CZ2016742A3 - A conjugate of monoclonal antibody nimotuzumab, its complex with radionuclides, a method of their preparation and their use - Google Patents

Abstract

The present invention relates to the monoclonal antibody conjugate nimotuzumab and the chelator of formula I, wherein the chelator of formula I is covalently bonded via isothiocyanate to the amino group of the lysine amino acid present in the monoclonal antibody nimotuzumab, a thiourea bridge. The invention further relates to a complex of the above-mentioned conjugate with radionuclides, to methods of preparing a conjugate and to a complex with radionuclides and to their use in the treatment of cancer.

Description

The present invention relates to a mitrazuzumab monoclonal antibody conjugate with a tetrazamacrocyclic ligand, a complex thereof with radionuclides, methods for their preparation and their use in the treatment of cancer.

BACKGROUND OF THE INVENTION

Monoclonal antibodies are immunoglobulins _(Mw 150 kDa) produced by the immune system as a response to the presence of substances (antigens) that are foreign organism. Antibodies are also produced against tumor cells because cancer cells on their surface often express antigens that the body considers foreign, or have receptors on the surface of the body, but they are found in substantially greater numbers. Thus, the antibodies can be used in the treatment of cancer, either alone or as a targeting group for delivery of other drugs to the tumor, such as radioisotopes for internal radiation, i.e., those isotopes whose radioactive decay is associated with with emission of β ^_ or particles that are capable of killing cells in the immediate vicinity of the radioisotope. This concept is used, for example, in the commercial Zevalin® formulation, which is used to treat non-Hodgkin's lymphoma (NHL). The active ingredient of Zevalin® is the rituximab antibody recognizing the B-cell surface CD-20 antigen, which is conjugated to a DTP A derivative (DTP A = diethylenetriaminepentaacetic acid) as a chelator for the therapeutic radioisotope ⁹⁰ Y (β-emitter).

It is known that macrocyclic ligands derived from DOTA (DOTA = 1,4,7,10-tetraazacy ₍ β-lododecane-1,4,7,10-tetraacetic acid; Scheme 1) are more suitable for in vivo use than DTP A derivatives. The reason is greater stability of the DOTA complex with metal in vivo, although DOTA complexes are less easily formed than with DTPA, but since in vivo stability is a more important parameter than ease of complexation, most of the currently clinically tested radiolabeled antibodies are based on conjugates with DOTA derivatives.

One of the most commonly used and technically simplest methods of conjugating ligands and antibodies is the reaction between the isothiocyanate on the ligand and the ε-amino groups of lysine on the antibody.

· »»

* · * * * · · · · · · · · · ·

In this way, up to about twenty ligand molecules can be bound to the antibody molecule, with a higher number of ligand groups improving radioisotope labeling, but also reducing the immunoreactivity of the whole conjugate. The optimum number is 3 to 5 ligand molecules per antibody molecule. The size of the ligand, its hydrophilicity, charge, etc. affect the immunoreactivity of the modified antibody, and therefore conjugates with different chelators may exhibit very different biological activity due to large differences in immunoreactivity.

Nimotuzumab (hR3, Theraloc, CIMAher) is a humanized antibody with affinity for the extracellular domain of the epidermal growth factor receptor (EGFR), which is highly present on the surface of many types of tumor cells. This antibody is therapeutically effective in itself and is being tested worldwide (Phase I - III) for various anticancer indications. It can also be used as a targeting molecule for delivery of other therapeutics such as metal radioisotopes. For this purpose nimotuzumab was conjugated to DOTA derivatives and labeled with ¹⁷⁷ Lu or ⁹⁰ Y (Comparison of [177Lu-DOTAO, Tyr3] octreotate and [177Lu-

- DOTAO, Tyr3] octreotide: which peptide is preferable for PRRT ?, J.P. Esser, E.P. Krenning, J.J.

M. Teunissen, P. P. M. Kooij, A. L. H. van Gameren, W. H. Bakker, D. J. Kwekkeboom, Eur. J. Nucl. Copper. Mol. Imaging (2006) 33: 1346-1351; Preparation and Preclinical Evaluation of 177Lu-Nimotuzumab Targeting Epidermal Growth Factor Receptor Overexpressing Tumors, Denis R. Beckford Vera, Sebastian Eigner, Catherine Eigner Henke, Ondrej Lebeda, Frantisek Melichar, Milos Beran, Nucl. Copper. Biol., 2012, 39 (1): 3–13; Pre-Clinical Evaluation of 177Lu-

- Nimotuzumab: a Potential Tool for Radioimmunotherapy of EGFR Overexpressing Tumors

Beckford Vera, DR, Eigner S., Beran M., Eigner Henke K., Laznickova A., Laznicek M., Melichar F and Chinol M, Cancer Biother. Radiopharm., 2011, 26 (3): 287-297.

The most commonly used ligand was the commercial ligand DOTA-NCS (Scheme 1). However, the published conjugation conditions lead to the loss of valuable ligand and often poorly reproducible results, particularly with respect to the number of ligand molecules bound in the conjugate. Thus, antibodies exhibit irregular changes in affinity for a given receptor, and thus ambiguous in vivo test results.

HOOC

HOOC

COOH

DOTA

Scheme 1

In the prior art, nimatuzumab antibody conjugates with DOTA derivatives are lacking, whose conjugation would be easy, reproducible and with a given number of ligand molecules per antibody molecule. These conjugates would result in higher and more reproducible biological activity in vitro and in vivo. Previous conjugation procedures utilize high pH and multiple excess ligands to bind the optimal number of ligand molecules to the antibody. However, these conditions lead to contamination of the reaction mixture with products of the hydrolysis of the -NCS group of ligands and hence higher consumption of ligand in the preparation of the conjugate. In addition to high ligand consumption, another disadvantage of DOTA-type ligands is the wide range of conjugated complexing units per antibody molecule, resulting in irregular changes in receptor affinity.

SUMMARY OF THE INVENTION

The present invention, which relates to a novel conjugate of a monoclonal antibody nimotuzumab with an -NCS group linked to a tetraazamacrocyclic ligand, simplifies and streamlines the conjugation reaction which results in a given number of ligand molecules bound to one antibody molecule and thereby reproducibly higher and reproducible bioactivity in vitro and in vitro in vivo complexes of this conjugate with radionuclides having utility in the treatment of cancer. The present invention also results in an unexpectedly high affinity of the resulting conjugate for receptors, resulting in potentially superior therapeutic outcomes for in vivo applications.

It is an object of the present invention to provide a conjugate of the monoclonal antibody nimotuzumab and at least one chelator of formula

* «

• • • •

HOOC

NCS

Wherein at least one chelator of formula (which is covalently bound by a thiourea bridge + - NH-C (= S) -NH-) formed by reaction of the isothiocyanate group of the chelator of formula III and the amino group of the lysine amino acid present in the monoclonal By monoclonal antibody is meant the entire nimotuzumab molecule, or Fab fragments thereof, for binding to tumor antigens.

In a preferred embodiment, the conjugate of the present invention comprises up to 10 chelator molecules of formula (II) covalently linked to a thiourea bridge formed by reacting the isothiocyanate group of the chelator of formula and the amino group of the lysine amino acid present in monoclonal antibody nimotuzumab; 2 to 4 linked chelator molecules of formula (I) · per nimotuzumab monoclonal antibody molecule.

The present invention also provides a process for preparing a conjugate of the monoclonal antibody nimotuzumab and a chelator of formula (If according to the present invention, wherein the nimotuzumab antibody solution of pH 8.0 to 9.2 is mixed with a chelator solution of formula pH 8.0 to 9.2, wherein the molar ratio of the monoclonal antibody nimotuzumab to the chelator of formula (II) is in the range of 1:10 to 1: 100, preferably in the range of 1:20 to 1:75, more preferably in the range of 1:45 to 1 : 55;

the resulting reaction mixture is reacted for at least 10 minutes, preferably for 10 to 600 minutes, more preferably for 50 to 250 minutes, most preferably for 100 to 120 minutes, at a temperature in the range of

The 4-4 (| ⁰ C, preferably in the range 15 to 30 ° C, more preferably 18 to 22 ° C, to form a conjugate of the monoclonal antibody and the chelator nimotuzumab formula (If the present invention.

Preferably, the pH of the reaction mixture is kept constant throughout the reaction, ranging from 8.0 to 10 ° C

9.2, more preferably in the range of from 8.3 to 8.7, most preferably in the range of from 8.5 to 8.6.

• ·

The antibody concentration is preferably in the range of 2.5 to 7 mg / ml of the resulting reaction mixture, more preferably in the range of 4.5 to 5 mg / ml of the resulting reaction mixture. The solvents used may not contain heavy metal ions at any step where the chelator of formula (I) and / or the conjugate of the monoclonal antibody nimotuzumab and the chelator of the invention is used. The progress of the conjugation reaction can preferably be monitored by HPLC.

The chelator of formula (1) and its preparation has been described in Rudovský J., Botta M., Hermami P., Coridze A., Aime S., Dalton Trans., 2006, 2323-2333. The preparation of its amine precursor (1,4,7,10-tetraazacyplododecane-4,7,10-triacetato-1 - [methyl [(4-aminophenyl) methyl] phosphinic} acid) is described in Rudovsky J., Kotek J , Hermann P., Lukes I., Mainero V., Aime S., Org.

Biomol. Chem., 2005, 3, 112-117. Conjugation of the chelator of formula (f) to simple amines, cyclodextrins and PAMAM dendrimers are described in Rudovsky J., Botta M., Hermann P., Hardcastle KI, Lukes I., Aime S., Bioconjugate Chem., 2006, 17, 975-987 ; Kotková Z., Helm L., Kotek J., Hermann P., Lukeš I., Dalton Trans., 2012, 41, 13509-13519. However, the published conjugation conditions lead to the loss of valuable ligand and often poorly reproducible results, particularly with respect to the number of ligand molecules bound in the conjugate. Thus, antibodies exhibit irregular changes in affinity for a given receptor, and thus ambiguous in vivo test results.

In one embodiment of the method for preparing the conjugate of the monoclonal antibody nimotuzumab and the chelator of formula (1) of the present invention, the solvent of the reaction mixture is water or water containing up to 20% by volume DMSO. Preferably the water contains up to 5 vol% DMSO, more preferably up to 1 vol% DMSO.

In one embodiment of the method for preparing the conjugate of the monoclonal antibody nimotuzumab and the chelator of formula (iy of the present invention), the reaction mixture further comprises a pH-maintaining buffer selected from phosphate buffer, acetate buffer, TRIS (tris (hydroxymethyl) aminomethane) buffer , HEPES (N - (2-hydroxyethyl) piperazine - N - (2-

- ethanesulfonic acid) buffer and EPPS (N - (2-hydroxyethyl) piperazine-N '- (3-propanesulfonic acid)) buffer. Preferably, a buffer in the range of 0.1 to 1.0 M is used. More preferably, a EPPS buffer in the range of 0.2 to 0.3 M is used.

In one embodiment of the method for preparing the conjugate of the monoclonal antibody nimotuzumab and the chelator of formula (I) according to the present invention, the step of purifying the reaction mixture is further followed, preferably by membrane filtration. Purification of the reaction mixture after conjugation of the ligand and antibody is performed; ···· ··· · * -.,>

'· · Í í i ···· ’··’ ...........

preferably carried out by means of a membrane for the separation of substances on the basis of their size. In this way, the high molecular weight conjugate (about 150 kDa) is separated from the low molecular weight substances, such as the unreacted chelator of formula (I), and the salts. The separation preferably takes place on membranes with a cut-off of 20 to 100 kDa, more preferably with a cut-off of 30 to 40 kDa. In a preferred embodiment, the resulting reaction mixture, after conjugation, is diluted to about twice the volume of the buffer used in the reaction, and the solution is applied to a column with an appropriate membrane. The column was then eluted with an aqueous solution of ammonium acetate (NFUOAc, 0.05 M).

Preferably, the purified conjugate solution is lyophilized. The lyophilized preparation can be stored for several months at 2-8 ° C without changing the quality of the prepared conjugate.

The present invention further provides a complex conjugate of a monoclonal antibody nimotuzumab and chelator of formula FTY with a radionuclide, which comprises a conjugate of a monoclonal antibody nimotuzumab and chelator formulas / Of the present invention, wherein at least one chelating agent of the formula / 1 / forming a coordination compound with a radionuclide selected from the group consisting of ⁴⁴ Sc, ⁴⁷ Sc, ⁶⁴ Cu, ⁶⁷ Cu, ⁹⁰ Y, ^H1 In, ¹⁸⁶ Re, ¹⁸⁸ Re, lanthanide radioisotopes, ²¹² Pb and ²¹³ Bi, preferably the radionuclide is selected from the group consisting of lanthanide radioisotopes, ⁴⁷ Sc, ⁶⁷ Cu, ^H1 In is ⁹⁰ Y, most preferably the radionuclide is ¹⁵³ Sm, ¹⁶⁶ Ho, ^1H In and ¹⁷⁷ Lu. Due to the presence of an acidic phosphorus group, the chelator of formula (I and its complexes is more hydrophilic than analogous complexes with DOTA or DOTA-NCS, and the complexation proceeds under milder conditions, faster and with higher yields.

The present invention also provides a process for preparing a nimotuzumab monoclonal antibody-chelator complex of formula (I) with a radionuclide of the present invention, wherein an aqueous solution of the radionuclide salt conjugate is mixed with an aqueous solution of nimotuzumab monoclonal antibody-chelator of formula / 1 according to the present invention, and the resulting reaction the mixture is reacted at a temperature of up to 50 ° C for at least 5 minutes. Preferably, the reaction temperature is in the range of from 10 to 50 ° C, more preferably in the range of from 20 to 45 ° C, most preferably in the range of from 35 to 40 ° C. The reaction time depends on the type of radionuclide and is preferably in the range of 30 minutes to 4 hours, more preferably in the range of 1 H to 2.5 h. In one embodiment, the radionuclide is ¹⁷⁷ Lu and the reaction time is in the range of 60 to 90 min. In another embodiment, the radionuclide is ¹⁶⁶ Ho and the reaction time ranges from 90 to 120 min. In another embodiment, the radionuclide is ⁹⁰ Y and the reaction time ranges from 60 to 90 min. In another embodiment, the radionuclide is ¹⁵³ Sm and the reaction time ranges from 90 to 120 min. In another embodiment, the radionuclide is ^H1 In and the reaction time ranges from 120 to 150 min. Preferably, especially in the case of emitters, after the reaction is complete, sodium ascorbate / ascorbic acid is added to the reaction mixture in an amount of 5 to 10% by weight. . of the resulting solution. Ascorbate / ascorbic acid serves as a stabilizer against radiolysis.

The radionuclide salt is understood to mean the water-soluble salt of the inorganic acid and the radionuclide cation, preferably the radionuclide salt is the radionuclide chloride.

In a preferred embodiment of the process for the preparation of the nimotuzumab monoclonal antibody-chelator complex of formula (Ir) with the radionuclide of the present invention, the reaction is carried out at a pH of 5 to 8, preferably a pH of 6 to 7. or HEPES buffer.

The present invention also provides a conjugate of the monoclonal antibody nimotuzumab and a chelator of formula - (I) - according to the present invention and / or a complex of this conjugate with a radionuclide according to the present invention for use in treating cancer, particularly for targeted treatment of tumors with EGFR overexpression. such as colorectal carcinomas, epidermal head and neck tumors, prostate carcinoma, pancreatic carcinoma, esophageal carcinoma, non-small cell lung carcinoma, breast carcinoma, ovarian carcinoma, and non-epithelial carcinomas such as gliomas.

t Brief description of the drawings

Giant. 1: Identification of immunoconjugates by SDS-PAGE; hR3 denotes the native nimotuzumab antibody, K1 denotes the antibody-chelator conjugate at a molar ratio of 1: 200 and K2 denotes the antibody-chelator conjugate at a molar ratio of 1:50.

Giant. 2: HPLC chromatogram of nimotuzumab conjugate, typical retention time is 9-10 min. The chromatogram shows no impurities with retention times of 11 to 12 min.

Giant. 3A: Radiochromatogram ¹⁷⁷ Lu-DOTA-nimotuzumab

Giant. 3B: Radiochromatogram Lu-chelator of formula (I) -nimotuzumab

Giant. 4: Radio-TLC chromatogram of the complex of conjugate ^{1777 L} -chelator of formula (I) -nimotuzumab.

Examples of terrestrial water of the invention

DOTA-NCS was purchased from Macrocyclics (USA). The chelator of the formula was synthesized according to previously published data (Rudovsky J., Botta M., Hermann P., Koridze A.,

...........

Aime S., Dalton Trans., 2006, 2323-2333). The monoclonal antibody nimotuzumab was purchased from Oncoscience AG (GER) and stored according to the supplier's instructions. Demineralized water was used in the experiments, which was further free of traces of heavy metal ions using Chelex resin, or highly pure TraceSelect® purity water. Buffer solutions were freed of traces of heavy metals using Chelex resin. Chelex chelating resin is handled according to the manufacturer's instructions (Sigma Aldrich). All the chemicals used were of a purity consistent with trace analysis. The laboratory dishes used were washed with 5 M aqueous HCl prior to use, then rinsed with high-purity water (TraceSelect®) and further dried without dust.

Example 1 Conjugation of nimotuzumab monoclonal antibody with DOTA-NCS

The conjugation of the monoclonal antibody nimotuzumab with DOTA-NCS was performed at a molar ratio of antibody: ligand = 1:50. A solution containing 2.5 mg of antibody was mixed with an aqueous buffer solution (0.2 M EPPS, pH 8.5 to 8.6) and an appropriate volume of an aqueous ligand solution in 0.2 M EPPS (concentration 10 mg / ml, pH 8). , 5 to 8.6). The amount of buffer added is chosen so that the total volume of the reaction mixture after mixing the components is 500 μΐ. The mixture is shaken on a Vortex (gentle shaking, ca. 450 rpm) for 2 h at room temperature. Immediately after the reaction time, the resulting conjugate was purified on PD- chromatography columns.

10. The column is first washed with aqueous NtLjOAc (2 x 10 mL and then 1 x 5 mL, 0.05 M, pH 7.0). Subsequently, the column is saturated with 1 ml solution (5 mg / ml) of native antibody. The excess antibody is rinsed with aqueous NH 4 OAc solution (2 x 10 mL and 1 x 5 mL, 0.05 M, pH 7.0). The column is now ready for conjugate purification. The individual fractions are collected into a set of eight clean and sterile Eppendorf plastic microtubes (1.5 ml). The reaction mixture is diluted with EPPS buffer (0.2 M, pH 8.5 to 8.6) to a total volume of 1 ml and the conjugate solution is applied to the column. After receiving the first fraction, 1 ml of an aqueous NH 4 OAc solution (0.05 M, pH 7.0) is sequentially applied to the column and the individual fractions are eluted. After elution of the 8th fraction, the column is washed with aqueous NH 4 OAc (2 x 10 mL, 0.05 M, pH 7.0). Fractions 3, 4 and 5 are transferred to a Vivaspin 6 vial. The vial volume is made up to 6 mL with NH 4 OAc aqueous solution (0.05 M, pH 7.0) and the conjugate solution is centrifuged for 30 min at 4000 rpm. The volume remaining at the top of the Vivaspin should be less than 400 μΐ. Aqueous 0.05 M NH 4 OAc (1 mL, pH 7.0) is added to the top of the vial and the solution is shaken for 5 min on Vortex at 1000 rpm. Subsequently, the solution is transferred to a clean and sterile Eppendorf plastic tube (1.5 ml size). The thus obtained pure conjugate solution is used for labeling or is stored in the form of a lyophilizate or in the form of a solution stored in a refrigerator at 2 to 8 ° C.

Example 2: Conjugation of nimotuzumab monoclonal antibody with a chelator of formula

The conjugation of the nimotuzumab monoclonal antibody with the chelator of formula Jt \ y is performed at a molar ratio of antibody: ligand = 1:50. A solution containing 2.5 mg of antibody was mixed with an aqueous buffer solution (0.2 M EPPS, pH 8.5 to 8.6) and an appropriate volume of an aqueous ligand solution in 0.2 M EPPS (concentration 10 mg / ml, pH 8). , 5 to 8.6). The amount of buffer added is chosen so that the total volume of the reaction mixture after mixing the components is 500 μΐ. The mixture is shaken on a Vortex (gentle shaking, about 450 rpm) for 2 h at room temperature. Immediately after the reaction time, the resulting conjugate was purified on PD-10 columns. The column is first washed with aqueous NH 4 OAc (2 x 10 mL and then 1 x 5 mL, 0.05 M, pH 7.0). Subsequently, the column is saturated with 1 ml solution (5 mg / ml) of native antibody. The excess antibody is rinsed with aqueous NH ₄ OAc (2 x 10 mL and 1 x 5 mL, 0.05 M, pH 7.0). The column is now ready for conjugate purification. The individual fractions are collected into a set of eight clean and sterile Eppendorf plastic microtubes (1.5 ml). The reaction mixture is diluted with EPPS buffer (0.2 M, pH 8.5 to 8.6) to a total volume of 1 ml and the conjugate solution is applied to the column. After the first fraction was collected, 1 ml of an aqueous NH ₄ OAc solution (0.05 M, pEl 7.0) was successively applied to the column and the individual fractions were eluted. After elution of the 8th fraction, the column is washed with aqueous NH 4 OAc solution (2 x 10 mL, 0.05 M, pH 7.0). Fractions 3,4 and 5 are transferred to a Vivaspin 6 vial. The vial volume is made up to 6 ml with aqueous NH ₄ OAc (0.05 M, pH 7.0) and the conjugate solution is centrifuged for 30 min at 4000 rpm. The volume remaining at the top of the Vivaspin vial should be less than 400 μ Do. To the top of the vial is added aqueous 0.05 M NH ₄ OAc (1 mL, pH 7.0) and the solution is shaken for 5 min on Vortex at 1000 oscillations / min. The solution is then transferred to a clean and sterile Eppendorf plastic microtube (size 1.5 ml). The thus obtained pure conjugate solution is used for labeling or is stored in the form of a lyophilizate or in the form of a solution stored in a refrigerator at 2 to 8 ° C.

Example 3: Lyophilization of conjugate of monoclonal antibody nimotuzumab and chelator of formula Conjugate solution immediately after purification according to the procedure described in Example 2 is lyophilized: To the conjugate solution, trehalose is added in an amount of 200 mmol per mg of conjugate and allowed to dissolve completely. Then the sample vial is placed in a liquid nitrogen bath and after freezing the vial is immediately placed in the prepared lyophilizer and the lyophilization process is started.

• Λ

After freeze-drying, the vial is closed with a rubber cap and stored in a refrigerator at 2 to 8 ° C until further processing.

Example 4: Conjugation of the nimotuzumab monoclonal antibody with a chelator of formula (s) in the presence of DMSO

The conjugation of the antibody to the chelator of the formula (iy is performed at a molar ratio of antibody: ligand = 1: 50. A solution containing 2.5 mg of antibody is mixed with an aqueous buffer solution (0.2 M EPPS, pH 8.5 to 8.6) and the appropriate volume of ligand solution in anhydrous DMSO (concentration 50-60 mg / ml) The amount of buffer added is chosen so that the total volume of the reaction mixture after mixing the components is 500 μΐ. min) for 2 h at room temperature After the reaction time, the resulting conjugate is purified on PD-10 columns, and the column is first washed with aqueous NHLjOAc (2 x 10 mL and then 1x5 mL, 0.05 M, pH 7). Subsequently, the column is saturated with 1 ml (5 mg / ml) of native antibody and the excess antibody is rinsed with aqueous NH 4 OAc (2 x 10 ml and 1 x 5 ml, 0.05 M, pH 7.0). The column is ready for purification of the conjugate Mint into a set of eight clean and sterile Eppendorf plastic microtubes (1.5 ml). The reaction mixture is diluted with EPPS buffer (0.2 M, pH 8.5 to 8.6) to a total volume of 1 ml and the conjugate solution is applied to the column. After receiving the first fraction, 1 ml of an aqueous NH 4 OAc solution (0.05 M, pH 7.0) is sequentially applied to the column and the individual fractions are eluted. After elution

The 8th fraction was washed with aqueous NH 4 OAc (2x10 mL, 0.05 M, pH 7.0). Fractions 3, 4 and 5 are transferred to a Vivaspin 6 vial. The vial volume is supplemented with 0.05 M NH ₄ OAc buffer per 6 ml and the conjugate solution is centrifuged for 30 min at 4000 rpm. The volume remaining at the top of the Vivaspin should be less than 400 μΐ. Aqueous 0.05 M NH 4 OAc (1 mL, pH 7.0) is added to the top of the vial and the solution is shaken for 5 min on Vortex at 1000 rpm. The solution is then transferred to a clean and sterile Eppendorf plastic tube (1.5 ml size). The pure conjugate solution thus obtained is immediately further processed, stored at 2 to 8 ° C for a maximum of 2 days or immediately lyophilized.

Example 5: Preparation of the DOTA-NCS-nimotuzumab conjugate complex with ¹⁷⁷ Lu Lu The purified DOTA-NCS-nimotuzumab conjugate prepared in Example 1 is transferred into an Eppendorf plastic microtube (1.5 ml volume) in an amount of 0.5 to 0.7 ml and NH 4 OAc (0.5 M, pH 7.0) was added to a final volume of 1 mL (including the radionuclide to be added). To this solution is added an aqueous solution of radioactive lutecium chloride in 0.04 M aqueous HCl, in an amount corresponding to a ratio of 100 MBq Lu per 1 mg

A .1

conjugate. The pH is checked and should be in the range of 6.0 to 7.0 without further adjustments. The reaction mixture is then incubated at 37 ° C for 1 h. After the incubation is complete, 5 mg of sodium ascorbate is added and the preparation is sterilized by filtration through a Millex-GV filter and divided into individual sterile vials for further use. Subsequently, quality control of the product is performed.

Example 6: Preparation of the nimotuzumab monoclonal antibody conjugate complex with a chelator of the formula ¹⁷⁷ Lu

The purified conjugate in an amount of 0.5 to 0.7 ml is transferred to an Eppendorf plastic microtube (1.5 ml volume) and supplemented with aqueous NH ₄ OAc (0.5 M, pH 7.0) so that the resulting volume of the solution ( including the radionuclide to be added) was 1 ml. To this solution is added an aqueous solution of radioactive lutecium chloride in 0.04 M HCl, in an amount corresponding to a ratio of 100 MBq Lu per 1 mg of conjugate. The pH is checked and should be in the range of 6.0 to 7.0 without further adjustments. The reaction mixture is then incubated at 37 ° C for 1 h. After the incubation is complete, 5 mg sodium ascorbate is added and the product is sterilized by filtration through a Millex-GV filter (filter for sterilizing protein solutions) and filled into individual sterile vials for further use. Subsequently, quality control is performed

1 * of the formulation which is further described in Examples 11 and 12.

Example 7: Determination of the number of chelate molecules of formula (f) per conjugate molecule

To determine the number of chelated molecules of formula (I) bound to the nimotuzumab monoclonal antibody molecule, a ¹⁷⁷ Lu carrier solution is used (i.e., a trace amount of ¹⁷⁷ Lu is added to the Lu ³⁺ solution at the desired concentration in 0.05 M aqueous HCl) in order to add about 10 MBq of ¹⁷⁷ Lu) to 10 ~ ⁷ moles of Lu ³⁺ . This solution is then added to the conjugate solution such that the molar ratios of antibody: Lu ³⁺ correspond to 1: 3 to 1:20. The pH of the reaction solution is adjusted to pH 5 by the addition of 0.5 M aqueous ammonium acetate. The reaction solution is then incubated at 42 ° C for 2h _M. After this time, a 0.01 M aqueous DTP A solution (pH 6) was added to the reaction mixture, and the mixture was allowed to react for an additional 15 min at room temperature. Binding of free ¹⁷⁷ Lu to DTPA completes labeling of the conjugate. Then, 2 μΐ of the mixture is applied to a TLC (silica gel impregnated glass fiber chromatography paper (iTLC-SG)) plate. The plate is developed in ascending order with a 1: 1 (v / v) mixture of 1 M (77 g dm ^-3 ) ammonium acetate and methanol. The activity distribution on the TLC is determined using a TLC scanner. In a given chromatographic system, the ¹⁷⁷ Lu-labeled conjugate remains at the start of the chromatographic track (Rf = 0) and the complex

17 * 7

Lu-DTPA moves with the front of the mobile phase (Rf ~ 1). The labeling yield is reported as a percentage of the activity at the start of the chromatogram relative to the sum of the activity over the whole chromatogram.

The number of chelator groups of formula XX per antibody molecule was calculated from the observed TLC labeling yields and the known antibody: metal ratio used according to the formula:

Chelator of formula (I) ·) = radiochemical yield (%) x (metal: conjugate ratio) x 100

Example 8: Confirmation of identity of prepared conjugate by electrophoresis method

Immunoconjugates are analyzed by SDS polyacrylamide gel electrophoresis under non-reducing and reducing conditions. SDS-PAGE is selected as an analytical method to verify the integrity of the nimotuzumab monoclonal antibody used both before and after ligand binding. Under non-reducing conditions, an entire IgG molecule with a molecular weight of ~ 150 kDa can be detected by SDS-PAGE. Under reducing conditions, the inter- and intramolecular disulfide bridges are cleaved by β-mercaptoethanol, thereby splitting the IgG molecule into subunits, 2 light (~ 25 kDa) and 2 heavy (~ 50 kDa) chains. This analysis allows effective control of the antibody molecule both before and after binding with the ligand before radiolabeling (Lu). SDS-PAGE verifies that the mAb molecule has not been degraded during the ligand binding process and several subsequent purification steps by centrifugation in Vivaspin.

Proteins are denatured prior to SDS-PAGE for 5 min at 100 ° C in sample buffer containing SDS. The reducing conditions are ensured by the presence of β-mercaptoethanol. Each well of the gel is loaded with 2 µg of proteins (according to the Bradford protein concentration values). Proteins are then separated using a NuPAGE 4? -12% gradient gel in a Cleaver Scientif. using commercial electrode buffer NuPAGE MES or NuPAGE MOPS. Gel staining of the separated proteins is performed with Coomassie Blue R-250.

Under non-reducing conditions, one gel band of ~ 150 kDa corresponding to the entire IgG molecule is expected to be detected in the gel. Under reducing conditions, two dominant bands of about 50 kDa and 25 kDa were detected. Giant. 1 demonstrates an exemplary SDS-PAGE of two prepared immunoconjugates (ΚΙ, K2). K1 denotes an antibody-chelator conjugate in a molar ratio of 1: 200 and K2 denotes an antibody-chelator conjugate in a molar ratio of 1:50. Conjugation of the antibody with the ligand also results in a visible slowing (shift) of the bands of these immunoconjugates over the original, unconjugated antibody molecule.

• 9. 9

Example 9: Confirmation of the identity of the prepared conjugate by HPLC

A sample is taken from the conjugate solution for HPLC in a volume of 50 to 100 μΐ into a 0.51 ml Eppendorf plastic microtube. Approximately 30 μΐ of solution was applied per analysis. The measurement is performed on an Agilent 1200 HPLC kit with UV and radiometric detection. The stationary phase consists of a TSK-Gel 3000 gel column (7, 300 mm, 10 µm) and a mobile phase of 0.01 M PBS with 0.05% DTPA.

Detection is done by VWD detector (UV, 280 nm) and also by radiometric detector Gábi Raytest. The measurement is isocratic for 20 min.

In this embodiment, the conjugate has a retention time of 9 to 10 min. Possible residues of the free ligand or other lower molecular weight impurities exhibit typical retention times of 11 to 12 min (FIG. 2).

Example 10: Determination of the immunoreactivity of the conjugate

The indirect non-competitive ELISA (Enzyme-Linked ImmunoSorbent Assay) is used to monitor the immunoreactivity of nimotuzumab. Human recombinant EGFR (Sino Biological Inc, China) is immobilized to the surface of the wells of immunoassays at constant concentration (100 ng / well in 0.01 M PBS, pH 7.4; PBS = phosphate buffered saline) ". Saturation of the free binding sites on the surface of the wells with 3% BSA solution (BSA = povine §erum-lbumin) in 0.01M PBS, pH 7.4 followed by incubation with primary antibody (nimotuzumab / DOTA-NCS-nimotuzumab / chelator of formula fYf-nimotuzumab) diluted in 1.5% BSA in 0.01 M PBS, pH 7.4. The resulting immunocomplex is visualized with an enzyme-labeled secondary antibody specific to the human IgG Fe fragment (1:20,000 dilution in 1.5% BSA in 0.01 M PBS, pH 7.4). The enzyme reaction product of the chromogenic substrate OPD (OPD = o-phenylenediamine) with horseradish peroxidase (HRP) is then detected spectrophotometrically with a maximum absorbance at 492 nm on a Tecan Sunrise instrument.

To quantify the immunoreactivity, the linear portion of the dependence obtained from the ELISA was used. In this region, the binding of the antibody to the antigen occurs by pseudo-first order kinetics. The experimentally obtained data can be obtained by approximation of the equation of lines and by the ratio of their slopes to obtain the immunoreactivity of the conjugate against the unmodified nimotuzumab form. The results are shown in Table 1.

Table 1: Comparison of the immunoreactivity of inactive nimotuzumab-chelator formulas and DOTA-NCS to human recombinant EGFR as determined by ELIS A and their comparison with the number of ligand molecules per immunoconjugate molecule.

Ligand in conjugate Number of ligand molecules per antibody molecule In vitro immunoreactivity (%) ' chelator formula (I / 1,8 60 DOTA NCS 2.3 36 DOTA NCS 2.6 44 DOTA NCS 3.9 36 chelator of formula (Iy 3.9 56 chelator of formula (G 6.9 41 DOTA NCS 7.6 18 chelator of formula XI) 7.9 30 DOTA NCS 8.3 26

Immunoreactivity was determined by ELIS A, relative to native nimotuzumab (= 100%).

Example 11: Determination of radiochemical purity of the radionuclide conjugate complex by HPLC

From a solution of the conjugate complex with a radionuclide, a sample is taken for HPLC in a volume of 50 to 100 μΐ into a 0.5 ml Eppendorf plastic microtube. Approximately 30 μΐ of solution is applied per analysis. The measurement is performed on an Agilent 1200 HPLC kit with UV and radiometric detection. The stationary phase consists of a TSK-Gel 3000 gel column (7.5θ300 mm, 10 µm) and a mobile phase of 0.01 M PBS with 0.05% aqueous DTPA. Detection is done by VWD detector (UV, 280 nm) and also by radiometric detector Gábi Raytest. The measurement is isocratic for 20 min. d

HPLC chromatograms of ¹⁷⁷ Lu-labeled nimotuzumab conjugates are shown in FIG. 3A and 3B. In this arrangement, the labeled conjugate has a typical retention time of 9 to 10 min. other labeled impurities 11 to 12 min.

Example 12: Radiochemical purity of the radionuclide conjugate complex by the TLC method The radiochemical purity of the radionuclide conjugate complex prepared according to the procedure of Example 6 is determined by thin layer chromatography (TLC). An aliquot is removed from the labeling mixture upon completion of incubation and before the addition of ascorbic acid to which 0.01 M DTPA solution (pH 6) is added and the mixture is allowed to react for an additional 15 min at room temperature. After this time, 2 μΐ of the reaction mixture is loaded onto silica gel impregnated glass fiber chromatography paper (iTLC-SG), which is ascending a mobile phase (1 M (77 g dm) ammonium acetate / methanol 1: 1 (v / v)). in)). The activity distribution on the developed TLC plate is detected by the TLC activity scanner. In a given chromatographic system, the radionuclide-labeled conjugate remains at the start of the chromatographic pathway (Rf = 0) and

177 &

the complex moves with the front of the mobile phase (Rf ~ 1) (Lu-DTPA see 8 [br. 4). Radiochemical purity corresponds to the percentage of activity at the start of the chromatogram relative to the sum of the activity over the whole chromatogram.

Example 13: Preparation of the nimotuzumab monoclonal antibody conjugate complex with a chelator of formula (I) with a ¹⁶⁶ Ho radionuclide

The purified conjugate prepared in Example 4 in an amount of 0.5 to 0.7 ml is transferred to an Eppendorf plastic microtube (1.5 ml volume) and supplemented with aqueous NH 4 OAc solution (0.5 M, pH 5.5) to give a final volume of the solution (including the radionuclide to be added) was 1 ml. To this solution is added an aqueous solution of radioactive holmit chloride in 0.2 M HCl, in an amount corresponding to a ratio of 100 x 2500 MBq of ¹⁶⁶ Ho per 1 mg of conjugate. The pH is checked and should be in the range of 5.0 to 6.0 without further adjustment. The reaction mixture is then incubated at 37 ° C for 1.5 h. After incubation, 5 mg sodium ascorbate is added and the product is sterilized by filtration through a Millex-GV filter (filter for sterilizing protein solutions) and filled into individual sterile vials. for further use. Subsequently, the quality control of the formulation is carried out as further described in Examples 11 and 12.

Example 14: Preparation of the nimotuzumab monoclonal antibody conjugate complex with a chelator of formula (I) with radionuclide ^{9 (1} Y)

The purified conjugate prepared in Example 2 in an amount of 0.5 to 0.7 ml is transferred to an Eppendorf plastic microtube (1.5 ml volume) and supplemented with aqueous NH 4 OAc solution (0.5 M, pH 7.0) to give a final volume. of the solution (including the radionuclide to be added) was 1 ml. To this solution is added an aqueous solution of radioactive yttrium chloride in 0.04 M HCl, in an amount corresponding to a ratio of 100-1000 MBq of ⁹⁰ Y per mg of conjugate. The pH is checked and should be in the range of 6.5 to 7.5 without further adjustment. The reaction mixture is then incubated at 37 ° C for 1 h. After the incubation is complete, 5 mg of sodium ascorbate is added and the product is sterilized by filtration through a Millex-GV filter (filter designed for

sterilization of protein solutions) and filled into individual sterile vials for further use.

Subsequently, the quality control of the formulation is carried out as further described in Examples 11 and 12.

Example 15: Preparation of the nimotuzumab monoclonal antibody conjugate complex with a chelator of formula (I) with a ¹⁵³ Sm radionuclide

The purified conjugate prepared in Example 2 in an amount of 0.5 to 0.7 ml is transferred to an Eppendorf plastic vial (1.5 ml volume) and supplemented with aqueous NH 4 OAc solution (0.5 M, pH 4.8) to give a final volume of the solution (including the radionuclide to be added) was 1 ml. To this solution is added an aqueous solution of Samarium Chloride Chloride in 0.2 M HCl in an amount corresponding to a ratio of 100 r-1000 MBq Sm per mg conjugate. The pH is checked and should be in the range of 4.5 to 5.5 without further adjustments. The reaction mixture is then incubated at 37 ° C for 1.5 h. After incubation, 5 mg of sodium ascorbate is added and the product is sterilized by filtration through a Millex-GV filter (filter for sterilizing protein solutions) and filled into individual sterile vials. for further use. Subsequently, the quality control of the formulation is carried out as further described in Examples 11 and 12.

Example 16: Preparation of the nimotuzumab monoclonal antibody conjugate complex with a chelator of formula (I) with a radionuclide ¹¹¹ In ¼

The purified conjugate prepared in Example 2 in an amount of 0.5 to 0.7 ml is transferred to an Eppendorf plastic microtube (1.5 ml volume) and supplemented with aqueous NH 4 OAc solution (0.5 M, pH 5.5) to give a final solution volume. (including the radionuclide to be added) was 1 ml. To this solution is added an aqueous solution of radioactive ^her InC13 in 0.2 M HC1 in an amount of 42 '.. 1 corresponding to a ratio of 100 MBq of In 1 000 to 1 mg of conjugate. The pH is checked and should be in the range of 5.0 to 6.0 without further adjustment. The reaction mixture is then incubated at 37 ° C for 2 h. After incubation, 5 mg sodium ascorbate is added and the product is sterilized by filtration through a Millex-GV filter (filter for sterilizing protein solutions) and filled into individual sterile vials for further use. Subsequently, the quality control of the formulation is carried out as further described in Examples 11 and 12.

Claims (10)

A conjugate of the nimotuzumab monoclonal antibody or Fab fragment thereof and at least one chelator of formula / 1? HOOC NCS Wherein the at least one chelator of formula (Dd) is covalently attached by a thiourea bridge formed by reaction of the isothiocyanate group of the chelator of formula (1) and the amino group of the amino acid lysine present in the monoclonal antibody nimotuzumab. The conjugate of claim 1, comprising up to 10 chelator molecules of formula (IJ) covalently bound by a thiourea linkage formed by the reaction of the isothiocyanate group of the chelator of formula / I and the amino group of the amino acid lysine present in the monoclonal antibody nimotuzumab. A method for preparing a conjugate according to claim 1 or 2, characterized in that the nimotuzumab solution of pH 8.0 to 9.2 is mixed with a chelator solution of formula (I / pH pH 8.0 to 9), 2, wherein the molar ratio of the monoclonal antibody nimotuzumab to the chelator of the formula χΐγ is in the range of 1:10 to 1: 100; the resulting reaction mixture is reacted for at least 10 minutes at a temperature in the range of 4 to 40 ° C to form a conjugate of the monoclonal antibody nimotuzumab and a chelator of formula (I) according to any one of the preceding claims. Process according to claim 3, characterized in that the solvent of the reaction mixture is water or water containing up to 20% by volume of DMSO. The method of claim 3 or 4, wherein the reaction mixture further comprises a buffer to maintain a constant pH value selected from the group consisting of phosphate buffer, acetate buffer, TRIS buffer, HEPES buffer and EPPS buffer. • · ······· · ···· · · · · · · · · · · · · Method according to any one of claims 3 to 5, characterized in that it further comprises the step of purifying the reaction mixture, preferably by membrane filtration. A nimotuzumab monoclonal antibody conjugate / radionuclide chelator complex comprising a conjugate of the nimotuzumab monoclonal antibody and a chelator of formula / Ijf according to any one of claims 1 to 2, wherein at least one chelator of formula / If forms a coordination compound with a radionuclide selected from the group comprising ⁴⁴ Sc, ⁴⁷ Sc, ⁶⁴ Cu, ⁶⁷ Cu, ⁹⁰ Y, ^1H In, ¹⁸⁶ Re, ¹⁸⁸ Re, lanthanide radioisotopes, ²¹² Pb and ²¹³ Bi, preferably the radionuclide is selected from the group consisting of lanthanide radioisotopes, ⁴⁷ Sc, ⁶⁷ Cu , 'ln and ⁹⁰ Y, most preferably the radionuclide is ¹⁵³ Sm, ¹⁶⁶ Ho, ¹¹ 4η and ¹⁷⁷ Lu. A process for the preparation of a complex according to claim 7 of a nimotuzumab monoclonal antibody-chelator of formula (I) with a radionuclide, characterized in that the aqueous solution of the radionuclide salt is mixed with an aqueous solution of nimotuzumab monoclonal antibody-chelator of formula (I) and to 50 ° C for at least 5 minutes. The process of claim 8 wherein the reaction is carried out at a pH in the range of 5 to 8. The nimotuzumab monoclonal antibody-chelator of formula / If conjugate according to claim 1 or 2 and / or the radionuclide conjugate complex of claim 7 for use in the treatment of cancer.