectral fluorescent probes to distinguish six original samples in just two flow cytometer detection channels has been documented.

Anti-CD71 and anti-MHCII antibodies were labelled with a combination of fluorescent polymers carrying a combination of two fluorophores (Examples <NUM>, <NUM> and <NUM>). One fluorophore is FITC, in which the fluorescence decreases in a lower pH environment, and the other fluorophore is Dyomics-<NUM>, which exhibits stable pH-independent fluorescence over the pH range of <NUM> to <NUM>. Isolated mouse splenocytes (BALB/c strain) were homogenised and washed in HBSS. They were then cooled and labelled with antibodies at <NUM> for <NUM> minutes. Subsequently, they were washed, transferred to a culture medium at <NUM> and analysed at a <NUM>-minute interval on a BD LSR-II flow cytometer. As a parameter, the emission ratio of FITC vs Dy-<NUM> was analysed over time.

<FIG> shows a significant change in the fluorophore emission ratio with increasing incubation time. As the antibody enters the cell, it is internalised into acidic organelles (lysosomes, late endosomes), where the quantum yield of FITC emission decreases while Dy-<NUM> emission remains at baseline. The rapidly internalising transferrin receptor (CD71) shows a rapid change in the fluorescence ratio, while the slowly internalising MHC-II shows a continuous slow decline.

In this way, it is possible to very accurately monitor the rate of internalisation of surface structures depending on the antibody used and to calculate and analyse the kinetics of absorption and transition to acidic intracellular compartments, either by flow cytometry or by quantitative imaging techniques.

The anti-mouse CD45 antibody (CapricoBio, USA) was labelled with a fluorescent polymer carrying Dy-485XL (Examples <NUM> and <NUM>), and the anti-mouse CD8 antibody (CapricoBio, USA) was labelled with a fluorescent polymer carrying Dy-395XL (Examples <NUM> and <NUM>). Spleen tissues were sectioned from sacrificed BALB/c mice, cut into small pieces, placed in OCT freezing medium (Sakura Finetek, Torrance, CA) and stored at -<NUM>. Tissue sections (<NUM> thick) were fixed in chilled acetone for <NUM> minutes, air dried, washed with cold PBS and blocked with <NUM>% normal horse serum (Jackson ImmunoResearch, West Grove, PA) for <NUM> minutes at room temperature (RT). Labelling with antibodies was carried out at <NUM> for <NUM> minutes with a prepared mixture of both antibodies simultaneously. After washing with cold PBS, they were fixed with <NUM>% paraformaldehyde and transferred to an aqueous medium containing DAPI (Sigma) and visualised on an Olympus FV-<NUM> microscope at the appropriate wavelengths (Dy-485XL Ex <NUM> and Em <NUM>/<NUM>, Dy-395XL Ex <NUM> and Em <NUM>/<NUM>).

<FIG> clearly shows green labelled lymphocyte populations (anti-CD45) and red labelled T-cells (anti-CD8), the nuclei of all cells in the preparation are labelled blue with DAPI.

Both user-labelled antibodies can be thus successfully used in combination for multiplex analysis of preparations for fluorescence microscopy.

The anti-JNK antibody was labelled with a conjugation kit that uses fluorescent polymers carrying the fluorophore Dy-<NUM> (Examples <NUM> and <NUM>).

Cell lysates were separated on <NUM>% SDS-PAGE, transferred onto nitrocellulose membranes and blocked in <NUM>% skimmed milk in PBS-T. The membrane was incubated with an unlabelled rabbit anti-JNK antibody or with an anti-JNK antibody labelled with a fluorescent polymer carrying the fluorophore Dy-<NUM>. The secondary antibody used to visualise the membrane labelled with an unlabelled antibody was IRDye <NUM> (anti-rabbit). Detection and quantification were carried out using an Odyssey infrared imaging system (LI-Cor Biosystems).

<FIG> shows that the antibody labelled directly with the conjugation kit (<FIG>) achieves identical results as the use of the secondary professionally labelled antibody (<FIG>).

The anti-mouse Thy <NUM> antibody was labelled with the conjugation kit using a fluorescent polymer carrying the fluorophore Dy-<NUM> (Examples <NUM> and <NUM>). A total of <NUM>·<NUM><NUM> mouse thymoma EL-<NUM> cells were injected subcutaneously into the right flank of NuNu mice, and the tumour growth was noted.

In vivo imaging was carried out on an OV100 Whole Mouse System (Olympus Corp. ) containing an MT-<NUM> light source and an Orca II ERG CCD camera (Hamamatsu, Japan). Animals were anaesthetised by intraperitoneal injection of <NUM>% Narkamon in the amount of <NUM>µl per mouse. Imaging of tumour growth was non-invasive; the accumulation of specific antibody in the tumour mass was observed through the skin covering the tumour. The fluorescence of the antibody specifically labelling the tumour cells was visualised using specific filters (Ex <NUM>, Em <NUM>/<NUM>). Images were taken separately for the fluorescence channel and the reflected light, then combined and stained in AnalySIS <NUM>. <NUM> software (SIS Systems).

Claim 1:
A fluorescent polymer, comprising a linear semitelechelic statistical copolymer to which at least one fluorescent label is bound in an amount in the range of from <NUM> to <NUM> mol %, based on the number of monomer units,
wherein the linear semitelechelic statistical polymer comprises polyacrylamide, polymethacrylamide, or poly(N-(<NUM>-hydroxypropyl)methacrylamide, wherein from <NUM> to <NUM> mol % of monomer units in the linear semitelechelic statistical copolymer are replaced by monomer units of general formula (I)
<CHM>
wherein
R is selected from the group consisting of a straight or branched carbon alkylenyl chain with a number of carbons from <NUM> to <NUM>; wherein R may be further substituted with one or more identical or different natural amino acid side chains;
Y is selected from the group consisting of a bond, -C(=O)-NH-, -NH-C(=O)-, -NH-C(=S)-NH-; wherein the molecular weight Mn of the linear semitelechelic copolymer is in the range of from <NUM>,<NUM> to <NUM>,<NUM>/mol;
and wherein the fluorophore has a molecular weight in the range of from <NUM> to <NUM>,<NUM>/mol, an excitation wavelength in the range of from <NUM> to <NUM> and an emission wavelength in the range of from <NUM> to <NUM>,<NUM>, and is covalently bound to the monomer unit of general formula (I) of the linear semitelechelic copolymer via terminal amine or thiocyanate or N-hydroxysuccinimide ester, wherein the groups formed following the fluorophore binding are subsequently part of the group Y;
and wherein the fluorescent polymer contains at one end of the polymer chain a functional group for binding to a protein structure, which functional group is selected from the group comprising esters, preferably N-hydroxysuccinimide ester or (C1-C4)alkyl esters, amides, preferably thiozolidine-<NUM>-thione amide, maleimide, azide, propargyl, preferably the functional group is azide or maleimide.