IT202100013634A1 - NEW REAGENTS FOR LIGHT MICROSCOPY - Google Patents

Description

Description of Patent for Industrial Invention having the title:

?NEW REAGENTS FOR OPTICAL MICROSCOPY?.

DESCRIPTION

Technical field

The present invention ? relating to the field of immunofluorescence microscopy and, in particular, to new immunofluorescent probes to be used in this technique.

Known technique

Immunofluorescence microscopy? a technique that allows you to view specific protein antigens in tissues or cells through the use of antibodies labeled with fluorescent molecules, also called immunofluorescent probes, exploiting the ability? of antibodies to specifically recognize and bind to a protein epitope outside or inside cells. There are two main methods for making the antigens of interest visible: direct and indirect immunofluorescence.

In direct immunofluorescence, antibodies directed against an antigen of interest and already? conjugated to a fluorophore they are reacted with a section of tissue or with cells where they bind specifically to the protein of interest. In the case of indirect immunofluorescence, on the other hand, the specific antibody for the antigen of interest is made to react with the sample to be analyzed and the binding sites between the antigen and the antibody are highlighted through the use of a second antibody, which binds to the constant portion of the primary antibody and ? conjugated to a fluorophore. In both cases, the stained sample is then viewed using a fluorescence microscope or confocal microscope. In particular, the sample ? illuminated with a beam of light of a specific wavelength that is absorbed by the fluorophore, which re-emits light of a longer wavelength than that of the absorbed light. The exciting light is separated from the emitted light, thanks to the use of a filter and therefore the fluorescent signal is visualized in correspondence with the antigen.

Compared to electron microscopy, this technique has the great advantage of being able to be used in ex vivo systems.

Primary or secondary antibodies commonly used in immunofluorescence are conjugated to the fluorophore via amino or sulfhydryl groups of side chain lysine or cysteine residues. However, binding to lysine residues, which is the most common technique, is used, does not allow to control the exact positioning and the number of fluorophore molecules that bind to the antibody, with consequent obtainment of a heterogeneous mixture of labeled antibodies (Wang et al, Protein Sci 2005, 14: 2436). Furthermore, the labeling of lysine residues near the of the binding site with the antigen pu? lead to steric hindrance of the antigen binding site and consequent loss of the ability to of binding of the antibody.

Another problem encountered in the labeling of antibodies, especially monoclonal ones, is the non-specific binding of the fluorophore to hydrophobic portions of the antibody. Consequently, when the labeled antibody is used in tissue or cell analysis techniques for microscopy, the non-specific binding is hydrolysed and the fluorophore diffuses into the incubation solution, generating a background signal leading to loss of specificity.

In order to overcome these drawbacks, site-specific labeling techniques have been developed through the introduction into the antibody of cysteines with free sulfhydryl groups at the extremity. C terminus, but are located remote from the antigen binding site.

Among the known marking techniques, the operating pH plays a fundamental role in the control of the selectivity in the conjugation of succinimide esters with terminal amino groups present on the antibody.

In detail, the pH values at which the amino groups present in a protein are divided into quantities? equal in protonated-inert form and in deprotonated-reactive form numerically correspond to the relative value pKn. At the aforementioned pH value the two protonated/deprotonated forms exist in conditions of rapid dynamic equilibrium.

In this regard, the methods of acylation of the terminal amine with respect to the amino groups of lysine residues are currently being studied in order to increase the selectivity of the terminal amine. of conjugation.

As previously mentioned, the amino groups present in an antibody can be grouped into two different types: the terminal ones (only two) and the numerous ?-amino groups of lysines. The pK values of the ?-amino end groups are generally between 7.5 and 13, while the corresponding end groups have an average value around 7.5.

With higher pH values, the majority of the amino groups are indistinctly deprotonated and consequently reactive.

In this condition, although the yield of the reaction is extremely high, the selectivity? of the coupling in respect of the terminal amino groups ? greatly reduced.

Against these conditions, the selectivity? of conjugation towards the terminal amino groups ? strongly limited, and vice versa ? high the possibility of uncontrolled conjugation of the lysine residues present on the side chain of the antibody.

In this regard, the unsolicited conjugations at the level of the ? amino groups of the lysines, certainly present in the protein to be derivatized, are extremely numerous.

It is easy to understand how this problem is only partially resolved to date.

In fact, there is a particularly felt need to have realistic measurements of the amino groups available for conjugation as a function of the pH values at which the antibody ? subjected during the reaction in such a way as to ensure high specificity? of conjugation.

To address the conjugation towards a particular typology of amino groups, in particular the terminal ones, discriminating instead the? Other typology, ie? the groups ? -aminos of lysine, does it mean to raise the selectivity? of the reaction and this is not ? an easy-to-solve problem. In recent years optical microscopy has made significant progress from an instrumental point of view, with a significant increase in performance in terms of lateral resolution. For example, super-resolution microscopes have been developed that exhibit 2 to 10 times the resolution of a regular light microscope. These tools make it possible to use optical microscopy to study subcellular structures, even very small and close together. For example, STED microscopy makes it possible to resolve nanoscopic objects of the order of 30-50 nm, while the various techniques based on stochastic reconstruction achieve a lateral resolution of 10-40 nm. Furthermore, the combination of stochastic and deterministic techniques (MINIFLUX, Hell 2016) currently allows to distinguish particles 1 nanometer large separated by 3 nanometers.

However, the technological advances obtained at the instrumental level have highlighted the great limitations of the fluorescent systems commonly used in immunofluorescence procedures, which have proved inadequate to allow the achievement of the full potential? of modern microscopes.

In fact, although the currently used fluorophores are designed to have optimal photo-physical characteristics, the antibodies to which they are bound have dimensions that are of the same order of magnitude as the sub-micrometric objects under observation. Also, as discussed above, the binding of the fluorophore to the antibody occurs at sites distant from the antigen binding site and therefore prevents the positioning of the fluorescent signal in close proximity to the antigen. of this.

In recent years, attempts have been made to improve the performance of the fluorescent recognition element through the development of alternative systems to antibodies, of smaller dimensions. smaller than these, mainly labeled aptamers and nanobodies.

However, the use of such fluorescent probes has several limitations, including the lack of availability? commercial. Furthermore, although the nanobodies are smaller in size than common immunofluorescence antibodies, they are not It is also possible in this case to obtain the positioning of the fluorophore at a short distance from the epitope. Aptamers, on the other hand, allow labeling a few ?ngstr?m from the epitope, but in turn have the limitation of being able to be used only for extra-cellular epitopes, while most of the epitopes of interest have an intracellular localization.

Is there therefore a need? to develop new immunofluorescent probes that overcome the problems discussed above.

Summary of the invention

The present inventors have developed a technique for the regioselective labeling of antibodies or Fab fragments at the level of the N-terminal amino group of the heavy and/or light chain and have found that antibodies selectively labeled through an amide linkage to molecules comprising groups fluorophores are stable and have homogeneous characteristics in terms of positioning and entity? of binding to the fluorophore.

Furthermore, the present inventors have found that when the fluorophore exhibits certain characteristics of flexibility? and lipophilicity, the antibodies or Fab fragments labeled according to the invention maintain avidity? for the antigen and are suitable for use in microscopy techniques, where they allow to obtain a surprisingly high level of resolution, thanks to the proximity? between the antigen binding site and the fluorophore and the selectivity? of fluorophore binding.

Also, in the case of Fab fragments, how will it be? demonstrated in the experimental examples, the inventors have found that the reduced dimensions of the conjugates obtained, about one sixth of those of conventional probes, not only generate a high density? and specificity? of staining, but allow localization of the probe in cellular regions where the conventional marker has no access, for example in cellular midbodies.

Therefore, a first object of the present invention ? an antibody or Fab, preferably a Fab, capable of emitting fluorescence upon light stimulation, wherein the amino group of the N-terminal amino acid of the light chain and/or the N-terminal amino acid of the heavy chain ? linked by an amide linkage to a molecule comprising a fluorophore group A, wherein said linkage constitutes at least 70% of the total binding of said molecule to said antibody or Fab.

A second object of the present invention? the use of an antibody or a Fab in accordance with the first object of the invention, as an immunofluorescent probe, preferably for direct immunofluorescence, in fluorescence microscopy techniques, preferably super resolution confocal fluorescence microscopy.

A third object of the present invention ? a method for the preparation of the antibody or Fab in accordance with the first object of the invention comprising the reaction between an antibody or Fab fragment and a molecule comprising a fluorophore group A and a group ?COOB, in which group B ? a group having electrophilic characteristics capable of reacting with the nucleophilic groups of proteins, preferably selected from: 1-pyrrolidinyl-2,5-dione and 1-pyrrolidinyl-3-suflonyl-2,5-dione having formula respectively

Description of the figures

Figures 1A and 1Abis represent the images of COS7 cells obtained by confocal microscopy following incubation with rabbit polyclonal Fab directed against the BARS protein prepared as described in example 1a and labeled with fluorophore CF568 as described in example 3a (INV ), in which the succinimide ester ? 1-pyrrolidinyl, 2,5-dione and 1-pyrrolidinyl, 3-suflonyl 2,5-dione, respectively.

Figures 1B and 1Bbis represent the images of COS7 cells obtained by confocal microscopy following incubation with rabbit polyclonal Fab directed against the BARS protein prepared as described in example 1a and labeled with fluorophore AF647 as described in example 3b (INV ), in which the succinimide ester ? 1-pyrrolidinyl, 2,5-dione and 1-pyrrolidinyl, 3-suflonyl 2,5-dione, respectively.

Figures 2A and 2Abis represent the images of Hela cells obtained by confocal microscopy following incubation with mouse monoclonal Fab directed against the protein alpha tubulin prepared as described in example 1b and labeled with fluorophore CF568 as described in example 3c ( INV), in which the succinimide ester ? 1-pyrrolidinyl, 2,5-dione and 1-pyrrolidinyl, 3-suflonyl 2,5-dione, respectively.

Figures 2B and 2Bbis represent the images of Hela cells obtained by confocal microscopy following incubation with rabbit polyclonal Fab directed against the AKAP9 protein prepared as described in example 1c and labeled with fluorophore CF647 as described in example 3e (COMPARISON ), in which the succinimide ester ? 1-pyrrolidinyl, 2,5-dione and 1-pyrrolidinyl, 3-suflonyl 2,5-dione, respectively. Figures 3A and 3B represent the images obtained by confocal microscopy of Hela cells following incubation with mouse monoclonal Fab directed against the protein alpha tubulin labeled with both fluorophore CF568 and with the control fluorophore AlexaFluor488 (AF488). The image of figure 3A ? was acquired at a wavelength included in the range 490 to 450nm at which the fluorescence emission comes from only the control AF488 fluorophore. The image of figure 3B ? was acquired at a wavelength included in the range from 575 to 620nm at which the fluorescence emission comes from the CF568 fluorophore alone.

Figures 4A and 4B represent the images obtained by confocal microscopy of Hela cells following incubation with mouse monoclonal Fab directed against the alpha tubulin protein labeled with both fluorophore CF568 and with the control fluorophore AlexaFluor488 (AF488). The image of figure 4A ? was acquired at a wavelength included in the range 490 to 450nm at which the fluorescence emission comes from only the control AF488 fluorophore. The image of figure 4B ? was acquired at a wavelength included in the range from 575 to 620nm at which the fluorescence emission comes from the CF568 fluorophore alone.

Figures 5A and 5B represent the images obtained by confocal microscopy of Hela cells following incubation with mouse monoclonal Fab directed against the alpha tubulin protein labeled with both the CF568 fluorophore and the AlexaFluor488 control fluorophore (AF488). The image of figure 5A ? was acquired at a wavelength included in the range 490 to 450nm at which the fluorescence emission comes from only the control AF488 fluorophore. The image of figure 5B ? was acquired at a wavelength included in the range from 575 to 620nm at which the fluorescence emission comes from the CF568 fluorophore alone.

Figures 6A and 6B represent the images obtained by confocal microscopy of Hela cells following incubation with mouse monoclonal Fab directed against the alpha tubulin protein labeled with both fluorophore AF647 and with the control fluorophore AlexaFluor488 (AF488).

The image of figure 6A ? was acquired at a wavelength included in the range 490 to 450nm at which the fluorescence emission comes from only the control AF488 fluorophore. The image of figure 6B ? was acquired at a wavelength included in the range from 660 to 700nm at which the fluorescence emission comes from the AF647 fluorophore alone.

Figures 7A and 7B represent the images obtained by confocal microscopy of Hela cells following incubation with mouse monoclonal Fab directed against the alpha tubulin protein labeled with both the AF647 fluorophore and the AlexaFluor488 control fluorophore (AF488). The image of figure 7A ? was acquired at a wavelength included in the range 490 to 450nm at which the fluorescence emission comes from only the control AF488 fluorophore. The image of figure 7B ? was acquired at a wavelength included in the range from 660 to 700nm at which the fluorescence emission comes from the AF647 fluorophore alone.

Figures 8A, 8B, 8C, 8D represent a comparison of images acquired by STORM technique on an advanced optical platform in SMLM regime (Single Molecule Localization Microscopy) of a microtubule labeled with fluorescent probes in accordance with the present invention.

Figure 9 represents a comparison between the resolution limit obtained by using probes according to the present invention and conventional probes. In detail, ? the mean diameter of a microtubule was measured.

The section of the microtubule ? been quantified by measuring the intensity profile? (i.e. number of locations) along a segment running perpendicular to the microtubule.

? been compared to the value of? amplitude in half? of the height of this profile, comparing both marking methods.

Figure 10 represents a graph elaborated with respect to an analysis carried out on a Fab characterized by a terminal amino group with pKa=7.8 and by a ?-amino group with pKa=8.89, potentially competing with each other.

The above figure shows the respective titration curves from which ? It is possible to evaluate the fractions of both terminal and ?-amino free amino groups at each pH value. Contextually ? represented a further curve representing the variations of the NH2-ter/NH2-lysine ratio and through which ? selectable an optimal pH for its maximization.

It is clear that the ratio between reactive end groups and amino groups of lysines tends to increase as the pH values decrease. Definitions

The flexibility index? Kier ((?), Kier molecular flexibility index, KierFlex,) ? a measure of flexibility? molecular, defined numerically by the parameters A, <1>kalfa and <2>kalfa according to the following relationship:

wherein <1>kalfa and <2>kalfa are parameters which include the contribution of rigid connections, ie cycles, unsaturations or heteroatoms, which do not allow the free rotation of the molecular planes along the interatomic axis of the elements constituting the molecule; And

A represents the number of ?corners? that is? of atoms of the molecule.

The value of each of the above descriptors ? calculated starting from simplified molecular structures, i.e. represented only by carbon-carbon connections, or carbon-heteroatom, thus excluding all carbon-hydrogen connections. Such structures, often defined in the literature as ?H-depleted molecular graph? (Kier, (1989), Quant.Struct.-Act.Relat. 8, 221-224), consist of number A of ?angles? (in vertex literature) and P of ?edges? (in literature paths).

A is determined by counting the angles of a molecular structure, as shown below:

The P value is used to calculate the indices <1>kalfa and <2>kalfa.

It is defined as ?degree? (in literature m) the number of consecutive connections considered as ?edge?. The index of k ? of degree 1 (m=1) if ? comprised only between 2 vertices, of degree 2 if it connects 3 vertices, and cos? Street. The degree (m) of an index (k) ? therefore referring to the maximum ?path? of the edge P, and is expressed as a superscript to the left.

As an example? the calculation of <m>P referred to the structures of 2,methyl-pentane and linear pentane is shown below:

The indices <1>kalfa and <2>kalfa respectively of first and second order, are normalized on the alpha factor, which allows to include the contribution of possible steric restrictions, different hybridizations (and therefore stiffness?

molecular) and stereo-electronic effects. The alpha values are tabulated for each carbon hybridization, and for the common heteroatoms included in the organic molecules, as shown below:

The <1>kalfa and <2>kalfa values are calculated as follows:

In the formula chosen for the definition of flexibility? of fluorophores, the kappa indices considered are only of first and second degree.

The logarithm of the water/octanol partition coefficient (AlogP) ? an approach for the predictive calculation of the water/octanol partition coefficient of small organic molecules.

The method allows to obtain the hydrophilicity (AlogP less than zero) and hydrophobic (or lipophilicity, ALogP greater than zero) value of a chemical structure, and uses the atomic contributions through a parametric model.

The logP value of each molecule is calculated according to the following formula:

and is called AlogP after the author?s name if calculated using the parameters reported in table 1 published in

G.M.J. Comput. Chem. 1986, 7, 565-577.

The partition coefficient of the molecule is considered as the sum of the contribution given by each atom of the structure, calculated on the basis of specific parameters. These parameters are listed in

mentioned above and determined on the basis of all the possible combinations of the chemical environment of each atom. So, the polarity? of the same atom is variable in function of the atoms to which there? tied up, why? every possible combination of connections between atoms to determine the numerical value of the parameter relating to each atom making up the molecule. The parameterized atoms are: H, C, O, S, N, Si, B, P, Se, F, I, Br, Cl. These parameters, relating to the AlogP calculation method, are extrapolated from the correlation of different partition coefficients experimentally measured on various model molecules. According to this method, the simulated value of logP, ie AlogP, pu? be determined uniquely for each molecule of interest, regardless of whether the calculated value is more? or less coinciding with the real coefficient.

TPSA (Topological Polar Surface Area) defines the polar surface area of a molecule using a computational calculation method.

TPSA ? based on a cumulative calculation of all the topological contributions of each molecular fragment; such contributions were described in Chem.Pharm.Bull. 1992, 40, 127-130 and

Chem.Pharm.Bull. 1994, 42, 976-978, and are summarized in the following table in which the PSA contrib indicates the topological contribution values of the indicated molecular fragments.

SURFACE CONTRIBUTIONS OF POLAR ATOMS

to define TPSA it is sufficient to calculate the sum of each contribution, according to the following formula:

TPSA therefore represents the sum of the surface area of the atoms of a molecule with an absolute value of partial charges greater than or equal to 0.2 (probably oxygen and nitrogen with any hydrogen attached to them) exposed to a solvent and ? expressed in ?? (?ngstrom<2>).

3D POLAR SASA defines the surface area of the molecule that ? accessible to the solvent.

This parameter provides the degree of hydration of the molecule which translates into greater stabilization of the conjugate, less tendency to aggregate because? hydrated and better emissive behavior due to higher solvation. Furthermore, the decreased tendency to interact with parts of the fragment also translates into a significant increase in greed why? the fluorophores do not collapse on the recognition regions of the FAB.

The algorithm for calculating the 3D polar SASA value allows to obtain the surface fraction of the molecule that cannot be included, due to steric issues, in the sphere of hydration of the solvent. The calculation is performed based on the Wan der Wals surfaces of both the solvent and the solute. The contribution of atomic fragments ? tabulated in Ferrara P. et al., (2002), PROTEINS: Structure, Function, and Genetics, 46, 24-33.

For the calculation of the polar 3D SASA value, the sum of the atomic contributions to the solvation of the molecule is considered. For the calculation of the numerical value, the solvation area is made to coincide with the effective energy value for this solvation, having made the initial assumption that these values are linearly dependent. The value is calculable

using the following algebraic expression:

where ?i ? the coefficient of the contribution ?weighted? of the atom i (see table below) and Ai(r) ? the fragment of area with which that atom contributes.

Ai(r) is calculated with the following expression:

It is calculated according to the following expression

?:

0 if

or

in all other cases.

Rprobe = 1.4 ?

pij = 0.8875 if the atoms under consideration are covalently bonded or

pij = 0.3516 in all other cases.

Rmin, Ri, pi and ?i are tabulated in Ferrara P. et al., cited above.

By calculating the formulas above, ? You can define the 3d polar SASA parameter uniquely.

For the purposes of the present invention, the flexibility index? Kier (?) and polar surface area (TPSA) were calculated with Spartan?15 software ( , Pipeline Pilot 2016 Biovia, MOE, CCG).

For the calculation of the total number of hydrogen bond acceptor atoms (Num_H_Acceptors) yes ? used Pipeline Pilot software (Dassault Syst?mes BIOVIA, Pipeline Pilot, R2 version, San Diego: Dassault Syst?mes, 2018 - Warr W.A. (2012) Scientific workflow systems: pipeline Pilot and KNIME. J. Comput. Aided Mol. Des ., 26, 801?804).

At this parameter? associated with the polarity? of the molecule, and therefore its solubility? in the aqueous medium, an important aspect given the reaction conditions described below.

The term ?fluorophore group? according to the present invention indicates an atom or a functional group capable of absorbing energy at a specific wavelength and of re-emitting fluorescence. The different types of fluorophores are characterized by different absorption and emission spectra

The term ?Fab? or ?Fab antibody fragment? according to the present invention means a proteolytic fragment of an antibody molecule comprising an entire light chain coupled to a heavy chain fragment containing the variable antigen-binding domain and a portion of the constant domain. Preferably, the Fab fragment according to the invention is monovalent, then ? a fragment that does not include the hinge region or can? result from the rupture of the sulfide bonds in a fragment comprising the hinge region and have one free sulfide group and only one binding site per antigen.

Different Fab fragments can be obtained depending on the proteolytic enzyme used for the digestion of the antibodies from which they derive.

For example, digestion of an antibody with papain generates monovalent fragments, digestion of the same with pepsin generates bivalent fragments, and digestion with ficin generates bivalent or monovalent fragments depending on the reaction conditions. For the purposes of the present invention the disulfide bonds of the divalent fragments are preferably reduced in order to obtain a monovalent Fab.

Description of the Invention

As described above, the present inventors have developed a method for the regioselective labeling of the N-terminal amino group of antibodies or Fab antibody fragments with a molecule comprising a fluorophore group. This method allows to obtain an immunofluorescence probe stable over time and with homogeneous characteristics in terms of positioning and intensity? of the fluorophore signal. The binding of the fluorophore at the level of the N-terminal amino groups allows its positioning at a very close distance, in the order of 1-2 nm, from the antigen of interest. Despite this proximity, the inventors have found that selecting the fluorophore and linker with specific characteristics prevents the fluorophore from interfering with the binding of the antibody or Fab to the antigen and keeps the avidity intact. binding to the epitope.

The antibodies and the labeled Fabs obtained through the method developed by the inventors can be used as direct fluorescent probes in optical microscopy, allowing to obtain a more high resolution compared to probes used in conventional direct or indirect fluorescence techniques. In particular, this their property? ? particularly advantageous in the case of super-resolution optical microscopy where the fluorescent probes available to date have numerous functional limitations and remain inadequate to obtain the full achievement of the potential? of microscope resolution. Furthermore, in the case of labeled Fabs, the small size of the probe object of the invention allows its localization in cellular regions where the conventional marker has no access, for example in cellular midbodies.

Therefore, a first object of the present invention ? an antibody or a Fab antibody fragment, preferably a Fab antibody fragment in which at least one amino group of the N-terminal amino acid of the light chain and/or of the N-terminal amino acid of the heavy chain ? linked by an amide linkage to a molecule comprising a fluorophore group A, wherein said linkage constitutes at least 70% of the total binding of said molecule to said antibody or Fab.

Preferably, said fluorophore group A ? the only fluorophore group bound to said antibody or Fab.

Preferably, the aforementioned bond ? selective at the level of the N-terminal amino groups, in particular it constitutes at least 80%, 85%, 90%, 95% or 98% of the total binding of said molecule with said antibody or Fab. The lack of non-specific binding sites or easily hydrolyzable binding prevents the formation of non-specific fluorescence when the labeled antibody or Fab is brought into contact with the test sample and increases the sensitivity of the label. and the? intensity? of the fluorescent signal that is concentrated near? of the antigen.

The aforementioned Fab fragment preferably derives from the digestion of an antibody with papain, pepsin or ficin, plus? preferably papain or ficina. The Fab fragment? preferably monovalent what? has only one antigen binding site.

The antibody or Fab according to the invention can be/derive from a monoclonal or polyclonal antibody, preferred among which ? a monoclonal antibody.

According to a preferred embodiment, the above polycolon antibody ? a rabbit polyclonal antibody, preferably of the IgG2b type and the aforementioned monoclonal antibody ? a mouse antibody, preferably of the IgG1 type.

The present inventors have found that the presence of a spacer group (linker) between the antibody/Fab and the fluorophore and/or the selection of fluorophores which have specific characteristics allows the avidity to be kept intact. of the antibody with the antigen and therefore to obtain fluorescent probes with high signal efficiency.

Preferably, in the antibody or Fab fragment according to the invention, said amino group of the N-terminal amino acid of the light chain and/or of the N-terminal amino acid of the heavy chain ? linked by an amide bond to a Z group of the formula:

-CO-(W)n-A

in which:

n ? 0 or 1,

TO ? a fluorophore group,

W? selected from C1-C10 alkyl, -CH2CH2(CH2CH2O)mCH2CH2-X-, or -CH2-CH2OCH2CH2-Z-Y-CH2CH2OCH2CH2-X-,

in which m ? between 0 and 8, X ? chosen between CO and NH, Y ? selected from CH2CH2OH, pyrimidine, methoxyphenyl, or a compound of formula

Furthermore, the present inventors have identified that particular characteristics of the Z group, in this case consisting of the fluorophore molecule A and the -CO-(W)n linker, allow to keep the avidity particularly high. of the antibody or antibody fragment against the antigen, despite the proximity of the fluorophore to the binding site.

Preferably, group Z has a total number of hydrogen bond acceptor atoms (Num_H_Acceptors) of the molecule between 8 and 16, preferably between 10 and 15.

Preferably, group Z also has a logarithm of the water/octanol partition coefficient (AlogP) between 3 and -5.

Furthermore, group Z preferably has a surface exposed to the solvent (3DpolarSASA, solvent accessible surface area) equal to or greater than 180 ??, preferably between 200 and 550 ?? and/or total polar surface area (TPSA) equal to or greater than 180 ??, preferably between 180?? and 500 ??, more? preferably between 190?? and 300 ??.

It is not excluded that group Z presents the aforementioned characteristics in combination with each other.

Therefore, according to a particularly preferred embodiment, group Z has:

- Num_H_Acceptors between 10 and 15;

- AlogP between 3 and -5;

- 3DpolarSASA between 200 and 550 ??

- TPSA including 190?? and 300 ??.

Furthermore, it is not excluded from the scope of this discussion that group Z presents, alternatively to the descriptors indicated above, the following characteristics:

- flexibility index? Kier ((?), KierFlex) between 7.5 and 15, preferably between 10 and 12,

- logarithm of the water/octanol partition coefficient (AlogP) less than or equal to 0, preferably between 0 and -10

- surface exposed to the solvent (3DpolarSASA, solvent accessible surface area) equal to or greater than 300 ??, preferably between 400 ?? and 450 ??, and/or

- total polar surface area (TPSA, total polar surface area) equal to or greater than 200 ??, preferably between 200?? and 220 ??.

Therefore, according to an alternative preferred embodiment, in the above antibody or Fab fragment, the Z group exhibits

- KierFlex between 10 and 12;

- AlogP between 0 and -10;

- 3DpolarSASA between 400 ?? and 450 ?? And

- TPSA between 200 ?? and 220 ??.

Preferably, in the aforementioned antibody or Fab, group A has a molar extinction coefficient (?) not lower than 80,000 and quantum yield not lower than 60%.

Preferably, in the above antibody or Fab, the fluorophore group A is chosen from the following groups:

- CF568 fluorophore

- fluorophores derived from xanthene or carbocyanins, preferably of the formula:

in which R? a group chosen from -OPO3H2 and -OH and R? ? chosen between -OSO3- and -F;

- 2?[(1E,3E)?5?[(2Z,3S)?3?(5?methoxy?5?oxopentyl)?3?methyl?5?sulfo?1?( 3?sulfopropyl)?2,3 ?dihydro?1H?indol?2?ylidene]penta?1,3?dien?1?yl]?3, 3?dimethyl?5?sulfo?1?(3?sulfopropyl)?3H?indol?2?yl ( AF647), having structure

- 2-(2,2,10,10-tetramethyl-4,8-bis(sulfonatomethyl)-2,10-dihydro-1H-pyrano[3,2-g:5,6-g']diquinolin-11- ium-6-yl)terephthalate (AF568), both regioisomers, having the structure:

- 2-[(1E,3Z)-3-[3-(3-carbamoylpropyl)phenyl]-5-[(2Z)-3,3-dimethyl-5-sulfonato-1-(3-sulfonatopropyl)-2, 3-dihydro-1H-indol-2-ylidene]penta-1,3-dien-1-yl]-5-chloro-3,3-dimethyl-7-(3-sulfonatopropyl)-3H-pyrrolo[2,3 -b]pyridin-7-ium;

- 2-[(E)-2-[(3E)-3-{2-[(2Z)-1-(5-carbamoylpentyl)-3,3-dimethyl-5-sulfonato-2,3-dihydro-1H -indol-2-ylidene]ethylidene}-2-(4-sulfonatophenoxy)cyclohex-1-en-1-yl]ethenyl]-3,3-dimethyl-1-(4sulfonatobutyl)-3H-indol-1-ium- 5-sulfonate;

- (1P)-2-(6-amino-3-iminiumyl-4,5-disulfonato-3H-xanthen-9-yl)-4-carbamoylbenzoate;

- 1-(5-carbamoylpentyl)-3,3-dimethyl-2-[(1E,3E)-5-[(2Z)-1,3,3-trimethyl-2,3-dihydro-1H-indol-2 -ylidene]penta-1,3-dien-1-yl]-3H-indol-1-ium;

- 2-[(1E,3E)-5-[(2Z)-1-(5-carbamoylpentyl)-3,3-dimethyl-5-sulfonato-2,3-dihydro-1H-indol-2-ylidene]- 3-methylpenta-1,3-dien-1-yl]-3,3-dimethyl-1-(4-sulfonatobutyl)-3H-indol-1-ium-5-sulfonate;

- 2-[(1E,3E)-5-[(1R,2Z)-1-(5-carbamoylpentyl)-3,3-dimethyl-5-sulfonato-2,3-dihydro-1H-indol-1-ium -2-ylidene]penta-1,3-dien-1-yl]-3,3-dimethyl-1-(4-sulfonatobutyl)-3H-indol-1-ium-5-sulfonate;

- 2-tert-butyl-4-[(1E)-3-[(2E)-1-(5-carbamoylpentyl)-3,3-dimethyl-5-sulfonato-2,3-dihydro-1H-indol-2 -ylidene]prop-1-en-1-yl]-9-ethyl-8,8-dimethyl-8H,9H-1?4-chromeno[7,6-b]pyridin-1-ylium;

- 4-[(1E)-3-[(2Z,3S)-3-(5-carbamoylpentyl)-3-methyl-6-sulfonato-1-(3-?)-2,3-dihydro-1H-indol -2-ylidene]prop-1-en-1-yl]-8,8-dimethyl-2-phenyl-6-(sulfonatomethyl)-9-(3-sulfonatopropyl)-8H,9H-1?4-chromeno[ 7,6-b]pyridin-1-ylium;

- 2-tert-butyl-4-[(1E,3E)-5-[(2E)-1-(5-carbamoylpentyl)-3,3-dimethyl-5-sulfonato-2,3-dihydro-1H-indol -2-ylidene]penta-1,3-dien-1-yl]-7-(diethylamino)-1 ?4-chromen-1-ylium;

- N,N-dimethyl-4-[(2E)-1,5,5-tris[4-(dimethylamino)phenyl]penta-2,4-dien-1-ylidene]cyclohexa-2,5-dien-1 -iminium;

- 2-[(1E,3E)-5-[(2Z)-1-(5-carbamoylpentyl)-3,3-dimethyl-5-sulfonato2,3-dihydro-1H-indol-2-ylidene]penta-1 ,3-dien-1-yl]-3,3-dimethyl-1-(4-sulfonatobutyl)-3H-indol-1-ium;

- 28R)-16-{2-[(3-carbamoylpropyl)(methyl)carbamoyl]phenyl}-3-oxa-9??,23-diazaheptacyclo[17.7.1.1?,?.0?,??.0? ,??.0??,??.0??,??] octacosa-1(27),2(17),4,9,13,15,18-heptaen-9-ylium;

- 2-[(1E,3E)-5-[(2Z)-1-(5-carbamoylpentyl)-3,3-dimethyl-2,3-dihydro-1H-indol-2-ylidene]penta-1,3 -dien-1-yl]-1,3,3-trimethyl-3H-indol-1-ium;

- 2-[(1E,3E)-5-[(1Z,2R)-1-(5-carbamoylpentylidene)-3,3-dimethyl-2,3-dihydro-1H-1??-indol-1-ylium -2-yl]penta-1,3-dien-1-yl]-3,3-dimethyl-1-(4-sulfonatobutyl)-3H-indol-1-ium;

- 4-carbamoyl-2-[13-(dimethylamino)-5-(dimethyliminiumyl)-2,2-dimethyl-2-silatricyclo[8.4.0.0?,?]tetradeca-1(10),3,6,8, 11,13-hexaen-9-yl]benzoate; And

- 2-[(1E,3E)-5-[(2Z)-1-(5-carbamoylpentyl)-3,3-dimethyl-5-sulfonato-2,3-dihydro-1H-indol-2-ylidene]penta -1,3-dien-1-yl]-3,3-dimethyl-1-(4-sulfonatobutyl)-3H-indol-1-ium;

- (7S,17R)-12-[4-(methoxycarbonyl)phenyl]-7,8,8,16,16,17-hexamethyl-2-oxa-6,18-diazapentacyclo[11.7.0.<03.11 >.0<5,9>.0<15,19>]icosa-1(13),3,5,9,11,14,19-heptaen-6-ium-4,20-disulfonate(AF532), having structure

- [10,10,22,22-tetramethyl-20-(sulfomethyl)-16-{2,3,4,5-tetrafluoro-6-[(4methoxy-4-oxobutyl)(methyl)carbamoyl]phenyl}-3 -oxa-9lambda4,23-diazaheptacyclo[17.7.1.1<5,9>.0<2,17>.0<4,15>.0<23,27>.0<13,28>]octacosa-1, 4,9(28),11,13,15,17,19(27),20-nonaen-12-yl]methanesulfonic acid (ASred), having the structure

- 2?({3?[12,20?bis(hydroxymethyl)?10,10,22,22?tetramethyl?3?oxa?9lamb da4,23?diazaheptacyclo[17.7.1.1<5,9>.0<2 ,17>.0<4,15>.0<23,27>.0<13,28>]octawhat?1,4,9( 28),11,13,15,17,19(27),20 ?onaen?16?yl]?2,5,6?trifluoro?4?[(4?methoxy-4?oxobutyl)(methyl)carbamoyl]phenyl}sulfanyl)ethane?1?sulfonic acid (S635), having the structure

- 3-(5,5-difluoro-7-(thiophen-2-yl)-5H-4?5,5?5-dipyrrolo[1,2-c,1,2-f][1,3,2 ]diazaborinin-3-yl)propanoic acid (BODIPY_558-568), having the structure:

- 4-(11-ethyl-2,2-dimethyl-4-(sulfomethyl)-3,4,8,9,10,11-hexahydro-2H-1?5-pyrido[3',2':6, 7]chromeno[3,2-g]quinolin-1-yl)butanoic acid (ATTO_665) having the structure:

- 3-(5,5-difluoro-7-((1E,3E)-4-phenylbuta-1,3-dien-1-yl)-5H-4?5,5?5-dipyrrolo[1,2- c,1,2-f][1,3,2]diazaborinin-3-yl)propanoic acid (BODIPY_581-591), having the structure:

- 5-(5,5-difluoro-7,9-bis(trifluoromethyl)-5H-4?5,5?5-dipyrrolo[1,2-c,1,2-f][1,3,2] diazaborinin-3-yl)pentanoic acid (BODIPY_FL_C5), having the structure:

- (2E)-2-((2E,4E)-5-(3-(5-carboxypentyl)-1,1-dimethyl-6,8-disulfo-1H-benzo[e]indol-3-ium-2 -yl)penta-2,4-dien-1-ylidene)-3-ethyl-1,1-dimethyl-6-sulfo-2,3-dihydro-1H-benzo[e]indole-8-sulfonate (CY_5- 5), having structure:

- 4-((2-(3,6-bis((3-sulfopropyl)amino)xanthen-9-yl)benzoyl)(methyl)amino)butanoic acid (DY-530), having the structure:

- 4-((4,5-dichloro-2-(2,7-dimethyl-3,6-bis((3-sulfopropyl)amino)xanthen-9-yl)benzoyl)(methyl)amino)butanoic acid (DY -546), having structure:

- 6-((2E)-2-((2E)-3-(1-(2-methoxyethyl)-3,3-dimethyl-6-sulfo-3H-1?5-indol-2-yl)prop- 2-en-1-ylidene)-3,3-dimethyl-5-sulfo-2,3-dihydro-1H-indol-1-yl)hexanoic acid (DY-548P1), having the structure

- 6-((2E)-2-((2E)-3-(1-(2-methoxyethyl)-3,3-dimethyl-6-sulfo-3H-1?5-indol-2-yl)prop- 2-en-1-ylidene)-3-methyl-5-sulfo-3-(3-sulfopropyl)-2,3-dihydro-1H-indol-1-yl)hexanoic acid (DY-549P1), having the structure:

- 6-((5Z)-5-(2-((4Z)-2-tert-butyl-7-(diethylamino)chromen-4-ylidene)ethylidene)-2,4,6-trioxo-3-(3 -sulfopropyl)-1,3-diazinan-1-yl)hexanoic acid (DY-550), having the structure:

- 2-(3,6-bis(dimethylamino)xanthen-9-yl)-5-((5

carboxypentyl)carbamoyl)benzoic acid (DY-554), having the structure:

- 2-(3,6-bis(diethylamino)xanthen-9-yl)-5-((5-carboxypentyl)carbamoyl)benzoic acid (DY-555), having the structure:

- 5-((5-carboxypentyl)carbamoyl)-2-(3-(diethylamino)-6-(ethyl(3-sulfopropyl)amino)xanthen-9-yl)benzoic acid (DY-556), having the structure:

.

- 2-(3,6-bis(ethyl(3-sulfopropyl)amino)xanthen-9-yl)-5-((5-carboxypentyl)carbamoyl)benzoic acid (DY-557), having the structure:

- 6-(((4-(3,6-bis(diethylamino)xanthen-9-yl)-3-sulfophenyl)sulfonyl)amino)hexanoic acid (DY-560), having the structure:

- 4-(pyrido[1,2,3-ij]quinolizino[9',1':6,7,8]chromeno[3,2-g]quinolin-9-yl)benzene-1,3-dicarboxylic acid (DY-580), having structure:

- 5-((2-(9-(ethyl(3-sulfopropyl)amino)-2,2,4-trimethyl-1-(3-sulfopropyl)chromeno[3,2-g]quinolin-6-yl)benzoyl )(methyl)amino)pentanoic acid (DY-585), having the structure:

- 6-(((3-methyl-4-(pyrido[1,2,3-ij]quinolizino[9',1':6,7,8]chromeno[3,2-g]quinolin-9-yl )phenyl)sulfonyl)amino)hexanoic acid (DY-590), having the structure:

- 3-(2-(2-(((4-(14,15-diethyl-4?5,14?5-pyrido[1,5,6-ij]pyrido[3',2':6,7 ]chromeno[2,3-f]quinolin-9-yl)-3-sulfophenyl)sulfonyl)amino)ethoxy)ethoxy)propanoic acid (DY-591), having the structure:

- 4-(1,11-bis(3-(aminosulfonyl)propyl)-2,2,10,10-tetramethyl-4,8-bis(sulfomethyl)pyrido[3',2':6,7]chromeno[ 3,2-g]quinolin-6-yl)benzene-1,3-dicarboxylic acid (DY-594), having the structure:

- 5-(((2-(1,11-bis(3-(aminosulfonyl)propyl)-2,2,10,10-tetramethyl-4,8-bis(sulfomethyl)pyrido[3',2':6 ,7]chromeno[3,2-g]quinolin-6-yl)phenyl)(hydroxy)methyl)(methyl)amino)pentanoic acid (DY-605), having the structure:

- 6-(5-((E)-2-(2-tert-butyl-7-(diethylamino)chromen-4-yl)ethenyl)-2,4,6-trioxo-3-(3-sulfopropyl)- 1,3-diazine-1-yl)hexanoic acid (DY-610), having the structure:

- 6-((2E)-2-((2E)-3-(7-amino-2-tert-butylchromen-4-yl)prop-2-en-1-ylidene)-3,3-dimethyl-5 -sulfo-2,3-dihydro-1H-indol-1-yl)hexanoic acid (DY-615), having the structure:

- 4-((2E)-2-((2E)-3-(2-tert-butyl-7-(diethylamino)chromen-4-yl)prop-2-en-1-ylidene)-3-methyl- 5-sulfo-1-(3-sulfopropyl)-2,3-dihydro-1H-indol-3-yl)butanoic acid (DY-630), having the structure:

- 6-((2E)-2-((2E)-3-(2-tert-butyl-7-(diethylamino)chromen-4-yl)prop-2-en-1-ylidene)-3,3- dimethyl-5-sulfo-2,3-dihydro-1H-indol-1-yl)hexanoic acid (DY-631), having the structure:

- 6-((2E)-2-((2E,4E)-5-(1-(2-methoxyethyl)-3,3-dimethyl-5-sulfo-3H-1?5-indol-2-yl) penta-2,4-dien-1-ylidene)-3-methyl-5-sulfo-3-(3-sulfopropyl)-2,3-dihydro-1H-indol-1-yl)hexanoic acid (DY-648P1) , having structure:

- 4-((2E)-2-((2E,4E,6E)-7-(1,1-dimethyl-3-(4-sulfobutyl)-1H-3?5-benzo[e]indol-2- yl)hepta-2,4,6-trien-1-ylidene)-1,1-dimethyl-1,2-dihydro-3H-benzo[e]indol-3-yl)butane-1-sulfonic acid (ICG) , having structure:

- 2-((Z)-2-((3Z)-3-(2-((2E)-1-(5-carboxypentyl)-3,3-dimethyl-5-sulfo-1,3-dihydro-2H -indol-2-ylidene)ethylidene)-2-(4-(oxidanidylsulfonyl)phenoxy)cyclohex-1-en-1-yl)ethenyl)-3,3-dimethyl-1-(4-(oxidanidylsulfonyl)butyl)- 3H-indol-1-ium-5-sulfonate (IRDye800CW), having the structure:

- ({10,10,22,22?tetramethyl?20?[(phosphonooxy)methyl]?16?{2,3,4,5?tetr afluoro?6?[(4?methoxy?4?oxobutyl)(methyl )carbamoyl]phenyl}?3?oxa?9l ambda4,23?diazaheptacyclo[17.7.1.1<5,9>.0<2,17>.0<4,15>.0<23,27>.0<13 ,28>]octacosa?1,4 ,9(28),11,13,15,17,19(27),20?nonaen?12?yl}methoxy)phosphonic acid (ABBERIOR star 635P), having structure

Particularly preferred among these are groups A selected from the group consisting of: CF568, AF647, AF568, AF532, Asred, S635 and ABBERIOR star 635P pi? preferably between AF647 and AF568.

How will I come? demonstrated in the following experimental examples fluorophores which do not present the values of KierFlex, AlogP, according to the invention indicated above, have proved to be non-functional, such as for example CF647, ATTO590 having structure

- and ACT647 having structure

- and ATTO565, having structure

- and ATTO647N, having structure

As shown, the antibody or Fab according to the present invention have particular advantages when used in fluorescence microscopy techniques. In fact, compared to traditional fluorescent probes, it allows for greater specificity to be obtained. and capacity? of resolution. This makes these reagents particularly suitable for use in super-resolution confocal fluorescence techniques, in which ? You can benefit the most from a dramatic increase in potential resolution (up to 5-fold) due to the decreased distance between the antigen of interest and the fluorophore.

The use of the probe? therefore mainly intended for biomedical research and for theranostics. Otherwise, the use of some commercial immunoconjugates? purely therapeutic, or in any case if intended for diagnostic use? intended for administration for human use, as described in Oncogene (2007; 26, 3734?3744 ). Therefore the use of the antibodies and Fab of the invention of the reagents object of this patent is to be understood exclusively? for ex-vivo use.

Therefore, a second object of the present invention ? the use of an antibody or a Fab in accordance with the first object of the invention as an immunofluorescent probe, preferably for direct immunofluorescence, in fluorescence microscopy techniques, preferably super resolution confocal fluorescence microscopy.

A third object of the present invention ? a method for the preparation of the above antibody or Fab antibody fragment, comprising the reaction between an antibody or Fab fragment and a molecule comprising a fluorophore group A and a -COOB group, and in which the group B is group having electrophilic characteristics capable of reacting with the nucleophilic groups of proteins, preferably selected from 1-pyrrolidinyl-2,5-dione and 1-pyrrolidinyl-3-suflonyl-2,5-dione.

Preferably, this method provides for the reaction between an antibody or Fab fragment and a molecule Z of formula

A-(W)n-CO-O-B,

in which

n ? 0 or 1,

TO ? a fluorophore group,

W? selected from C1-C10 alkyl, -CH2CH2(CH2CH2O)mCH2CH2-X-, or -CH2-CH2OCH2CH2-ZY-CH2CH2O?CH2CH2-X-, wherein m ? between 0 and 8, X ? chosen between CO and NH, Y ? selected from CH2CH2OH, pyrimidine, preferably 2,3 pyrimidin-111, methoxyphenyl, preferably paramethoxyphenyl or a compound of formula

and group B? group having electrophilic characteristics capable of reacting with the nucleophilic groups of proteins, preferably selected from 1-pyrrolidinyl-2,5-dione and 1-pyrrolidinyl-3-suflonyl-2,5-dione.

having formula respectively

And

According to a preferred embodiment, the method of the invention, after the reaction between said antibody and said molecule, provides for a step in which the reaction product is incubated for a period of time of at least 10 minutes, preferably first at a temperature of 37°C preferably for at least 10, preferably between 10 and 30 minutes, then at a temperature of 4°C preferably for a period of at least 8 hours, more? preferably between 8 and 15 hours, pi? preferably 12 hours in the presence of a low molecular weight water-soluble primary amine in a concentration preferably of at least 0.3M, preferably between 0.3M and 5M, more? preferably between 0.3 and 3M, pi? preferably 2M, and a surfactant in quantity? preferably at least 0.1% by volume, preferably between 0.1% and 1% by volume.

Preferably the above method comprises the following steps:

a) Preparation of a solution of said antibody or Fab fragment in a buffer having a pH between 6 and 8, preferably between 6 and 7.5; more preferably 6.5. Preferably, said buffer selected from MES, HEPES and phosphate;

b) Preparation of a solution of a molecule comprising a group, a fluorophore A group and a ?COOB group as defined above, in the minimum quantity? necessary of an anhydrous organic solvent, preferably selected from DMSO and DMF;

c) Mixing of the two solutions prepared in a) and b. Preferably, the two solutions are mixed slowly, in the dark and at a temperature ranging between 20 and 38°C, preferably between 25 and 37°C, preferably for a period of at least 10 minutes, plus? preferably between 15 and 120 minutes, plus? preferably for 60 minutes. The two solutions are preferably mixed in such a ratio that the number of equivalents ratio of the antibody or Fab and that of the molecule containing the fluorophore is between 0.5 and 2, preferably 1.

d) Addition to the solution resulting from step c.) of an aqueous solution, of a water-soluble primary amine, and of a surfactant, preferably in a quantity such as to obtain a final primary amine concentration of at least 0.3M, preferably between 0.3M and 5M, plus? preferably between 0.3 and 3M, pi? preferably of 2M and of surfactant of at least 0.1% by volume, more? preferably between 0.1% and 1% by volume and a pH between 8 and 10, preferably 8.6, and incubation for at least 10 minutes of the mixture as follows? obtained. Preferably, the mixture resulting from stage d) is first incubated at a temperature of 37°C for at least 10, preferably between 10 and 30 minutes, then at a temperature of 4°C preferably for a period of at least 8 hours, more? preferably between 8 and 15 hours, pi? preferably 12 hours.

Preferably, between stage c) and d) or, after stage d), the method further comprises incubating the mixture of stage c) or d) first at a temperature ranging from 20 to 38°C, preferably from 25 to 37°C. ?C, for a period preferably of at least 5 minutes, preferably between 10 and 20 minutes, plus? preferably 15 minutes and then at a temperature between 4? and 20?C for a period preferably of at least 1 hour, plus? preferably between 5 and 15 hours, still ? more preferably 12 hours.

Preferably, in the above method the water-soluble primary amine of step d) is? choice between ethanolamine or glycine.

Preferably, also in combination with the above embodiments, in the above method the surfactant is? a non-ionic surfactant, preferably selected from those containing one or more? unit? polyethylene glycol, such as TritonX and Tween-20; even more preferably ? TritonX, which in addition to the units? of polyethylene glycol also has a hydrophobic fragment, consisting of a hydrocarbon group of the aryl-4-(1,1,3,3-tetramethylbutyl) type.

Preferably, in the above method the mole ratio between the antibody or the Fab fragment and the A-(CH2)n-CO-O-B molecule? of 2 to 4 for an antibody or Fab concentration range of 2.5-4.0 mg/mL.

Preferably, the above method further comprises a step e) in which the antibody or Fab bound to the linker and fluorophore obtained from step d) is purified. Preferably said step e) comprises a purification by chromatography with molecular exclusion, and the identification of the fractions containing the fluorescent conjugate takes place by means of thin layer chromatography acquired by laser scanning. The Fab fragment used in the method according to the present invention can be obtained by techniques well known to the person skilled in the art, for example by digestion of an antibody with papain or ficin.

EXAMPLES

Example 1- Preparation of Fab fragments

1a - Preparation of Fab obtained from a rabbit polyclonal antibody directed to an intracellular protein, C-terminal-binding protein/brefeldin A-ADP ribosylated substrate (CtBP/BARS).

The starting material for the entire procedure was rabbit polyclonal antibody directed against the CtBP/BARS protein and obtained from a commercial formulation (IgG 50-200 ?g, 50% by volume glycerol, 0.1-1 mL of physiological buffer pH 6.80-7.20, bovine albumin 0.1% by weight, sodium azide 5 mM) or from rabbit serum.

The antibody ? was dialyzed against 20 mM phosphate buffer at pH 7.00 and incubated 12 hours at 4°C with 200 mL of sepharose resin conjugated to protein A. The heterogeneous mixture? was mixed by slow inversion and subsequently washed with 5 volumes of 20 mM phosphate buffer pH 7.00. The antibody ? was eluted from the resin by addition of 3 volumes of 100 mM citrate buffer pH 2.80. The eluate? was immediately neutralized during elution with 0.5 volumes of 1 M borate buffer pH 9.00. The product obtained? was dialyzed against 20 mM phosphate buffer pH 6.50 and concentrated to 125 µL. 3.12 ?g of papain immobilized on agarose were suspended in 12.5 ?L of phosphate buffer pH 7.00 containing 25 mM cysteine hydrochloride and 25 mM sodium EDTA, and were incubated with 125 ?L of antibody obtained from the previous process and shaken by inversion for 5 hours at 37?C. The supernatant? was separated from the resin by filtration and incubated for 10 minutes with 200 ?L of protein A. The supernatant ? was again separated from the resin by filtration and dialysed extensively against phosphate buffer and concentrated to 4 mg/mL.

1b - Preparation of Fab obtained from a mouse monoclonal antibody directed against the intracellular protein ?-tubulin.

The starting material for the entire procedure was mouse monoclonal antibody directed to the intracellular protein ?-tubulin of commercial formulation (IgG 50-200 ?g, 50% by volume glycerol, 0.1-1 mL of physiological buffer at pH 6.80-7.20, bovine albumin 0.1% by weight, sodium azide 5 mM) or from ascites fluid. The antibody ? was dialysed against 20 mM phosphate buffer at pH 9.00 with 1.5 M NaCl and incubated 12 hours at 4?C with 200 ?L sepharose resin conjugated to protein A. The heterogeneous mixture was ? was stirred by slow inversion and subsequently washed with 5 volumes of 20 mM phosphate buffer pH 9.00 with 1.5 M NaCl. The antibody ? was eluted from the resin by addition of 3 volumes of 100 mM citrate buffer pH 2.80. The eluate? was immediately neutralized during elution with 0.5 volumes of 1 M borate buffer pH 9.00. The product obtained? was dialyzed against 20 mM phosphate buffer pH 6.50 and concentrated to 125 µL. 3.12 ?g of phycin immobilized on agarose were suspended in 12.5 ?L of phosphate buffer pH 7.00 containing 25 mM cysteine hydrochloride and 25 mM sodium EDTA, and were incubated with 125 ?L of antibody obtained from the previous process and shaken by inversion for 5 hours at 37?C. The supernatant? was separated from the resin by filtration and incubated for 10 minutes with 200 ?L of protein A. The supernatant ? was again separated from the resin by filtration and dialysed extensively against phosphate buffer and concentrated to 4 mg/mL.

1c ? Preparation of Fab obtained from a rabbit polyclonal antibody directed to the protein A-kinase anchoring protein 9 (AKAP9).

Such Fab ? been obtained by applying the modalities? operations described in example 1a.

Example 2 ? Characterization of fluorophores

Kier, AlogP, TPSA and 3DploarSASA and Num_H_Acceptors values of the following fluorophores AF568, AF647, CF568, AF532, Asred, S635, ATTO565, ABBERIOR star 635P, ATTO590, ATTO647, ATTO647N were measured.

Specifically: the? index of flexibility? Kier (?) and polar surface area (TPSA) were calculated with Spartan?15 software (Wavefunction Inc., Pipeline Pilot 2016 Biovia, MOE, CCG); the logarithm of the water/octanol partition coefficient (AlogP) ? was calculated as described above, using the parameters reported in table 1 published in Ghose, A.K.; et al., cited above; the surface area of the molecule accessible to the solvent 3DpolarSASA ? was calculated as described above, using the parameters reported in table I published in Ferrara P. et al., cited above; the number of hydrogen bond acceptor atoms (Num_H_Acceptors ) ? was calculated with the Pipeline Pilot software (Dassault Syst?mes BIOVIA, Pipeline Pilot, R2 version, San Diego: Dassault Syst?mes, 2018 - Warr W.A. (2012) Scientific workflow systems: pipeline Pilot and KNIME. J. Comput. Aided Mol des., 26, 801?804)

For a pi? end of analysis of the properties? physico-chemical properties of the molecules under examination, these were standardized using a multi-step procedure which included: i) in silico construction of the 3-D molecule;

ii) completion of the molecule with a methyl ester (to replace FAB); iii) optimization of the protonation/deprotonation state of ionizable groups at a pH of 7.4, (OPLS3, Schr?dinger, Inc., New York, NY, 2013); iv) conformational analysis with the MCMM method (Chang, G., Guida, W. C., and Still, W. C. (1989) J Am Chem Soc 111, 4379-4386) and selection of the energetically favored confirmator. As? optimized, the molecules were analyzed.

The values obtained for each of the fluorophores are given below:

- CF568: KierFlex 9.17; AlogP -0.219; 3DpPolarSASA 214.048 ?? and TPSA

210.51 ??;Num_H_Acceptors 11

- AF532: KierFlex 6.61; AlogP -0.577; 3DpPolarSASA 246.006 ?? and TPSA 192.68 ??; Num_H_Acceptors 10

- Asred: KierFlex 10.98; AlogP 1.408; 3DpPolarSASA 189.951 ?? and TPSA 193.24??; Num_H_Acceptors 11

- S635: KierFlex 11.81; AlogP 3.506; 3DpPolarSASA 235.506 ?? and TPSA 193.24??; Num_H_Acceptors 11

- AF647: KierFlex 13.76; AlogP -4.428; 3DpPolarSASA 541.24 ?? and TPSA 294.87??, Num_H_Acceptors 15

- ABERRIOR Star 635P: KierFlex 12.13; AlogP 1.512; 3DpPolarSASA 329.16 ?? and TPSA 226.55??;Num_H_Acceptors 13

- ACT590: KierFlex 9.18; AlogP 5.36; 3DpPolarSASA 147.00 ?? and TPSA 81.91 ??; Num_H_Acceptors 6

- ACT647: KierFlex 8.38; AlogP 7.40; 3DpPolarSASA 46.73 ?? and TPSA 52.86??; Num_H_Acceptors 4

- ACT 565: KierFlex 6.66, AlogP 3.93; 3DpPolarSASA 135.06 ?? and TPSA 81.91 ??; Num_H_Acceptors 6

- ACT 647N: KierFlex 8.38, AlogP 7.54; 3DpPolarSASA 44.16 ?? and TPSA 52.86??; Num_H_Acceptors 4

Example 3 ? Marking of Fab fragments

3a - Labeling of the Fab obtained in example 1a by means of CF568 fluorophore,

Two solutions were prepared.

The first comprised 10 µg of Fab prepared as described in example 1a in 2.5 µl of 100 mM phosphate buffer at pH 6.50.

The second included 8<-10 >mol of succinimide ester of the fluorophore CF568 in 200 ?L of anhydrous DMF and the fluorophore concentration of the solution was verified by visible spectrophotometry (Principles of Fluorescence Spectroscopy Third Edition, Joseph R. Lakowicz)

The two solutions were mixed slowly and incubated at 37°C in the dark for one hour.

To the resulting mixture were added 80?l of an aqueous solution at pH 8.6 containing 2M ethanolamine and 0.1% by volume of Tween 20, which was ? was first maintained in the dark at 37°C for 15 minutes and then at 4°C overnight.

The separation of the fluorescent bio-conjugate from the excess of free fluorophore? been performed by molecular exclusion chromatography (Sephadex G25 Fine, GE Healthcare), and the identification of the eluted fractions containing the fluorescent conjugate ? occurred via thin-layer chromatography displayed by laser scanning, the absence of the free probe ? been verified by laser scanning. In detail, the product purified by chromatography ? was eluted in 50 µl fractions.

450 nanoliters of each fraction were loaded onto reverse phase TLC (C18) and the plate developed in H2O/CH3CN eluent (1:1 vol/vol) with the addition of 0.01% by volume of a saturated aqueous solution of ammonia. The slab? dried in the dark at room temperature and then scanned with an Amersham Typhoon Imaging Systems reader.

The acquisition conditions were the following: laser line at 561nm or 633nm; acquisition bands: 580nm with 30nm bandwidth, 680nm with 30nm bandwidth. Scan ? processed for an area of 5 cm X 5 cm, with a pixel size of 100 micron<2 > and the filtered emission ? was detected by means of a phototube set at 450 volts. The fractions in which detected spots at RF=0 were collected and processed for the next step. The fractions showing spots at RF=0 and RF=0.8 or only at RF=0.8 were discarded.

The degree of labeling of the Fab, quantified by UV-visible spectrophotometry? found to be between 1.8 and 2.2. The algebraic expression used to calculate the degree of marking (DOL) ? the following:

where the values Amax and A280 indicate, respectively, the maximum intensity? recorded in the region of absorption of the fluorophore and the maximum intensity? recorded in the region of the protein: these values are obtained directly from the UV-VIS spectrum for each conjugate. Otherwise, the ?prot ? estimated around 71000 M<-1>cm<-1>, while C280 and ?max are supplied by the manufacturer of the fluorescent molecule.

The conjugate ? was stabilized by the addition of 20 ?L of an aqueous solution of 0.1 wt% albumin and 5 mM sodium azide, and ? was brought to the storage and use concentration of 0.1 mg/mL via centrifugal vacuum evaporator.

where the values Amax and A280 indicate, respectively, the maximum intensity? recorded in the region of absorption of the fluorophore and the maximum intensity? recorded in the region of the protein: these values are obtained directly from the UV-VIS spectrum for each conjugate. Otherwise, the ?prot ? estimated around 71000 M<-1>cm<-1>, while C280 and ?max are supplied by the manufacturer of the fluorescent molecule.

The conjugate ? was stabilized by the addition of 20 ?L of an aqueous solution of 0.1 wt% albumin and 5 mM sodium azide, and ? was brought to the storage and use concentration of 0.1 mg/mL via centrifugal vacuum evaporator.

3b ? Labeling of the Fab obtained in Example 1a by means of AF647 fluorophore

For the labeling of the Fab obtained in the example 1a by fluorophore AF647 yes? proceeded as described in example 3a.

3c ? Labeling of the Fab obtained in Example 1b by CF568 fluorophore

For the labeling of the Fab obtained in example 1b by means of the CF568 fluorophore yes? proceeded as described in example 3a.

3d ? Labeling of the Fab obtained in Example 1b by means of AF647 fluorophore

For the labeling of the Fab obtained in the example 1b by fluorophore AF647 yes? proceeded as described in example 3a.

3e ? Labeling of the Fab obtained in example 1c by means of CF647 fluorophore

For the labeling of the Fab obtained in the example 1c by fluorophore CF647 yes? proceeded as described in example 3a.

3f ? Labeling of the labeled Fab obtained in Example 3c by AlexaFluo488 fluorophore

For the labeling of the labeled Fab obtained in example 3c by means of the AlexaFluo488 fluorophore, yes? proceeded as described in example 3a. 3g ? Labeling of the labeled Fab obtained in the 3d example with AlexaFluo488 fluorophore,

For the labeling of the labeled Fab obtained in the 3d example using the AlexaFluo488 fluorophore, yes? proceeded as described in example 3a. 3h - Labeling of the Fab obtained in example 1b by means of AF 532 fluorophore

For the labeling of the Fab obtained in the example 1b by fluorophore AF 532 yes? proceeded as described in example 3a.

3i - Labeling of the labeled Fab obtained in example 1b by Aberrior star 635P fluorophore

For the labeling of the labeled Fab obtained in the example 1b with the Aberrior star 635P fluorophore yes? proceeded as described in example 3a.

EXAMPLE 4 - Confocal microscopy analysis

Confocal microscopy experiments were carried out using the fluorescent Fabs obtained respectively in example 3a (INV), 3b (INV), 3c (INV) and 3e (CONTROL).

In particular, HeLa were fixed in formaldehyde, neutralized and permeabilized with a solution of 50 mM ammonium chloride, 0.05 wt% saponin and incubated with a 0.1 wt% bovine albumin solution. Subsequently the samples were left to hybridize with 15 ?L of a phosphate buffer containing 0.05% saponin and 0.1% albumin by weight and 150ng of the above fluorescent Fabs. The marker in excess ? was washed with 1 mL of phosphate buffered saline (PBS) pH 6.80-7.20 and mounted on 20 ?L of Mowiol?.

The mycoscope used? a Leica SP5-II inverted confocal (Leica Microsystems, Milan, Italy). Cell samples were embedded in Mowiol polymer on 0.7 mm thick fluorescence slides. The images were acquired by an immersion objective d? oil 100 X, having numerical aperture 1.40 (Leica Microsystems). The excitation of the fluorophores ? was obtained respectively, by laser line at 561 nm and 647 nm, by white laser source, with pulsed emission (SuperK, Leica. The fluorescent emissions of each fluorophore were filtered by AOBS in a range from 560nm to 650nm.

In the images obtained with the rabbit polyclonal Fab directed against the BARS protein labeled with the CF568 fluorophore, prepared as described in Example 3a (INV), ? It is possible to observe nuclear and Golgi organelle staining, and drastically less cytosolic staining (see FIG1A and FIG1Abis, which refer to images obtained with labeled Fab by reaction with 1-pyrrolidinyl, 2,5-dione and 1-pyrrolidinyl succinimide ester, 3 suflonyl 2,5-dione, respectively). The localization of the fluorescent fragment ? in perfect agreement with the data obtained by indirect immunofluorescence.

In the images obtained with the rabbit polyclonal Fab directed against the AF647 fluorophore-labelled BARS protein, prepared as described in example 3b (INV), ? It is possible to observe nuclear and Golgi organelle staining, and drastically less cytosolic staining (see FIG1B and FIG1Bbis, which refer to images obtained with labeled Fab by reaction with succinimide ester 1-pyrrolidinyl, 2,5-dione and 1- pyrrolidinyl, 3-suflonyl 2,5-dione, respectively). Furthermore, the intensity? any signals? lower than that obtained in example 3a (see Fig 1A and Fig1Abis), congruently with the lower quantum yield of the used fluorophore. The localization of the fluorescent fragment ? also in this case in perfect agreement with the data obtained by indirect immunofluorescence.

In the images obtained with the mouse monoclonal Fab directed against the CF568 fluorophore-labeled alpha tubulin protein, prepared as described in example 3c (INV), ? it is possible to observe a staining of the cytoskeleton, specifically of the microtubules (see FIG2A and FIG2Abis, which refer to images obtained with Fab labeled by reaction with succinimide ester 1-pyrrolidinyl, 2,5-dione and 1-pyrrolidinyl, 3 suflonyl 2,5 -dione, respectively). The localization of the fluorescent fragment ? in perfect agreement with the data obtained by indirect immunofluorescence.

In the images obtained with the rabbit polyclonal Fab directed against the AKAP9 protein labeled with the fluorophore CF647, prepared as described in Example 3e (COMPARISON), ? It is possible to observe an incorrect localization of the fluorescent conjugate, as nuclear as well as cytosolic and/or mitochondrial staining is observed (see FIG 2B and FIG 2Bbis, which refer to images obtained with labeled Fab by reaction with 1-pyrrolidinyl succinimide ester, 2, 5-dione and 1-pyrrolidinyl, 3-suflonyl 2,5-dione, respectively). The localization of the fluorescent fragment ? in total disagreement with the data obtained from the literature and by indirect immunofluorescence, which instead report a localization of the target protein only on the Golgi organelle.

As can be seen from the results of the examples above, Fab conjugated through a linker with specific length and flexibility characteristics? to CF568 and AF647 fluorophores, having specific characteristics such as: KierFlex between 7.5 and 15; AlogP less than or equal to 0; 3DpolarSASA equal to or greater than 300, are stable, have homogeneous characteristics in terms of fluorophore placement and maintain avidity? for the antigen. In particular, to the 3D polar SASA parameter which supplies the degree of hydration of the molecule, we owe a greater stabilization of the conjugate, a lesser tendency to aggregate because? hydrated and better emissive behavior due to higher solvation. Furthermore, the decreased tendency to interact with parts of the fragment also translates into a significant increase in greed why? the fluorophores do not collapse on the Fab recognition regions.

EXAMPLE 5 - Comparison between a conventional labeling (indirect immunofluorescence) and that produced by fluorescent Fabs, object of the present invention.

Conventional marking? was carried out with a fragment of mouse monoclonal antibody, of the IgG1 type, in human HeLa cells fixed, permeabilized and subjected to a common immunofluorescence procedure. Human HeLa cells were fixed, permeabilized and subjected to an immunofluorescence procedure as described below. The sample preparation procedure ? completely analogous to indirect immunofluorescence. The cells were grown on a 1cmX1cm quartz glass slide, in RPMI medium (Dulbecco) supplemented with 10% by volume of bovine serum, penicillin, glutamine. The cells are fixed by treatment with 4% formaldehyde, in buffered saline, pH 7.00, for 10 minutes. The excess of aldehyde ? was neutralized with 50mM ammonium chloride solution, buffered to pH 7.00. The fixed samples are washed in phosphate buffered saline, and permeabilized in 0.05% by weight vegetable saponin solution. The samples were incubated for one hour with Fab directed against the alpha-tubulin protein conjugated with fluorophore CF568 prepared as described in Example 3c (samples 1-3) or fluorophore AF647 prepared as described in Example 3d (samples 4- 5) and the excess present? removed by 3 washings in phosphate buffered saline. Any non-specific adsorption sites of the markers (primary and secondary) in the cell samples, ? was prevented by incubation with 0.1 wt% bovine albumin solution in physiological saline.

For each sample? a commercial probe was added consisting of the AlexaFluor488 marker conjugated to rabbit polyclonal antibody, directed against mouse monoclonal antibody of the IgG1 type.

The signal emitted by the fragment object of the invention, ? been detected by acquisition of fluorescence emission in the specific wavelength range for the type of fluorophore: in the case of CF568, the acquisition range ? in the range from 575nm to 620nm; while in the case of AF647, the? interval of acquisition? in the range from 660nm to 700nm. The control fluorophore, AlexaFluor bound to the secondary antibody directed against the Fab, can be excited separately from the other two, so its emission, collected in the range from 490nm to 450nm, if collected limited to the time in which it? excited individually, pu? be considered independent and therefore free of light contamination between the channels. ? therefore it is possible to make a quantitative and functional comparison between a conventional labeling (indirect immunofluorescence) and that produced by fluorescent Fabs, object of the present invention.

3 samples labeled with the CF568 probe and with the AlexaFluor488 commercial probe and 2 samples labeled with the AF647 probe and with the AlexaFluor488 commercial probe were prepared.

Comparing respectively the figure 3A with the figure 3B, the 4A with the 4B, the 5A with the 5B, the 6A with the 6B and the 7A with the 7B, it is evident not only the complete functionality? of the proposed marker, coherently with the calculated data, both for the CF568 probe and for the AF647 probe, but an aspect of considerable importance emerges from this analysis as regards the superiority? functional of the invention.

As can be seen from figures 3B, 4B, 5B, 6B and 7B which represent the images in which the signal emitted by the Fab object of the invention is? been detected by acquiring fluorescence emission in the specific wavelength range for the CF568 or AF647 fluorophore, the small size of the probe object of the invention, about one sixth of that of the conventional probe, is not only able to generate a? high density? of staining, but also allow localization of the probe in cellular regions where the conventional marker has no access. The area of interest lies in the midbodies.

On the contrary, as can be seen from figures 3A, 4A, 5A, 6A and 7A which represent the images in which the signal emitted by the Fab object of the invention is? been detected by acquiring fluorescence emission in the specific wavelength range for the commercial fluorophore AF488, the conventional probe does not have access to the area where the midbodies are located.

These regions are connections between cells that are reduced to a thin filament in the final stages of the cytokinesis process. Responsible for this bottleneck are the actin filaments that form a contractile filament that constrict the cytoskeleton and, creating a bottleneck, make s? that the two daughter cells separate. In the examples shown below it can be seen that only the probe object of the patent, ? able to penetrate and report, in detail, the morphological state of the aforementioned midbodies.

Cell division? currently a process of considerable interest why? involved in metastatic and neoplastic mechanisms, so the use of the Fab according to the invention to visualize cellular compartments not visible with traditional labeling can be an ideal example of the superiority? functional of these reagents in the field of cell imaging.

Therefore such fluorescent Fabs are useful in microscopy techniques allowing to obtain a high level of resolution and to visualize cellular compartments not visible with traditional staining.

In this regard, figures 8A, 8B, 8C, 8D represent a comparison between the images obtained with currently commercially available probes and probes in accordance with the present invention.

The samples were incubated for one hour with Fab directed against the protein ?-tubulin, conjugated with the fluorophore AF 647 prepared as described in Example 3d.

In parallel, to other samples ? A probe consisting of a commercial anti-?-tubulin antibody randomly conjugated with the AF 647 fluorophore was added.

The signal emitted by the fragment object of the invention (figures 8A, 8B) ? was compared with the signal emitted by conventionally labeled antibodies (figures 8C, 8D) in order to quantify the uncertainty limit with which tubulin is localized and, consequently, the microtubule diameter measured.

As visible in Figures 8C, 8D, the size of the microtubular section evidenced by the use of the conventional antibody ? greater than the real one. Such an increase? associated with the greater localization uncertainty caused by the diffusion of the light signal emitted and corresponding to a region of indefinite space within which the uncertainty on the real position of the microtubule is distributed.

On the contrary, as can be seen from figures 8A, 8B which represent the images in which ? been detected the signal emitted by the Fab object of the invention, the smaller diameter microtubular ? index of a lower dispersion of the fluorescence emission and, therefore, of a higher resolution limit.

In detail, the distribution of the fluorescent signal along the intersection of the microtubule represents the indetermination of the staining which is? significantly lower when the Fab object of the present invention is used.

Again, the fact that the Fab object of the invention is positioned at about 1-2 nm from the tubulin causes a lower dispersion of the fluorescent signal.

Figure 9 shows once again the superiority? function of the present invention consistently with the above data. In fact, the average value of the diameter relating to each measured segment distances itself from the real value by about 2 nm with Fab in accordance with the present invention. Conversely, localizations obtained with conventional probes (indicated by Ab in the figure) oscillate by about 8 nm around the mean value.

In detail, the average value detected by the use of Fab in accordance with the present invention ? equal to 28 nm, in parallel the average value detected through the use of probes of the convention type? equal to 42 nm.

Considering the effective diameter of a microtubule equal to 25 nm, the significant increase in the resolution limit by using probes in accordance with the present invention is evident.

In parallel, as can be seen from figure 10, ? A screening was developed using 10 conventionally used fluorophores in super-resolution microscopy and having different structures.

In detail, the loss of affinity? of the Fab conjugated to its own antigen pu? be verified directly by confocal microscopy, evaluating known cell morphologies.

From this analysis? found that four of the ten structures tested prevent the binding of the conjugate to the antigen; There? corresponds to the loss of the expected morphology (in this case the cytoskeleton), since the presence of the fluorophore totally prevents it from binding to the antigen.

At the same time, in order to evaluate the influence exerted by the pH values of the reaction on the possible overlapping of the behavior of the Fabs with regard to the derivatization of the two categories of amino groups previously described, ? an in silico prediction study of the relative pKa values was performed (figure 10).

As known, the mean pK values of the terminal amino and amino groups are approximately 7.5 and 13, respectively.

This difference would formally allow to discriminate between the various amino groups simply by working on the operating pH. ? calculable approximately that 3 units? of pK of difference (for example pK 7.5 against pK 10.5) between a terminal amino group and an amino group of lysine, involves a high percentage difference in the protonation state between the two amino groups compared.

For example, at a pH = 7.4 which ? close to the pK of the terminal NH2 group, 44.3% of these are deprotonated and available, while only 0.08% of the amino groups are available.

In any case, the simultaneous presence of amino groups (terminus against aminos) which compete towards a fluorophore functionalized with a succinimide group, requires a realistic evaluation of the percentages of deprotonated amino groups available for conjugation at each pH value in which around the requirement for adequate specificity is satisfied.

The prediction of the pKa values of the amino groups and? was performed with a hybrid quantum mechanics/molecular mechanics method (ab initio QM/MM), using two applications, Qsite (Murphy, R. B.; Philipp, D. M.; Friesner, R. A., "A mixed quantum mechanics/molecular mechanics (QM/MM) method for large-scale modeling of chemistry in protein environments," J. Comp. Chem., 2000, 21, 1442-1457; Philipp, D. M.; Friesner, R. A., "Mixed ab initio QM/MM modeling using frozen orbitals and tests with alanine dipeptide and tetrapeptide," J. Comp. Chem., 1999, 20, 1468-1494) and Potein Tirtration Curve, respectively contained in the Small-Molecule Drug Discovery Suite (Small-Molecule Drug Discovery Suite 2018-2, Schr? dinger, LLC, New York, NY, 2018) and Biologics Suite (Biologics Suite 2018-2,

Schrödinger, LLC, New York, NY, 2018).

In terms of prediction of the pKa values, a first analysis had as particular object two antibodies widely used in experimental practice and for which ? the related 3-D structure is available:

i) the mouse IgG1 isotype k monoclonal FAB antibody fragment (anti-c-myc clone 9E10, PDB ID: 2OR9 ? Table 1) and

ii) the rabbit monoclonal FAB M204 antibody fragment (Table 2).

Table 2 - M204

Table 1 - ID: 2OR9

In both cases, the present inventors have observed some lysine residues characterized by pKa values not included, as expected, between 10 and 13, but attested on values more? bass of less than 9, not resulting cos? distant from those of the terminal amino groups whose average value ? around 7.5. A second important observation arises from the analysis of the mouse antibody, whose terminal amino group of the light chain has, conversely, a significantly lower pKa? higher than expected (7.5) settling on the value of 9.96. These observations have highlighted conditions whose occurrence? of clear importance for our purpose. The analysis? then it was extended to the prediction of the pKa values of the amino groups to other 18 antibodies (for a total of 20, ie about 20% of the mouse and rabbit antibodies with a known amino acid sequence), whose 3D structure? been determined. Were these antibodies selected according to the diversity maximization criterion? of amino acid sequence, cos? to exclude a possible structural amino acid bias (Table 3).

Table 3

This analysis demonstrates that in most of the FABs considered, the respective pKa of the two chains (H, L) have average values around 7.5. In a smaller number of cases, however, as for the cited anti-c-myc antibody clone 9E10, one of the two terminal NH2 groups (that of the light chain, L), has significantly higher pKa values. elevated.

When this occurs, it is obvious that the terminal amino group actually available for selective derivatization e.g. at pH ?7.4, is only 1 (that of the heavy chain).

What about selectivity? of the reaction, it ? linked to the particular operating pH.

The following example illustrates the case of a FAB characterized by a terminal amino group with pKa= 7.8 and a ?-amino group with pKa=8.89, potentially in competition. Figure 10 shows the titration curves from which ? It is possible to evaluate the percentage fractions of the free amino groups (in the figure, NH2-ter) and of the ?-amino groups (in the figure indicated as NH2-lysine) at each pH.

In parallel, ? reported a curve (NH2-ter/NH2-lysine) representing the variations of the ratio NH2-ter/NH2-lysine and from which ? possible to select an optimal pH for its maximization.

In accordance with the object of the present invention, ? clear as it is necessary to identify reaction conditions such as to guarantee the selectivity? of the derivatization reaction of the Fab fragment with an activated fluorophore.

In this regard, the conjugation reaction includes numerous steps of which only the final one ? irreversible and, conversely, the others are reversible and governed by conditions of equilibrium and by the availability? of the reactive substrate, the latter dependent on the pH values.

From the study of the aminolysis reaction of the ester, it can be seen that the latter proceeds by means of general basic catalysis, making it clear that the reversibility? dynamics exists exclusively for the first reaction steps (consisting in the deprotonation of the amino group and in the formation of a tetravalent intermediate). The latter, however, evolves irreversibly in the final product by means of an expulsion of the hydroxy-succinimide group followed by a substantially simultaneous formation of the amide bond with the fluorophore.

Ultimately, the pH of exercise can? be chosen so as to ensure selectivity? with respect to the terminal amino groups without affecting the total yield of the reaction.

Claims (7)

CLAIMS 1) Antibody or Fab fragment, wherein at least one amino group of the N-terminal amino acid of the light chain and/or the N-terminal amino acid of the heavy chain ? amide bonded to a molecule comprising a fluorophore A group, wherein said linkage constitutes at least 70% of the total binding of said molecule to said antibody or Fab, preferably at least 80%, 85%, 90%, or 98% of the total binding of said molecule to said antibody or Fab, wherein said antibody is monocolonal or said Fab derives from a monoclonal antibody and said amino group of the N-terminal amino acid of the light chain and/or of the N-terminal amino acid of the heavy chain ? linked by an amide bond to a Z group of the formula: -CO-W-A in which: TO ? a fluorophore group chosen from: - CF568 fluorophore - fluorophores derived from xanthene, or carbocyanins, preferably of the formula: in which R? a group chosen from ?OPO3H2 and ?OH and R? ? chosen between -OSO3<- >and ?F; - 2-[(1E,3E)-5-[(2Z,3S)-3-(5-methoxy-5-oxopentyl)-3-methyl-5-sulfo-1-(3-sulfopropyl)-2,3 -dihydro-1H-indol-2-ylidene]penta-1,3-dien-1-yl]-3,3-dimethyl-5-sulfo-1-(3-sulfopropyl)-3H-indol-2-yl ( AF647), having structure - 2-(2,2,10,10-tetramethyl-4,8-bis(sulfonatomethyl)-2,10-dihydro-1H-pyrano[3,2-g:5,6-g']diquinolin-11- ium-6-yl)terephthalate (AF568, both regioisomers), having structure - 2-[(1E,3Z)-3-[3-(3-carbamoylpropyl)phenyl]-5-[(2Z)-3,3-dimethyl-5-sulfonato-1-(3-sulfonatopropyl)-2, 3-dihydro-1H-indol-2-ylidene]penta-1,3-dien-1-yl]-5-chloro-3,3-dimethyl-7-(3-sulfonatopropyl)-3H-pyrrolo[2,3 -b]pyridin-7-ium; - 2-[(E)-2-[(3E)-3-{2-[(2Z)-1-(5-carbamoylpentyl)-3,3-dimethyl-5-sulfonato-2,3-dihydro-1H -indol-2-ylidene]ethylidene}-2-(4-sulfonatophenoxy)cyclohex-1-en-1-yl]ethenyl]-3,3-dimethyl-1-(4-sulfonatobutyl)-3H-indol-1- ium-5-sulfonate; - (1P)-2-(6-amino-3-iminiumyl-4,5-disulfonato-3H-xanthen-9-yl)-4-carbamoylbenzoate; - 1-(5-carbamoylpentyl)-3,3-dimethyl-2-[(1E,3E)-5-[(2Z)-1,3,3-trimethyl-2,3-dihydro-1H-indol-2 -ylidene]penta-1,3-dien-1-yl]-3H-indol-1-ium; - 2-[(1E,3E)-5-[(2Z)-1-(5-carbamoylpentyl)-3,3-dimethyl-5-sulfonato-2,3-dihydro-1H-indol-2-ylidene]- 3-methylpenta-1,3-dien-1-yl]-3,3-dimethyl-1-(4-sulfonatobutyl)-3H-indol-1-ium-5-sulfonate; - 2-[(1E,3E)-5-[(1R,2Z)-1-(5-carbamoylpentyl)-3,3-dimethyl-5-sulfonato-2,3-dihydro-1H-indol-1-ium -2-ylidene]penta-1,3-dien-1-yl]-3,3-dimethyl-1-(4-sulfonatobutyl)-3H-indol-1-ium-5-sulfonate; - 2-tert-butyl-4-[(1E)-3-[(2E)-1-(5-carbamoylpentyl)-3,3-dimethyl-5sulfonato-2,3-dihydro-1H-indol-2-ylidene ]prop-1-en-1-yl]-9-ethyl-8,8-dimethyl-8H,9H-1 ?<4>-chromeno[7,6-b]pyridin-1-ylium; - 4-[(1E)-3-[(2Z,3S)-3-(5-carbamoylpentyl)-3-methyl-6-sulfonato-1-(3-sulfonatopropyl)-2,3-dihydro-1H-indol -2-ylidene]prop-1-en-1-yl]-8,8-dimethyl-2-phenyl-6-(sulfonatomethyl)-9-(3-sulfonatopropyl)-8H,9H-1?<4>- chromeno[7,6-b]pyridin-1-ylium; - 2-tert-butyl-4-[(1E,3E)-5-[(2E)-1-(5-carbamoylpentyl)-3,3-dimethyl-5-sulfonato-2,3-dihydro-1H-indol -2-ylidene]penta-1,3-dien-1-yl]-7-(diethylamino)-1?<4>-chromen-1-ylium; - N,N-dimethyl-4-[(2E)-1,5,5-tris[4-(dimethylamino)phenyl]penta-2,4-dien-1-ylidene]cyclohexa-2,5-dien-1 -iminium; - 2-[(1E,3E)-5-[(2Z)-1-(5-carbamoylpentyl)-3,3-dimethyl-5-sulfonato-2,3-dihydro-1H-indol-2-ylidene]penta -1,3-dien-1-yl]-3,3-dimethyl-1-(4-sulfonatobutyl)-3H-indol-1-ium; - 28R)-16-{2-[(3-carbamoylpropyl)(methyl)carbamoyl]phenyl}-3-oxa-9??,23-diazaheptacyclo[17.7.1.1?,?.0?,??.0? ,??.0??,??.0??,??] octacosa-1(27),2(17),4,9,13,15,18-heptaen-9-ylium; - 2-[(1E,3E)-5-[(2Z)-1-(5-carbamoylpentyl)-3,3-dimethyl-2,3-dihydro-1H-indol-2-ylidene]penta-1,3 -dien-1-yl]-1,3,3-trimethyl-3H-indol-1-ium; - 2-[(1E,3E)-5-[(1Z,2R)-1-(5-carbamoylpentylidene)-3,3-dimethyl-2,3-dihydro-1H-1?<5>-indol-1 -ylium-2-yl]penta-1,3-dien-1-yl]-3,3-dimethyl-1-(4-sulfonatobutyl)-3H-indol-1-ium; - 4-carbamoyl-2-[13-(dimethylamino)-5-(dimethyliminiumyl)-2,2-dimethyl-2-silatricyclo[8.4.0.0?,?]tetradeca-1(10),3,6,8, 11,13-hexaen-9yl]benzoate; And - 2-[(1E,3E)-5-[(2Z)-1-(5-carbamoylpentyl)-3,3-dimethyl-5-sulfonato-2,3-dihydro-1H-indol-2-ylidene]penta -1,3-dien-1-yl]-3,3-dimethyl-1-(4-sulfonatobutyl)-3H-indol-1-ium; - (7S,17R)-12- [4-(methoxycarbonyl)phenyl]-7,8,8,16,16,17-hexamethyl-2-oxa-6,18-diazapentacyclo[11.7.0.<03.11 >.0<5,9>.0<15,19>]icosa-1(13),3,5,9,11,14,19-heptaen-6-ium-4,20-disulfonate(AF532), having structure - [10,10,22,22-tetramethyl-20-(sulfomethyl)-16-{2,3,4,5-tetrafluoro-6-[(4-methoxy-4-oxobutyl)(methyl)carbamoyl]phenyl} -3-oxa-9lambda4,23-diazaheptacyclo[17.7.1.1<5,9>.0<2,17>.0<4,15>.0<23,27>.0<13,28>]octawhat? 1,4,9(28),11,1 3,15,17,19(27),20-nonaen-12-yl]methanesulfonic acid (ASred), having the structure - 2?({3?[12,20?bis(hydroxymethyl)?10,10,22,22?tetramethyl?3?oxa?9lamb da4,23?diazaheptacyclo[17.7.1.1<5,9>.0<2 ,17>.0<4,15>.0<23,27>.0<13,28>]octawhat?1,4,9( 28),11,13,15,17,19(27),20 ?onaen?16?yl]?2,5,6?trifluoro?4?[(4?methoxy-4?oxobutyl)(methyl)carbamoyl]phenyl}sulfanyl)ethane?1?sulfonic acid (S635), having the structure - 3-(5,5-difluoro-7-(thiophen-2-yl)-5H-4?5,5?5-dipyrrolo[1,2-c,1,2-f][1,3,2 ]diazaborinin-3-yl)propanoic acid (BODIPY_558-568), having the structure: - 4-(11-ethyl-2,2-dimethyl-4-(sulfomethyl)-3,4,8,9,10,11-hexahydro-2H-1?5-pyrido[3',2':6, 7]chromeno[3,2-g]quinolin-1-yl)butanoic acid (ATTO_665) having the structure: - 3-(5,5-difluoro-7-((1E,3E)-4-phenylbuta-1,3-dien-1-yl)-5H-4?5,5?5-dipyrrolo[1,2- c,1,2-f][1,3,2]diazaborinin-3-yl)propanoic acid (BODIPY_581-591), having the structure: - 5-(5,5-difluoro-7,9-bis(trifluoromethyl)-5H-4?5,5?5-dipyrrolo[1,2-c,1,2-f][1,3,2] diazaborinin-3-yl)pentanoic acid (BODIPY_FL_C5), having the structure: - (2E)-2-((2E,4E)-5-(3-(5-carboxypentyl)-1,1-dimethyl-6,8-disulfo-1H-benzo[e]indol-3-ium-2 -yl)penta-2,4-dien-1-ylidene)-3-ethyl-1,1-dimethyl-6-sulfo-2,3-dihydro-1H-benzo[e]indole-8-sulfonate (CY_5- 5), having structure: - 4-((2-(3,6-bis((3-sulfopropyl)amino)xanthen-9-yl)benzoyl)(methyl)amino)butanoic acid (DY-530), having the structure: - 4-((4,5-dichloro-2-(2,7-dimethyl-3,6-bis((3-sulfopropyl)amino)xanthen-9-yl)benzoyl)(methyl)amino)butanoic acid (DY -546), having structure: - 6-((2E)-2-((2E)-3-(1-(2-methoxyethyl)-3,3-dimethyl-6-sulfo-3H-1?5-indol-2-yl)prop- 2-en-1-ylidene)-3,3-dimethyl-5-sulfo-2,3-dihydro-1H-indol-1-yl)hexanoic acid (DY-548P1), having the structure - 6-((2E)-2-((2E)-3-(1-(2-methoxyethyl)-3,3-dimethyl-6-sulfo-3H-1?5-indol-2-yl)prop- 2-en-1-ylidene)-3-methyl-5-sulfo-3-(3-sulfopropyl)-2,3-dihydro-1H-indol-1-yl)hexanoic acid (DY-549P1), having the structure: - 6-((5Z)-5-(2-((4Z)-2-tert-butyl-7-(diethylamino)chromen-4-ylidene)ethylidene)-2,4,6-trioxo-3-(3 -sulfopropyl)-1,3-diazinan-1-yl)hexanoic acid (DY-550), having the structure: - 2-(3,6-bis(dimethylamino)xanthen-9-yl)-5-((5-carboxypentyl)carbamoyl)benzoic acid (DY-554), having the structure: - 2-(3,6-bis(diethylamino)xanthen-9-yl)-5-((5-carboxypentyl)carbamoyl)benzoic acid (DY-555), having the structure: - 5-((5-carboxypentyl)carbamoyl)-2-(3-(diethylamino)-6-(ethyl(3-sulfopropyl)amino)xanthen-9-yl)benzoic acid (DY-556), having the structure: - 2-(3,6-bis(ethyl(3-sulfopropyl)amino)xanthen-9-yl)-5-((5-carboxypentyl)carbamoyl)benzoic acid (DY-557), having the structure: - 6-(((4-(3,6-bis(diethylamino)xanthen-9-yl)-3-sulfophenyl)sulfonyl)amino)hexanoic acid (DY-560), having the structure: - 4-(pyrido[1,2,3-ij]quinolizino[9',1':6,7,8]chromeno[3,2-g]quinolin-9-yl)benzene-1,3-dicarboxylic acid (DY-580), having structure: - 5-((2-(9-(ethyl(3-sulfopropyl)amino)-2,2,4-trimethyl-1-(3 sulfopropyl)chromeno[3,2-g]quinolin-6-yl)benzoyl)(methyl)amino)pentanoic acid (DY-585), having the structure: - 6-(((3-methyl-4-(pyrido[1,2,3-ij]quinolizino[9',1':6,7,8]chromeno[3,2-g]quinolin-9-yl )phenyl)sulfonyl)amino)hexanoic acid (DY-590), having the structure: - 3-(2-(2-(((4-(14,15-diethyl-4?5,14?5-pyrido[1,5,6-ij]pyrido[3',2':6,7 ]chromeno[2,3-f]quinolin-9-yl)-3-sulfophenyl)sulfonyl)amino)ethoxy)ethoxy)propanoic acid (DY-591), having the structure: - 4-(1,11-bis(3-(aminosulfonyl)propyl)-2,2,10,10-tetramethyl-4,8-bis(sulfomethyl)pyrido[3',2':6,7]chromeno[ 3,2-g]quinolin-6-yl)benzene-1,3-dicarboxylic acid (DY-594), having the structure: - 5-(((2-(1,11-bis(3-(aminosulfonyl)propyl)-2,2,10,10-tetramethyl-4,8-bis(sulfomethyl)pyrido[3',2':6 ,7]chromeno[3,2-g]quinolin-6-yl)phenyl)(hydroxy)methyl)(methyl)amino)pentanoic acid (DY-605), having the structure: - 6-(5-((E)-2-(2-tert-butyl-7-(diethylamino)chromen-4-yl)ethenyl)-2,4,6-trioxo-3-(3-sulfopropyl)- 1,3-diazine-1-yl)hexanoic acid (DY-610), having the structure: - 6-((2E)-2-((2E)-3-(7-amino-2-tert-butylchromen-4-yl)prop-2-en-1-ylidene)-3,3-dimethyl-5 -sulfo-2,3-dihydro-1H-indol-1-yl)hexanoic acid (DY-615), having the structure: - 4-((2E)-2-((2E)-3-(2-tert-butyl-7-(diethylamino)chromen-4-yl)prop-2-en-1-ylidene)-3-methyl- 5-sulfo-1-(3-sulfopropyl)-2,3-dihydro-1H-indol-3-yl)butanoic acid (DY-630), having the structure: - 6-((2E)-2-((2E)-3-(2-tert-butyl-7-(diethylamino)chromen-4-yl)prop-2-en-1-ylidene)-3,3- dimethyl-5-sulfo-2,3-dihydro-1H-indol-1-yl)hexanoic acid (DY-631), having the structure: - 6-((2E)-2-((2E,4E)-5-(1-(2-methoxyethyl)-3,3-dimethyl-5-sulfo-3H-1?5-indol-2-yl) penta-2,4-dien-1-ylidene)-3-methyl-5-sulfo-3-(3-sulfopropyl)-2,3-dihydro-1H-indol-1-yl)hexanoic acid (DY-648P1) , having structure: - 4-((2E)-2-((2E,4E,6E)-7-(1,1-dimethyl-3-(4-sulfobutyl)-1H-3?5-benzo[e]indol-2- yl)hepta-2,4,6-trien-1-ylidene)-1,1-dimethyl-1,2-dihydro-3H-benzo[e]indol-3-yl)butane-1-sulfonic acid (ICG) , having structure: - 2-((Z)-2-((3Z)-3-(2-((2E)-1-(5-carboxypentyl)-3,3-dimethyl-5-sulfo-1,3-dihydro-2H -indol-2-ylidene)ethylidene)-2-(4-(oxidanidylsulfonyl)phenoxy)cyclohex-1-en-1-yl)ethenyl)-3,3-dimethyl1-(4-(oxidanidylsulfonyl)butyl)-3H- indol-1-ium-5-sulfonate (IRDye800CW), having the structure: - ({10,10,22,22?tetramethyl?20?[(phosphonooxy)methyl]?16?{2,3,4,5?tetr afluoro?6?[(4?methoxy?4?oxobutyl)(methyl )carbamoyl]phenyl}?3?oxa?9l ambda4,23?diazaheptacyclo[17.7.1.1<5,9>.0<2,17>.0<4,15>.0<23,27>.0<13 ,28>]octacosa?1,4 ,9(28),11,13,15,17,19(27),20?nonaen?12?yl}methoxy)phosphonic acid (ABBERIOR star 635P), having structure W? selected from C1-C10 alkyl, -CH2CH2(CH2CH2O)mCH2CH2-X-, or -CH2-CH2OCH2CH2-Q-Y-CH2CH2OCH2CH2-X-, in which m ? between 0 and 8, X ? chosen between CO and NH, Y ? selected from CH2CH2OH, pyrimidine, methoxyphenyl, or a compound of formula 2) Antibody or Fab fragment according to claim 1, wherein group Z has a total number of hydrogen bond acceptor atoms (Num_H_Acceptors) of the molecule in the range from 8 to 16, preferably from 10 to 15. 3. Antibody or Fab fragment according to claims 1 to 2, wherein group Z has a logarithm of the water/octanol partition coefficient (AlogP) in the range 3 to -5. 4) Antibody or Fab fragment according to claims 1 to 3, wherein group Z has a surface exposed to the solvent (3DpolarSASA, solvent accessible surface area) equal to or greater than 180 ?? , preferably between 200 and 550 ?? and/or total polar surface area (TPSA) equal to or greater than 180 ??, preferably between 180 ?? and 500 ??, more? preferably between 190 ?? and 300 ??. 5) Antibody or Fab fragment according to claims 1 to 4, wherein group Z has: - flexibility index? Kier ((?), KierFlex) between 7.5 and 15, preferably between 10 and 12, - logarithm of the water/octanol partition coefficient (AlogP) less than or equal to 0, preferably between 0 and -10 - surface exposed to the solvent (3DpolarSASA, solvent accessible surface area) equal to or greater than 300 ??, preferably between 400 ?? and 450 ??, and/or - total polar surface area (TPSA, total polar surface area) equal to or greater than 200 ??, preferably between 200?? and 220 ??. 6) Antibody or Fab fragment according to claims from 1 to 5, wherein said group A has a molar extinction coefficient (?) not lower than 80000 and quantum yield not lower than 60%. 7) Antibody or Fab fragment according to claim 1 to 6, wherein said group A ? chosen between: - CF568 fluorophore - 2-[(1E,3E)-5-[(2Z,3S)-3-(5-methoxy-5-oxopentyl)-3-methyl-5-sulfo-1-(3-sulfopropyl)-2,3 -dihydro-1H-indol-2-ylidene]penta-1,3-dien-1-yl]-3,3-dimethyl-5-sulfo-1-(3-sulfopropyl)-3H-indol-2-yl ( AF647), having structure - 2-(2,2,10,10-tetramethyl-4,8-bis(sulfonatomethyl)-2,10-dihydro-1H-pyrano[3,2-g:5,6-g']diquinolin-11- ium-6-yl)terephthalate (AF568, both regioisomers), having structure - (7S,17R)-12-[4-(methoxycarbonyl)phenyl]-7,8,8,16,16,17-hexamethyl-2-oxa-6,18-diazapentacyclo[11.7.0.<03.11 >.0<5,9>.0<15,19>]icosa 1(13),3,5,9,11,14,19-heptaen-6-ium-4,20-disulfonate(AF532), having structure - [10,10,22,22-tetramethyl-20-(sulfomethyl)-16-{2,3,4,5-tetrafluoro-6-[(4-methoxy-4-oxobutyl)(methyl)carbamoyl]phenyl} -3-oxa-9lambda4,23-diazaheptacyclo[17.7.1.1<5,9>.0<2,17>.0<4,15>.0<23,27>.0<13,28>]octacosa- 1,4,9(28),11,13,15,17,19(27),20-nonaen-12-yl]methanesulfonic acid (ASred), having the structure - 2?({3?[12,20?bis(hydroxymethyl)?10,10,22,22?tetramethyl?3?oxa?9lamb da4,23?diazaheptacyclo[17.7.1.1<5,9>.0<2 ,17>.0<4,15>.0<23,27>.0<13,28>]octawhat?1,4,9(2 8),11,13,15,17,19(27), 20?onaen?16?yl]?2,5,6?trifluoro?4?[(4?methoxy?4oxobutyl)(methyl)carbamoyl]phenyl}sulfanyl)ethane?1?sulfonic acid (S635), having the structure - ({10,10,22,22?tetramethyl?20?[(phosphonooxy)methyl]?16?{2,3,4,5?tetr afluoro?6?[(4?methoxy?4?oxobutyl)(methyl )carbamoyl]phenyl}?3?oxa?9l ambda4,23?diazaheptacyclo[17.7.1.1<5,9>.0<2,17>.0<4,15>.0<23,27>.0<13 ,28>]octacosa?1,4 ,9(28),11,13,15,17,19(27),20?nonaen?12?yl}methoxy)phosphonic acid (ABBERIOR star 635P), having structure