ANTIBODY-DRUG CONJUGATES AND THEIR USE IN THERAPY

Abstract

The invention relates to cytotoxic conjugates and antibody-drug conjugates, and to their use in therapy, in particular for treating HER2+ cancers.

Claims

1. A cytotoxic conjugate of formula (I) below: ##STR00054## in which: the attachment head is represented by either one of the two formulae below: ##STR00055## the linker arm is a cleavable linker arm chosen from the formulae below: ##STR00056## the spacer is represented by the formula below: ##STR00057## X is Br, Cl, I or F; m is an integer ranging from 1 to 10.

2. The cytotoxic conjugate as claimed in of claim 1, wherein X is Br and m is equal to 4 or 5.

3. The cytotoxic conjugate of claim 1, wherein the cytotoxic drug is chosen from methotrexate, IMiDs, duocarmycin, combretastatin, calicheamicin, monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), maytansine, DM1, DM4, SN38, amanitin, pyrrolobenzodiazepine, pyrrolobenzodiazepine dimer, pyrrolopyridodiazepine, pyrrolopyridodiazepine dimer, a histone deacetylase inhibitor, a tyrosine kinase inhibitor, and ricin, preferably MMAE.

4. The cytotoxic conjugate of claim 1, of formula (Ia) below: ##STR00058##

5. An antibody-drug conjugate of formula (II) below: ##STR00059## which: A is an anti-HER2 antibody or antibody fragment; the attachment head is represented by either one of the two formulae below: ##STR00060## the linker arm is a cleavable linker arm represented by the formula below: ##STR00061## the spacer is represented by the formula below: ##STR00062## X is Br, Cl, I or F; m is an integer ranging from 1 to 10; n is an integer ranging from 1 to 4.

6. The antibody-drug conjugate of claim 5, wherein X is Br and m is equal to 4 or 5.

7. The antibody-drug conjugate of claim 5, wherein the cytotoxic drug is chosen from methotrexate, IMiDs, duocarmycin, combretastatin, calicheamicin, monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), maytansine, DM1, DM4, SN38, amanitin, pyrrolobenzodiazepine, pyrrolobenzodiazepine dimer, pyrrolopyridodiazepine, pyrrolopyridodiazepine dimer, a histone deacetylase inhibitor, a tyrosine kinase inhibitor, and ricin, preferably MMAE.

8. The antibody-drug conjugate of claim 5, wherein A is trastuzumab.

9. The antibody-drug conjugate of claim 5, of formula (IIa) below: ##STR00063##

10. A composition comprising one or more antibody-drug conjugate(s) of claim 5.

11. The composition of claim 10, wherein at least 50%, preferably at least 65%, of the antibody-drug conjugates have an n equal to 4.

12. The composition of claim 10, wherein A is an antibody and the average Drug-to-Antibody Ratio (average DAR) is between 3.5 and 4.5, preferably between 3.8 and 4.2, for example equal to 4.0 ±0.2.

13. The composition as claimed in of claim 10, further comprising paclitaxel, docetaxel, doxorubicin, cyclophosphamide, an aromatase inhibitor such as anastrozole and/or an antibody used in anti-cancer immunotherapy such as an anti-PD1 antibody.

14. The antibody-drug conjugate of claim 4, for use as a medicament.

15. The antibody-drug conjugate of claim 4, for use in the treatment of an HER2+ cancer, preferably HER2+ breast cancer.

16. A process for preparing a cytotoxic conjugate of claim 1, comprising a step which consists in coupling an attachment head of formula: ##STR00064## with a compound of formula ##STR00065## in which: the linker arm is a cleavable linker arm chosen from the formulae below: ##STR00066## the spacer is represented by the formula below: ##STR00067## X is Br, Cl, I or F; m is an integer ranging from 1 to 10, and preferably equal to 4 or 5.

17. A process for preparing an antibody-drug conjugate of claim 5, comprising the following steps: (i) preparing a cytotoxic conjugate according to the process of claim 16, and (ii) reacting the cytotoxic conjugate obtained in step (i) with an anti-HER2 antibody or an anti-HER2 antibody fragment.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0121] FIG. 1 represents the HIC (hydrophobic interaction chromatography) profile of a McSAF-pyridine antibody-drug conjugate composition according to the invention. The figure shows that the composition is enriched in DAR4, with around 69% DAR4.

[0122] FIG. 2 represents an SEC (size exclusion chromatography) analysis of a McSAF-pyridine antibody-drug conjugate composition according to the invention.

[0123] The figure shows that the composition is extremely homogeneous, with more than 99% monomer.

[0124] FIG. 3 represents the distribution of the DARs of 14 independent bioconjugations on the 1 mg scale. This demonstrates the high reproducibility of the bioconjugation for obtaining the McSAF-pyridine antibody-drug conjugate.

[0125] FIG. 4 represents the distribution of the DARs of various bioconjugations on various scales (1 mg, 2.5 mg and 5 mg scales). This demonstrates the high reproducibility of the bioconjugation for obtaining the McSAF-pyridine antibody-drug conjugate irrespective of the scale.

[0126] FIG. 5 represents the distribution of the DARs of a representative bioconjugation for obtaining the McSAF-pyridine antibody-drug conjugate by native mass spectrometry analysis. The figure demonstrates the chemical nature of the conjugate and shows that the composition is enriched in DAR4, with around 80% DAR4.

[0127] FIG. 6 represents the recognition of the HER2 antigen by McSAF-pyridine, T-DM1 and trastuzumab.

[0128] FIG. 7 represents the in vitro cytotoxicity of McSAF-pyridine on cells expressing HER2, or cells not expressing HER2, compared to T-DM1 and MMAE alone.

[0129] FIG. 8 represents the amount of MMAE released in human plasma by LC-MS/MS, comparing McSAF-pyridine with an ADC prepared using maleimide coupling chemistry (McSAF-maleimide1). This figure reflects the stability of the McSAF-pyridine antibody-drug conjugate.

[0130] FIG. 9 represents the change in the average DAR in solution at 37° C. of McSAF-pyridine compared to an ADC prepared using maleimide coupling chemistry (McSAF-maleimide1). This figure reflects the stability of the McSAF-pyridine antibody-drug conjugate.

[0131] FIG. 10 represents the change in the average DAR in solution in the presence of HSA (human serum albumin) at 37° C. of McSAF-pyridine compared to an ADC prepared using maleimide coupling chemistry (McSAF-maleimide1). This figure reflects the stability of the McSAF-pyridine antibody-drug conjugate.

[0132] FIG. 11 represents the change in the average DAR in solution at 40° C. over 28 days of McSAF-pyridine compared to an ADC prepared using maleimide coupling chemistry (McSAF-maleimide1), as measured by the HIC method. This figure reflects the stability of the McSAF-pyridine antibody-drug conjugate.

[0133] FIG. 12 represents the change in the DAR4 percentage in solution at 40° C. over 28 days of McSAF-pyridine compared to an ADC prepared using maleimide coupling chemistry (McSAF-maleimide1), as measured by the HIC method. This figure reflects the stability of the McSAF-pyridine antibody-drug conjugate.

[0134] FIG. 13 represents the change in the monomer percentage in solution at 40° C. over 28 days of McSAF-pyridine compared to an ADC prepared using maleimide coupling chemistry (McSAF-maleimide1), as measured by the SEC method.

[0135] FIG. 14 represents the change in tumor volume of mice treated with 5 mg/kg of McSAF-pyridine, 5 mg/kg of T-DM1 or a vehicle without ADC.

[0136] FIG. 15 represents the distribution of the tumor volumes of all of the mice treated with 5 mg/kg of McSAF-pyridine, 5 mg/kg of T-DM1 or a vehicle without ADC at day 70. This figure reflects the homogeneity of the anti-tumor response to the antibody-drug conjugate at 5 mg/kg.

[0137] FIG. 16 represents the change in tumor volume of mice treated with 1 mg/kg of McSAF-pyridine, 1 mg/kg of T-DM1 or a vehicle without ADC.

[0138] FIG. 17 represents the distribution of the tumor volumes of all of the mice treated with 1 mg/kg of McSAF-pyridine, 1 mg/kg of T-DM1 or a vehicle without ADC at day 70. This figure reflects the different anti-tumor responses to the antibody-drug conjugate and to T-MD1 at 1 mg/kg.

[0139] FIG. 18 represents the denaturing mass spectrometry profile of the McSAF-pyridine conjugate. The figure shows the presence of a completely reconstructed and conjugated species (LHHL DAR4).

[0140] FIG. 19 represents the HIC (hydrophobic interaction chromatography) profile of a McSAF-pyridine retroamide antibody-drug conjugate composition according to the invention. The figure shows that the composition is enriched in DAR4, with around 68% DAR4.

[0141] FIG. 20 represents the denaturing mass spectrometry profile of the McSAF-pyridine retroamide conjugate. The figure shows the presence of a completely reconstructed and conjugated species (LHHL DAR4).

[0142] FIG. 21 represents the distribution of the DARs of a representative bioconjugation for obtaining the McSAF-pyridine retroamide antibody-drug conjugate by native mass spectrometry analysis. The figure demonstrates the chemical nature of the conjugate and shows that the composition is enriched in DAR4, with around 75% DAR4.

[0143] FIG. 22 represents the distribution of the DARs of 5 independent bioconjugations on the 250 pg scale. This demonstrates the high reproducibility of the bioconjugation for obtaining the McSAF-pyridine retroamide antibody-drug conjugate.

EXAMPLE

[0144] Example 1A: Synthesis of a Cytotoxic Conjugate According to the Invention (Pyridine)

[0145] General reaction scheme

##STR00029##

[0146] Detailed Reaction Scheme

[0147] Preparation of benzyl isonicotinate (2)

##STR00030##

[0148] Isonicotinic acid (1) (5.00 g; 40.614 mmol; 1.0 eq) was solubilized in thionyl chloride (15 mL; 206.77 mmol; 5.1 eq) and refluxed overnight. After returning to room temperature, the excess thionyl chloride was removed by evaporation under reduced pressure and then the residue obtained was dissolved in anhydrous dichloromethane (55 mL). Benzyl alcohol was added (4.2 mL; 40.614 mmol; 1.0 eq) and the mixture was stirred at reflux for 10 h. After returning to room temperature, the reaction medium was neutralized with a saturated sodium hydrogen carbonate solution and extracted with dichloromethane (3×100 mL). The organic phases were combined, washed with a saturated sodium chloride solution, dried over magnesium sulfate and concentrated under reduced pressure. The product obtained was purified by flash chromatography (SiO.sub.2, cyclohexane/ethyl acetate 50:50) to give (2) (6.97 g; 80%) in the form of a colorless oil.

[0149] .sup.1H NMR (300 MHz, DMSO) δ 8.80 (dd; J=6.1; 1.6 Hz; 2H.sub.1,5), 7.86 (dd; J=6.1; 1.6 Hz, 2H.sub.2,4), 7.56-7.29 (m; 5H.sub.9-13), 5.39 (s; 2H.sub.7).

[0150] .sup.13C NMR (75 MHz, DMSO) δ 165.0 (1C.sub.6); 151.3 (2C.sub.1,5); 137.2 (1C.sub.3); 136.1 (1C8); 129.0 (2C.sub.10, 12); 128.8 (1C.sub.11); 128.6 (2C.sub.9,13); 123.0 (2C.sub.2,4); 67.4 (1C.sub.7).

[0151] HRMS (ESI): neutral mass calculated for C.sub.13H.sub.11NO.sub.2 [M]: 213.0790; observed 213.0796.

[0152] Preparation of benzyl 2,6-bis(hydroxymethyl)isonicotinate (3)

##STR00031##

[0153] Benzyl isonicotinate (2) (2.48 g; 11.630 mmol; 1.0 eq) was dissolved in methanol (43 mL), stirred at 50° C. and concentrated sulfuric acid (320 μL; 6.016 mmol; 0.52 eq) was added. A solution of ammonium persulfate (26.500 g; 116.000 mmol; 10.0 eq) in water (43 mL) was added in two steps: a first rapid addition of 30 drops, a white suspension forming, then dropwise rapidly for 5 min. The reaction ramps up to 75° C., and then the yellow solution obtained was stirred at 50° C. for an additional 1 h. After returning to room temperature, the methanol was evaporated under reduced pressure. 50 mL of ethyl acetate were added and the medium was neutralized by addition of a saturated sodium hydrogen carbonate solution. The aqueous phase was extracted with ethyl acetate (3x100 mL) and the combined organic phases were washed with a saturated sodium chloride solution, dried over magnesium sulfate, and then concentrated under reduced pressure. The crude product was purified by flash chromatography

[0154] (Si02, dichloromethane/methanol, 95:5) to give (3) (1.56 g; 49%) in the form of a beige solid.

[0155] .sup.1H NMR (300 MHz, DMSO) δ 7.81 (s; 2H.sub.2,4); 7.55-7.32 (m; 5H.sub.9-13); 5.60 (t; J=5.9 Hz; 2H.sub.15,17); 5.40 (s; 2H.sub.7); 4.59 (d; J=5.9 Hz; 4H.sub.14,16).

[0156] .sup.13C NMR (75 MHz, DMSO) δ 165.0 (1C.sub.6); 162.8 (2C.sub.1,5); 138.0 (1C.sub.3); 135.7 (1C.sub.8); 128.6 (2C.sub.10, 12); 128.4 (1C.sub.11); 128.3 (2C.sub.9,13); 117.0 (2C.sub.2,4); 66.9 (1C.sub.7); 63.9(2C.sub.14,16).

[0157] HRMS (ESI): neutral mass calculated for C.sub.15H.sub.15NO.sub.4 [M]: 273.1001; observed 273.1001.

[0158] Preparation of 2,6-bis(hydroxymethyl)isonicotinic acid (4)

##STR00032##

[0159] Benzyl 2,6-bis(hydroxymethyl)isonicotinate (3) (1.33 g; 4.867 mmol; 1.0 eq) was dissolved in methanol (50 mL) and the solution was degassed with argon for 15 min. 10% by weight palladium-on-charcoal (133 mg) was added and the reaction medium was stirred at room temperature under a hydrogen atmosphere for 2 h. The reaction medium was filtered on dicalite (methanol rinsing). The filtrate was concentrated under reduced pressure to give (4) (849 mg; 95%) in the form of a beige solid.

[0160] .sup.1H NMR (300 MHz, DMSO) δ 7.78 (s; 2H.sub.2,4); 5.54 (s broad; 2H.sub.9,11); 4.59 (5; 4H.sub.8,10).

[0161] .sup.13C NMR (75 MHz, DMSO) δ 166.7 (1C.sub.6); 162.5 (2C.sub.1,5); 139.4 (1C.sub.3); 117.3 (2C.sub.2,4); 64.0 (2C.sub.8,10).

[0162] HRMS (ESI): neutral mass calculated for C.sub.8H.sub.9NO.sub.4[M]: 183.0532; observed 183.0526.

[0163] Preparation of methyl 6-((2,6-bis(hydroxymethyl)pyridin-4-yl)amidohexanoate (5)

##STR00033##

[0164] 2,6-bis(hydroxymethyl)isonicotinic acid (4) (50 mg; 0.273 mmol; 1 eq) was dissolved in anhydrous N,N-dimethylformamide (3.0 mL), the solution was cooled to 0° C., and then HATU (156 mg; 0.410 mmol; 1.5 eq) and 2,6-lutidine (147.0 μL; 1.260 mmol; 4.7 eq) were added. The activation solution was stirred at 0° C. for 15 min and then methyl 6-aminohexanoate (59 mg; 0.322 mmol; 1.2 eq) was added. The walls of the flask were rinsed with 2 mL of anhydrous N,N-dimethylformamide and the reaction medium was stirred at room temperature for 15 h. The reaction mixture was diluted in ethyl acetate, washed three times with a saturated sodium chloride solution, dried over magnesium sulfate, filtered and concentrated under reduced pressure. The product was purified by flash chromatography (dichloromethane/methanol, 90:10) to give (5) (76 mg; 91%) in the form of an off-white solid.

[0165] .sup.1H NMR (300 MHz, DMSO) δ 8.79 (t; J=5.6 Hz; 11-.sub.17); 7.71 (s; 2H.sub.2,4); 5.50 (t; J=5.8 Hz; 2H16,18); 4.57 (d; J=5.8 Hz; 4H.sub.15,17); 3.57 (s; 3H.sub.14); 3.25 (m; 2H.sub.8); 2.30 (t; J=7.4 Hz; 2H.sub.12); 1.62-0 1.45 (m; 4H.sub.9,11); 1.37-1.21 (m; 2H.sub.10).

[0166] .sup.13C NMR (75 MHz, DMSO) δ 173.3 (1C.sub.13); 165.1 (106); 161.8 (21,5); 142.9 (1C.sub.3); 115.8 (2C.sub.2,4); 64.1 (2C.sub.15,17); 51.2 (1C.sub.14); 38.5 under DMSO (1C.sub.8); 33.2 (1C.sub.12); 28.6 (1C.sub.9); 25.9 (1C-H); 24.2 (1C.sub.10).

[0167] HRMS (ESI): neutral mass calculated for C.sub.15H.sub.22N.sub.2O.sub.5[M]: 310.1529; observed 310.1526.

[0168] Preparation of methyl 6-(2,6-bis(bromomethyl)pyridin-4-yl)amidohexanoate (6)

##STR00034##

[0169] Methyl 6-((2,6-bis(hydroxymethyl)pyridin-4-yl)amidohexanoate (5) (55 mg; 0.177 mmol; 1 eq) was suspended in anhydrous acetonitrile (10.5 mL) and then phosphorus tribromide (50 pL; 0.532 mmol; 3.0 eq) was added dropwise. The reaction medium was stirred at 45° C. for 2 h. The solution was cooled to 0° C., neutralized with water (10 mL) and extracted with ethyl acetate (3×15 mL). The combined organic phases were washed with a saturated sodium chloride solution, dried over magnesium sulfate and concentrated under reduced pressure. The product was purified by flash chromatography (SiO.sub.2, cyclohexane/ethyl acetate 60:40) to give (6) (57 mg; 74%) in the form of a white solid.

[0170] .sup.1H NMR (300 MHz; DMSO) δ 8.83 (t, J=5.6 Hz;1H.sub.7); 7.84 (s; 2H.sub.2,4); 4.74 (s; 4H.sub.15,16); 3.57 (s, 3H.sub.14); 3.31-3.20 (m; 2H.sub.8); 2.31 (t; J=7.4 Hz; 2H.sub.12); 1.64-1.45 (m; 4H.sub.9,11); 1.39-1.22 (m, 2H.sub.10).

[0171] .sup.13C NMR (75 MHz, DMSO) δ 173.3 (1C.sub.13); 163.8 (1C.sub.6); 157.5 (2C.sub.1,5); 144.2 (1C.sub.3); 120.8 (2C.sub.2,4); 51.2 (1C.sub.14); 38.9 under DMSO (1C.sub.8); 34.1 (2C.sub.15,16); 33.2 (1C.sub.12); 28.5 (1C.sub.9); 25.9 (1C.sub.11); 24.2 (1C.sub.10).

[0172] HRMS (ESI): neutral mass calculated for C.sub.15H.sub.20Br.sub.2N.sub.2O.sub.3 [M]: 433.9841; observed 433.9832.

[0173] Preparation of 6-(2,6-bis(bromomethyl)pyridin-4-yl)amidohexanoic acid (.sup.7)

##STR00035##

[0174] Methyl 6-(2,6-bis(bromomethyl)pyridin-4-yl)amidohexanoate (6) (57 mg; 0.131 mmol; 1.0 eq) was dissolved in tetrahydrofuran (4 mL) and a solution of hydrated lithium hydroxide (8 mg; 0.327 mmol; 2.5 eq) in water (4 mL) was added slowly. The reaction medium was stirred at room temperature for 8.5 h. The tetrahydrofuran was evaporated under reduced pressure and the aqueous residue was treated with an aqueous 1N hydrochloric acid solution and extracted with ethyl acetate (3x10 mL). The combined organic phases were washed with a saturated sodium chloride solution, dried over magnesium sulfate and concentrated under reduced pressure. The product was purified by flash chromatography (dichloromethane/methanol, 90:10) to give (7) (44 mg; 80%) in the form of an off-white solid.

[0175] .sup.1H NMR (300 MHz; DMSO) δ 12.00 (s; 1H.sub.14); 8.83 (t; J=5.5 Hz; 1H.sub.7); 7.84 (s; 2H.sub.2,4); 4.74 (s; 4H.sub.15,17); 3.31-3.21 (m; 2H.sub.8); 2.21 (t; J=7.3 Hz; 2H.sub.12); 1.60 -1.46 (m; 4H.sub.9,11); 1.39-1.25 (m; 2H.sub.10).

[0176] .sup.13C NMR (75 MHz; DMSO) δ 174.4 (1C.sub.13); 163.8 (1C.sub.6); 157.5 (2C.sub.1,5); 144.1 (1C.sub.3); 120.8 (2C.sub.2,4); 39.0 under DMSO (1C.sub.8); 34.1 (2C.sub.15,16); 33.6 (1C.sub.12); 28.6 (1C.sub.9); 26.0 (1C.sub.11); 24.2 (1C.sub.10).

[0177] HRMS (ESI): m/z calculated for C.sub.14H.sub.19Br.sub.2N.sub.2O.sub.3 [M+H].sup.+: 420.9757; observed 420.9752.

[0178] Preparation of 6-(2,6-bis(bromomethyl)pyridin-4-yl)amido-N-hexanamide-valine-citrulline-p-aminobenzylcarbamate-MMAE (8)

##STR00036##

[0179] Under an inert atmosphere, in the dark and under anhydrous conditions, 6-(2,6-bis(bromomethyl)pyridin-4-yl)amidohexanoic acid (7) (13.2 mg; 0.0313 mmol; 2.28 eq) was dissolved in anhydrous acetonitrile (1.2 mL) and then N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) (21.2 mg; 0.0857 mmol; 6.25 eq) was added. The activation medium was stirred at 25° C. for 1 h 20. A solution of valine-citrulline-p-aminobenzylcarbamate-MMAE trifluoroacetic acid salt (17.0 mg; 0.0137 mmol; 1.0 eq), dissolved in anhydrous N,N-dimethylformamide (300 μL) in the presence of N,N-diisopropylethylamine (9.4 μL; 0.0537 mmol; 3.92 eq), was added to the activation medium. The reaction medium obtained was stirred at 25° C. for 1 h. The mixture was diluted twofold with N,N-dimethylformamide and purified by semi-preparative high-pressure liquid chromatography (t.sub.R=22.1 min; on a Gilson PLC 2050 system [ARMEN V2 (pump) and ECOM TOYDAD600 (UV detector)], UV detection at 254 nm at 25° C.; Waters XBridge™ C-18 column; 5 μm (250 mm×19.00 mm); elution with 0.1% trifluoroacetic acid (by volume) in water (solvent A), and acetonitrile (solvent B); gradient 20 to 100% B over 32 min then 100% B for 6 min at 17.1 mL/min) to give (8) (18.2 mg; 87%) in the form of a white solid.

[0180] .sup.1H NMR (300 MHz, DMSO) δ (ppm) 10.04-9.95 (m; 1H); 8.94-8.79 (m; 1 H); 8.20-8.06 (m; 2H); 7.98-7.87 (m; 1 H); 7.84 (s; 2H); 7.81 (s; 1 H); 7.70-7.61 (m; 1 H); 7.58 (d; J=8.2 Hz; 2H); 7.38-7.11 (m; 6H); 6.07-5.92 (m; 1 H); 5.47-5.37 (m; 1 H); 5.15-4.96 (m; 1 H); 4.73 (s; 4H); 4.54-4.29 (m; 2H); 4.32-4.12 (m; 1 H); 4.05-3.92 (m; 1 H); 3.30-3.08 (m; 9H); 3.06-2.93 (m; 2H); 2.91-2.77 (m; 2H); 2.24-2.05 (m; 2H); 2.21-2.11 (m; 3H); 2.02-1.88 (m; 1 H); 1.60 -1.44 (m; 5H); 1.36-1.13 (m; 4H); 1.08-0.93 (m; 6H); 0.93-0.67 (m; 28H).

[0181] HRMS (ESI): m/z calculated for C.sub.72H.sub.111Br.sub.2N.sub.12O.sub.14 [M+H]+: 1525.6704; observed 1525.6700.

Example 1B: Synthesis of a Cytotoxic Conjugate According to the Invention (Pyridine Retroamide)

[0182] General reaction scheme

##STR00037## ##STR00038##

[0183] Detailed reaction scheme

[0184] Preparation of benzyl 2,6-bis(((tert-butyldimethylsilyl)oxy)methyl)isonicotinate (9)

##STR00039##

[0185] Benzyl 2,6-bis(hydroxymethyl)isonicotinate (3) (1.56 g; 5.708 mmol; 1.0 eq) was dissolved in anhydrous dichloromethane (12 mL), 2,6-lutidine (3.6 mL; 28.542 mmol; 5.0 eq) was added and the solution was cooled to 0° C. tert-Butyldimethylsilyl trifluoromethanesulfonate (5.5 mL; 23.974 mmol; 4.2 eq) was added dropwise in 10 min, and then the reaction medium was stirred under argon at room temperature (20° C.) for 19 h. The medium was cooled to 0° C. and then neutralized by addition of a saturated sodium hydrogen carbonate solution. The aqueous phase was extracted with dichloromethane (3x100 mL) and the combined organic phases were washed with a saturated sodium chloride solution, dried over magnesium sulfate, filtered and then concentrated under reduced pressure. The crude product was purified by flash chromatography (SiO.sub.2, cyclohexane/ethyl acetate 90:10) to give (9) (2.50 g; 87%) in the form of a beige solid.

[0186] .sup.1H NMR (300 MHz, DMSO) δ 7.83 (s; 2H.sub.2,4); 7.50-7.36 (m, 5H.sub.9-13); 5.38 (s; 2H.sub.7); 4.79 (s; 4H.sub.14,21); 0.90 (s; 18H.sub.18-20,25-27); 0.09 (s; 12H.sub.16,15,22,23).

[0187] Preparation of 2,6-bis(((tert-butyldimethylsilyl)oxy)methyl)isonicotinic acid (10)

##STR00040##

[0188] Benzyl 2,6-bis(((tert-butyldimethylsilyl)oxy)methyl)isonicotinate (9) (2.50 g; 4.982 mmol; 1.0 eq) was dissolved in 60 mL of a methanol/ethyl acetate mixture (5:1) and the solution was degassed with argon for 15 min. 10% by weight palladium-on-charcoal (250 mg, 10% m/m) was added and the reaction medium was stirred at room temperature (20° C.) under a hydrogen atmosphere for 5 h. The reaction medium was filtered on dicalite (methanol rinsing). The filtrate was concentrated under reduced pressure to give (10) (1.93 g; 94%) in the form of a white solid.

[0189] .sup.1H NMR (300 MHz, DMSO) δ 7.78 (s; 2H.sub.2,4); 4.78 (s; 4H-.sub.18,15), 0.92 (s; 18H.sub.12-14,19-21); 0.10 (s; 12H9,10,16,17).

[0190] Preparation of benzyl (2,6-bis(((tert-butyldimethylsilyi)oxy)methyl)pyridin-4-yl)carbamate (11)

##STR00041##

[0191] 2,6-bis(((tert-Butyldimethylsilyl)oxy)methyl)isonicotinic acid (10) (519.4 mg; 1.262 mmol; 1.0 eq) was dissolved in anhydrous tetrahydrofuran (4 mL). The solution was cooled to −10° C. Triethylamine (227.0 μL; 1.637 mmol; 1.3 eq) and then ethyl chloroformate (180.7 μL; 1.890 mmol; 1.5 eq) were added with stirring. The reaction medium was stirred under argon at —10° C. for 1 h 15. Then, a solution of sodium azide (139.8 mg; 2.142 mmol; 1.7 eq) in water (300 μL) was added. The reaction medium was stirred at 0° C. for 1 h 45. The triethylamine salts were filtered off, then the filtrate was concentrated under reduced pressure (maximum bath temperature 40° C., maximum vacuum 100 mbar). The residue was then taken up in 10 mL of water and the product was extracted with ethyl acetate (3×10 mL). The combined organic phases were washed with a saturated sodium chloride solution, dried over magnesium sulfate and concentrated under reduced pressure. To give the acyl azide in the form of a yellow oil. It was dissolved in toluene (14 mL), and then benzyl alcohol (522 μL; 5.044 mmol; 4.0 eq) was added. The reaction medium was stirred at 90° C. for 18 h. The toluene was evaporated under reduced pressure. The product was purified by flash chromatography (SiO.sub.2, cyclohexane/ethyl acetate 90:10) to give (11) (535.6 mg; 82%) in the form of a yellow oil.

[0192] .sup.1H NMR (300 MHz, CDCI3) δ 7.50-7.32 (m; 7H.sub.2,4,16-14); 6.85 (s; 1H.sub.6); 5.23 (s; 2H.sub.8); 4.75 (s; 4H.sub.15,22); 0.96 (s; 18H.sub.19-21,26-28); 0.12 (s; 12H.sub.16,17,23,24).

[0193] Preparation of 2,6-bis(((tert-butyldimethylsilyl)oxy)methyl)pyridin-4-amine (12)

##STR00042##

[0194] Benzyl (2,6-bis(((tert-butyldimethylsilyl)oxy)methyl)pyridin-4-yl)carbamate (11) (528.6 mg; 1.020 mmol; 1.0 eq) was dissolved in methanol (30 mL). The solution was degassed with argon for 15 min. Then, 10% by weight palladium-on-charcoal (57.4 mg, 10% m/m) was added. The reaction medium was stirred under a hydrogen atmosphere at room temperature (20° C.) for 16 h. The palladium on charcoal was filtered on dicalite (methanol rinsing) and then the filtrate was concentrated under reduced pressure. The product was obtained in salified form (nitrogen of the pyridine). This was taken up in water (160 mL), and then a 10% solution of sodium hydroxide in water was added at 0° C. until a pH of 9 was obtained. The product was then extracted with dichloromethane (5×50 mL). The combined organic phases were washed with a saturated sodium chloride solution, dried over magnesium sulfate and concentrated under reduced pressure to give (12) (312.2 mg; 80%) in the form of a yellow oil.

[0195] .sup.1H NMR (300 MHz; DMSO) δ 6.44 (s; 2H.sub.2,4); 6.04 (s; 2H.sub.6); 4.47 (s; 4H.sub.7,14);

[0196] 0.91 (s; 18H.sub.11-13,18-20); 0.07 (s; 12H.sub.8,9,15;16).

[0197] Preparation of methyl 6-((2,6-bis(((tert-butyldimethylsilyl)oxy)methyl)pyridin-4-yl)amino)-6-oxohexanoate (13)

##STR00043##

[0198] 2,6-bis(((tert-Butyldimethylsilyl)oxy)methyl)pyridin-4-amine (12) (146.7 mg; 0.383 mmol; 1.0 eq) was dissolved in anhydrous dichloromethane (2.8 mL). The solution was cooled to 0° C. and triethylamine (106.7 pL; 0.765 mmol; 2.0 eq) and then methyl adipoyl chloride (59.7 μL; 0.383 mmol; 1.0 eq) were added dropwise. The reaction medium was stirred at room temperature (20° C.) for 15 h 30. Anhydrous dichloromethane (1 mL) was then added to the reaction medium, as was methyl adipoyl chloride (26.8 μL; 0.172 mmol; 0.5 eq). The reaction medium was stirred at room temperature (20° C.) for 1 h 30. Then, anhydrous dichloromethane (1 mL) was added to the reaction medium, as were methyl adipoyl chloride (59.7 μL; 0.383 mmol; 1.0 eq) and triethylamine (26.7 μL; 0.191 mmol; 0.5 eq). The reaction medium was stirred at room temperature (20° C.) for 4 h. The reaction was halted by addition of a saturated sodium hydrogen carbonate solution (3 mL) at 0° C. and extracted with dichloromethane (3×10 mL). The combined organic phases were dried over magnesium sulfate and concentrated under reduced pressure. The product was purified by flash chromatography (SiO2, cyclohexane/ethyl acetate 50:50) to give (13) (144.0 mg; 72%) in the form of a colorless oil.

[0199] .sup.1H NMR (300 MHz, DMSO) δ 10.25 (s; 1H.sub.6); 7.58 (s; 2H.sub.2,4); 4.63 (s; 4H.sub.14,21); 3.58 (s; 3H13); 2.41-2.28 (m; 4118,11); 1.62-1.51 (m; 4H.sub.9,10); 0.92 (s, 18H.sub.18-20,25-27); 0.09 (s; 12H.sub.15,16,22,23).

[0200] Preparation of methyl 6-((2,6-bis(hydroxymethyl)pyridin-4-yl)amino)-6-oxohexanoate (14)

##STR00044##

[0201] Methyl 6-((2,6-bis(((tert-butyldimethylsilyl)oxy)methyl)pyridin-4-yl)amino)-6-oxohexanoate (13) (136.9 mg; 0.261 mmol; 1.0 eq) was dissolved in trifluoroacetic acid (3 mL). The reaction medium was stirred at 30° C. for 17 h. The trifluoroacetic acid was evaporated under reduced pressure. The residue was taken up in ethyl acetate (10 mL) and a saturated sodium hydrogen carbonate solution (10 mL) was added. The emulsion was stirred vigorously. The product was extracted with ethyl acetate (5×10 mL). The combined organic phases were dried over magnesium sulfate and concentrated under reduced pressure. The product was purified by flash chromatography (SiO.sub.2, dichloromethane/methanol 80:20) to give (14) (72.2 mg; 93%) in the form of a colorless oil.

[0202] .sup.1H NMR (300 MHz, DMSO) δ 10.24 (s; 1H.sub.6); 7.57 (s; 2H.sub.2,4); 5.37 (t; J=5.8

[0203] Hz; 2H.sub.15,17); 4.45 (d; J=5.8 Hz; 4H.sub.14,16); 3.58 (s; 3H.sub.13); 2.40-2.29 (m; 4118,11); 1.72-1.45 (m; 4H.sub.9,10).

[0204] Preparation of methyl 6-((2,6-bis(bromomethyl)pyridin-4-yl)amino)-6-oxohexanoate (15)

##STR00045##

[0205] Methyl 6-((2,6-bis(hydroxymethyl)pyridin-4-yl)amino)-6-oxohexanoate (14) (93.5 mg; 0.316 mmol; 1.0 eq) was dissolved in anhydrous acetonitrile (6 mL) and then phosphorus tribromide (90 μL; 0.958 mmol; 3.0 eq) was added slowly. The reaction medium was stirred at 45° C. for 2 h. The solution was cooled to 0° C., neutralized with water (5 mL) and extracted with ethyl acetate (3×10 mL). The combined organic phases were washed with a saturated sodium chloride solution, dried over magnesium sulfate and concentrated under reduced pressure. The product was obtained in salified form (nitrogen of the pyridine). This was taken up in water, and then a 10% solution of sodium hydroxide in water was added at 0° C. until a pH of 9 was obtained. The product was then extracted with dichloromethane (3×20 mL). The combined organic phases were washed with a saturated sodium chloride solution, dried over magnesium sulfate and concentrated under reduced pressure. The product was purified by flash chromatography (SiO.sub.2,dichloromethane/methanol 90:10) to give (15) (80.8 mg; 62%) in the form of a slightly pink solid.

[0206] .sup.1H NMR (300 MHz, CDCI.sub.3) δ 7.87 (s; 1H.sub.6); 7.64 (s; 2H.sub.2,4); 4.49 (s; 4H.sub.14,15); 3.71 (s; 3H.sub.13); 2.53-2.32 (m; 4H.sub.8,11); 1.84-1.66 (m; 4H.sub.9,10).

[0207] Preparation of 6-((2,6-bis(bromomethyl)pyridin-4-yl)amino)-6-oxohexanoic acid (16)

##STR00046##

[0208] Methyl 6-((2,6-bis(bromomethyl)pyridin-4-yl)amino)-6-oxohexanoate (15) (35.2 mg; 0.083 mmol; 1.0 eq) was dissolved in tetrahydrofuran (2.2 mL) and a solution of hydrated lithium hydroxide (8.7 mg; 0.208 mmol; 2.5 eq) in water (2.1 mL) was added. The reaction medium was stirred at room temperature (20° C.) for 3 h. The reaction medium was acidified at 0° C. with an aqueous 0.1 N hydrochloric acid solution and then extracted with ethyl acetate (3×20 mL). The combined organic phases were washed with a saturated sodium chloride solution, dried over magnesium sulfate and concentrated under reduced pressure lo to give (16) (33.4 mg; 99%) in the form of a crystalline white solid.

[0209] .sup.1H NMR (300 MHz, DMSO) δ 12.06 (s; 1H.sub.13); 10.46 (s; 1H.sub.6); 7.66 (s; 2H.sub.2,4); 4.63 (s; 4H.sub.14,15); 2.39-2.33 (m; 2H.sub.8); 2.26-2.20 (m; 2H.sub.11); 1.67-1.45 (m; 4H.sub.9,10).

[0210] Preparation of 6-((2,6-bis(bromomethyl)pyridin-4-yl)amino)-6-oxohexanamide-valine-citrulline-p-aminobenzylcarbamate-MMAE (17)

##STR00047##

[0211] Under an inert atmosphere, in the dark and under anhydrous conditions, 6-((2,6-bis(bromomethyl)pyridin-4-yl)amino)-6-oxohexanoic acid (16) (3.1 mg; 7.60 μmol; 1.8 eq) was dissolved in anhydrous acetonitrile (110 μL) and then isobutyl 1,2-dihydro-2-isobutoxy-1-quinolinecarboxylate (IIDQ) (2.28 μL; 7.68 μmol; 1.8 eq) was added. The activation medium was stirred at 25° C. for 30 min.

[0212] A solution of valine-citrulline-p-aminobenzylcarbamate-MMAE trifluoroacetic acid salt (5.3 mg; 4.28 μmol; 1.0 eq), dissolved in anhydrous N,N-dimethylformamide (200 μL) in the presence of N,N-diisopropylethylamine (2.98 μL; 17.11 μmol; 4.0 eq) and previously stirred at room temperature (20° C.) for 30 min, was added to the activation medium. The reaction medium obtained was stirred at 25° C. for 1 h 20. The mixture was diluted twofold with acetonitrile and purified by semi-preparative high-pressure liquid chromatography (t.sub.R=23.5 min; on a Gilson PLC 2050 system [ARMEN V2 (pump) and ECOM TOYDAD600 (UV detector)], UV detection at 254 nm at 25° C.; Waters XBridge™ C-18 column; 5 μm (250 mm×19.00 mm); elution with 0.1% trifluoroacetic acid (by volume) in water (solvent A), and acetonitrile (solvent B); gradient 30 to 60% B over 26 min then 60% to 100% B over 2 min and 100% B for 3 min at 17.1 mL/min) to give (17) (2.7 mg; 42%) in the form of a white lyophilizate.

[0213] .sup.1H NMR (300 MHz, CDCI.sub.3): δ 7.97 (5; 1 H); 7.58-7.41 (m; 1 H); 7.40-7.29 (m; 5H); 5.42-5.14 (m; 1 H); 5.00-4.85 (m; 2H); 4.76-4.64 (m; 1 H); 4.63-4.45 (m; 5H); 4.23-4.01 (m; 1 H); 4.02-3.72 (m; 2H); 3.60-3.43 (m; 3H); 3.43-3.35 (m; 3H); 3.34-3.25 (m; 4H); 3.23-3.08 (m; 4H); 3.07-2.97 (m; 3H); 2.95-2.81 (m; 3H); 2.62-2.29 (m; 6H); 1.84-1.40 (m; 21 H); 1.35-1.11 (m; 6H); 1.10-0.92 (m; 17H); 0.93-0.69 (m; 16H); 0.71-0.53 (m; 3H).

[0214] HRMS (ESI): m/z calculated for C.sub.71 H.sub.109Br.sub.2N.sub.12O.sub.14 [M+H]+: 1511.6547; observed 1511.6557.

Example 2A: Synthesis of an Antibody-Drug Conjugate According to the Invention (Pyridine)

[0215] Code of the synthesized product: McSAF-pyridine (corresponding to formula

[0216] (Ila), also called hereinafter “antibody-drug conjugate according to the invention” or generally “ADC”).

[0217] Antibody used: trastuzumab.

[0218] Preparation of solutions

[0219] Bioconjugation buffer: 1X saline buffer, for example phosphate, borate, acetate, glycine, tris(hydroxymethyl)aminomethane, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid in a pH range of between 6 and 9, with a final NaCI concentration of between 50 and 300 mM and a final EDTA concentration of io between 0.1 and 10 mM. For example, 1X phosphate buffer at a pH of 8.3, with a final NaCI concentration of 180 mM and a final EDTA concentration of 1 mM.

[0220] Trastuzumab at a concentration of between 1 and 10 mg/mL in the bioconjugation buffer, for example 5 mg/mL.

[0221] Reducing agent: Solution of a reducing agent chosen from dithiothreitol, (3-mercaptoethanol, tris(2-carboxyethyl)phosphine hydrochloride, tris(hydroxypropyl)phosphine at a concentration of between 0.1 and 10 mM in the bioconjugation buffer. For example, a 1 mM solution of tris(2-carboxyethyl)phosphine hydrochloride in the bioconjugation buffer.

[0222] Linker solution: cytotoxic conjugate (8) at a concentration of between 0.1 and 10 mM in a mixture of organic solvents chosen from dimethyl sulfoxide, N,N-dimethylformamide, methanol, tetrahydrofuran, acetonitrile, N,N-dimethylacetamide, dioxane. For example, a 1 mM solution in a mixture of organic solvents composed of 20% N,N-dimethylformamide and 80% methanol.

[0223] Bioconjugation reaction

[0224] Under an inert atmosphere, the reducing agent (4 to 100 eq, for example 8 eq) was added to the trastuzumab in the bioconjugation buffer (2.5 mg; 1 eq) and the reaction medium was incubated between 15 and 40° C., for example 37° C., for 0.25 to 3 h, for example 2 h, and then the linker solution (4 to 100 eq, for example 12 eq) was added under an inert atmosphere and the reaction medium was agitated between 15 and 40° C., for example 37° C., for 0.5 to 15 h, for example 2.5 h. This reaction was duplicated, in parallel, as many times as necessary to obtain the desired final amount of ADC, i.e. five times.

[0225] Purification of the ADC

[0226] The reaction mixture was purified on a PD-10 (GE Healthcare) using Gibco PBS pH 7.4 buffer as many times as necessary to remove residual chemical reagents, i.e. purified two times.

[0227] Results

[0228] The steps described above made it possible to obtain 7.43 mg of McSAF-pyridine (57%).

Example 2B: Synthesis of an Antibody-Drug conjugate According to the Invention (Pyridine Retroamide)

[0229] Code of the synthesized product: McSAF-pyridine retroamide (corresponding to formula (Ila), also called hereinafter “antibody-drug conjugate according to the invention” or generally “ADC”).

[0230] Antibody used: trastuzumab.

[0231] Preparation of solutions

[0232] Bioconjugation buffer: 1X borate buffer at a pH of 8.3, with a final NaCI concentration of 25 mM and a final EDTA concentration of 1 mM.

[0233] Trastuzumab at a concentration of 5 mg/mL in the bioconjugation buffer.

[0234] Reducing agent: Solution of tris(2-carboxyethyl)phosphine hydrochloride at a concentration of 1 mM in the bioconjugation buffer.

[0235] Linker solution: cytotoxic conjugate (17) at a concentration of 2 mM in a mixture of organic solvents composed of 70% N,N-dimethylformamide and 30% methanol.

[0236] Bioconjugation reaction

[0237] The solution of trastuzumab in the bioconjugation buffer (0.25 mg; 1 eq) was placed under argon. The reducing agent (8 eq) was then added and the reaction medium was incubated at 37° C. for 2 h. Then, the linker solution (17 eq) was added under argon and the reaction medium was agitated at 37° C. for 2 h 30.

Example 3A: analyses of the antibody-drug conjugate (McSAF-pyridine)

[0238] HIC (hydrophobic interaction chromatography) analysis

[0239] Materials and method

[0240] The McSAF-pyridine ADC was diluted to 1 mg/mL with PBS pH 7.4 before being filtered through 0.22 pm. 50 pg of products were injected onto an MAbPac

[0241] HIC-Butyl, 5 pm, 4.6 x 100 mm, column (ThermoScientific), connected to a Waters Alliance HPLC system (e2695) equipped with a PDA (e2998) set for detection at 280 nm. The McSAF-pyridine ADC was eluted at a flow rate of 1 mL/min with a gradient from 100% buffer A (1.5 M ammonium sulfate, 50 mM monobasic sodium phosphate, 5% isopropanol (v/v), pH 7.0) to 20% buffer B (50 mM monobasic sodium phosphate, 20% isopropanol (v/v), pH 7.0) in 2 minutes then to 85% buffer B in 30 minutes and then this gradient was maintained for 1 min. The temperature was maintained at 25° C. throughout the separation.

[0242] The results are shown in FIG. 1 and in table 1 below.

TABLE-US-00001 TABLE 1 McSAF-pyridine DAR 0 DAR 1 DAR 2 DAR 3 DAR 4 DAR 5 Retention time (min) 6.1 9.73 12.59 17.2 20.61 25.56 Area (%) 2.85 0.1 0.57 10.93 68.51 17.04 Average DAR 3.93

[0243] SEC (size exclusion chromatography) analysis

[0244] Materials and method

[0245] The McSAF-pyridine ADC was diluted to 1 mg/mL with PBS pH 7.4 before being filtered through 0.22 μm. 50 .sub.i—ig of products were injected onto an AdvanceBio SEC 2.7 μm, 7.8×300 mm, column (Agilent Technologies), connected to a Waters Alliance HPLC system (e2695) equipped with a PDA (2998) set for detection at 280 nm, with a MALLS Wyatt (miniDawn™) detector and with a Wyatt (Optilab® T-rEX) refractive index detector. The McSAF-pyridine ADC was eluted at a flow rate of 1 mL/min with an isocratic buffer C (1 mM monobasic sodium phosphate, 155 mM sodium chloride, 3 mM dibasic sodium phosphate, 3 mM sodium azide, pH 7.0) in 24 minutes. The temperature was maintained at 25° C. throughout the separation.

[0246] The results are shown in FIG. 2 and in table 2 below.

TABLE-US-00002 TABLE 2 McSAF-pyridine Soluble aggregates Oligomers Monomers Retention time (min) 3.73 6.46 7.27 Area (%) 0.03 0.83 99.14

[0247] Reproducibility

[0248] The reproducibility results are presented in FIG. 3 (reproducibility over 14 independent bioconjugations (scale 1 mg of trastuzumab employed), analysis by HIC) and in FIG. 4 (reproducibility on different scales, HIC analysis).

[0249] Conclusion: the bioconjugation is perfectly reproducible on the same scale and on different scales.

[0250] Native MS (native mass spectrometry) analysis

[0251] Materials and method

[0252] Mass spectrometric analysis was performed on a Vion IMS Qtof mass spectrometer coupled to an Acquity UPLC H-Class system from Waters (Wilmslow, UK). Prior to MS analysis, the samples (20 μg) were desalted on a BEH SEC 2.1×150 mm 300 A desalting column with an isocratic gradient (50 mM ammonium acetate, pH 6.5) at 40 4/min. A bypass valve was programmed to allow solvent to enter the spectrometer only between 6.5 and 9.5 min. MS data were acquired in positive mode with an ESI source over an m/z range of 500 to 8000 at 1 Hz and were analyzed using the UNIFI 1.9 software and the MaxEnt algorithm for deconvolution.

[0253] The results are shown in FIG. 5 and in table 3 below.

TABLE-US-00003 TABLE 3 McSAF- pyridine DAR 0 DAR 1 DAR 2 DAR 3 DAR 4 DAR 5 MW (Da).sup.1 n.o..sup.2 n.o..sup.2 n.o..sup.2 152165 153535 154903 Area (%) 0 0 0 8.20 79.57 12.23 Average 4.04 DAR .sup.1molecular mass of the G0F/G0F glycoform .sup.2not observed [00048]

[0254] Denaturing HRMS (denaturing high resolution mass spectrometry) analysis

[0255] The mass spectrometric analysis of the McSAF-pyridine ADC was performed on a Bruker maXis mass spectrometer coupled to a Dionex Ultimate 3000 RSLC system. Prior to MS analysis, the samples (5 μg) were desalted on a MassPREP desalting column (2.1x10 mm, Waters) heated to 80° C. using an aqueous 0.1% formic acid solution as solvent A and a 0.1% solution of formic acid in acetonitrile as solvent B at 500 μL/min. After 1 min, a linear gradient of 5 to 90% B in 1.5 min was applied. MS data were acquired in positive mode with an ESI source over an m/z range of 900 to 5000 at 1 Hz and were analyzed using the DataAnalysis 4.4 software (Bruker) and the MaxEnt algorithm for deconvolution. The DAR by species was determined using the intensity of the observed peaks.

[0256] The results are shown in FIG. 18 and in table 4 below.

TABLE-US-00004 TABLE 4 LHHL LHH HH Intensity (%) MM (Da).sup.1 Intensity (%) MM (Da).sup.1 Intensity (%) MM (Da).sup.1 DAR 0 n.o..sup.2 n.o..sup.2 n.o..sup.2 DAR 1 n.o..sup.2 n.o..sup.2 n.o..sup.2 DAR 2 n.o..sup.2 n.o..sup.2 n.o..sup.2 DAR 3 n.o..sup.2 n.o..sup.2 100 105295 DAR 4 86 153533 100 130089 n.o..sup.2 DAR 5 14 154903 n.o..sup.2 n.o..sup.2 Average DAR 4.14 4.00 3.00 LH L Intensity (%) MM (Da).sup.1 Intensity (%) MM (Da).sup.1 DAR 0 n.o..sup.2 100 23439 DAR 1 n.o..sup.2 n.o..sup.2 DAR 2 100 76765 n.o..sup.2 DAR 3 n.o..sup.2 n.o..sup.2 DAR 4 n.o..sup.2 n.o..sup.2 DAR 5 n.o..sup.2 n.o..sup.2 Average DAR 2.00 0.00 Species H was not observed. .sup.1molecular mass of the G0F/G0F glycoform .sup.2not observed [00049]

Example 3B: analyses of the antibody-drug conjugate (McSAF-pyridine retroamide)

[0257] HIC (hydrophobic interaction chromatography) analysis

[0258] Materials and method

[0259] The McSAF-pyridine retroamide ADC was analyzed by carrying out the protocol described in example 3A.

[0260] The results are shown in FIG. 19 and in table 5 below.

TABLE-US-00005 TABLE 5 McSAF-pyridine retroamide DAR 0 DAR 1 DAR 2 DAR 3 DAR 4 DAR 5 DAR 6 Retention time (min) 6.39 n.o..sup.1 12.37 17.22 20.73 25.46 30.42 Area (%) 0.98 n.o..sup.1 1.36 7.05 67.74 21.97 0.89 Average DAR 4.10 .sup.1not observed

[0261] Denaturing HRMS (denaturing high resolution mass spectrometry) analysis

[0262] The McSAF-pyridine retroamide ADC was analyzed by carrying out the protocol described in example 3A.

[0263] The results are shown in FIG. 20 and in table 6 below.

TABLE-US-00006 TABLE 6 LHHL LH L Intensity (%) MM (Da).sup.1 Intensity (%) MM (Da).sup.1 Intensity (%) MM (Da).sup.1 DAR 0 n.o..sup.2 n.o..sup.2 100 23439 DAR 1 n.o..sup.2 n.o..sup.2 n.o..sup.2 DAR 2 n.o..sup.2 100 76737 n.o..sup.2 DAR 3 n.o..sup.2 n.o..sup.2 n.o..sup.2 DAR 4 87 153473 n.o..sup.2 n.o..sup.2 DAR 5 13 154849 n.o..sup.2 n.o..sup.2 Average DAR 4.13 2.00 0.00 The LHH, HH and H species were not observed. .sup.1molecular mass of the G0F/G0F glycoform .sup.2not observed [00050]

[0264] Native MS (native mass spectrometry) analysis

[0265] Materials and method

[0266] Mass spectrometric analysis was performed as described in example 3A.

[0267] The results are shown in FIG. 21 and in table 7 below.

TABLE-US-00007 TABLE 7 McSAF- pyridine retroamide DAR 0 DAR 1 DAR 2 DAR 3 DAR 4 DAR 5 MW (Da) n.o..sup.3 n.o..sup.3 n.o..sup.3 152240.sup.1 153478.sup.2 154841.sup.2 Area (%) 0 0 0    4.8   74.5   20.7 Average 4.16 DAR .sup.1molecular mass of the G0F/G1F glycoform [00051].sup.2molecular mass of the G0F/G0F glycoform [00052].sup.3not observed

[0268] Reproducibility

[0269] The reproducibility results are presented in FIG. 22 (reproducibility over 5 independent bioconjugations (scale 250 μg of trastuzumab employed), analysis by HIC).

Example 4: In vitro evaluation of the antibody-drug conjugate (McSAF-pyridine)

[0270] Binding to the HER-2 antigen: recognition of the HER2 antigen on a positive line (BT-474) and a negative line (MCF-7), comparison with a reference trastuzumab-cytotoxic drug conjugate described in the prior art, called trastuzumab emtansine (hereinafter “T-DM1”).

[0271] Materials and method

[0272] The cells were obtained from the ATCC (BT-474 and MCF-7). An aliquot of frozen cells was thawed rapidly in a water bath at 37° C. and washed twice with culture medium (F12/DMEM supplemented with 8% FCS, 100 μg/mL of sodium penicillin G, 100 μg/mL of streptomycin sulfate) and placed in a 150 cm.sup.2 cell culture flask at a density of at least 10 000 cells/cm.sup.2. The cells were kept at 37° C. in a humid atmosphere with 5% CO.sub.2 for at least a week.

[0273] The cells were then collected and 100 000 to 500 000 cells in 80 μL were incubated with 20 μL of ADCs (McSAF-pyridine or T-DM1) for 0.5-1 h at 4° C. at concentrations of ADCs ranging from 0.1 to 20 μg/mL (8 concentrations—0.1; 0.5; 1; 2.5; 5; 10; 15; 20 μg/mL) or with an antibody targeting HER2 (trastuzumab, positive control) at the same concentrations. The cells were washed three times with labeling buffer (1X PBS-2 mM EDTA-0.5% BSA) at 0° C. and incubated with a secondary antibody (100 μ1.sub.— to 1/100th, F(ab').sub.2-goat anti-human IgG Fc-PE, Life Technologies, # H10104) for 0.5 — 1 h at 4° C. After incubation with the secondary antibodies, the cells were washed twice with labeling buffer at 0° C.

[0274] After washing, the cells were centrifuged and resuspended in 100-500 μL of staining buffer at 0° C. before analysis by flow cytometry. The relative mean fluorescence emission (MFI) of the probes used was determined for each sample on a flow cytometer and analyzed by software such as FCS Express 5 Flow Cytometry (De Novo Software).

[0275] Results

[0276] The results, presented in FIG. 6, show that the recognition of HER2 by McSAF-pyridine is similar to the recognition of HER2 by the native antibody (trastuzumab) and T-DM1, and is dependent on the presence of HER2.

[0277] Cytotoxicity (MTS assay): cytotoxic effect of McSAF-pyridine on a positive line and a negative line compared to T-DM1.

[0278] Materials and method

[0279] The cells were obtained from the ATCC (BT-474 and MCF-7). An aliquot of frozen cells was thawed rapidly in a water bath at 37° C. and washed twice with io culture medium (F12/DMEM supplemented with 8% FCS, 100 μg/mL of L-glutamine, 100 μg/mL of sodium penicillin G, 100 μg/mL of streptomycin sulfate) and placed in a 150 cm.sup.2 cell culture flask at a density of at least 10 000 cells/cm.sup.2. The cells were kept at 37° C. in a humid atmosphere with 5% CO.sub.2 for at least a week.

[0280] The cells were then collected and deposited in 96-well plates at densities of between 1.25 and 2.5×10.sup.3 cells per well for the cytotoxicity assays. The cells were incubated for 48 hours at 37° C. before the addition of the ADCs tested and of the vehicle (PBS). The DMSO percentages never exceeded 0.5%. The ADCs tested were added at the following final concentrations: 225; 75; 25; 8.33; 2.78; 0.926; 0.309; 0.103; 0.034; 0.011 nM; and incubated for 72 h (+/−2 h).

[0281] Cell viability was determined on deposition of the cells (D-2), before the addition of the ADCs tested (D0) and 72 h after the addition of the compounds tested (D3), by measuring the amount of cellular ATP using the CellTiter-Glo® Luminescent Cell Viability Assay kit (Promega) according to the supplier's recommendations for use. Luciferase activity was measured on a luminometer (PerkinElmer® EnVision™).

[0282] Each concentration of compound was carried out in triplicate and two independent experiments were performed.

[0283] Results

[0284] The results, presented in FIG. 7, show a cytotoxic effect of McSAF-pyridine which is higher compared to T-DM1 and dependent on the presence of HER2.

Example 5: Stability of the Bioconjugation

[0285] Plasma stability : quantification by LC-MS/MS of the amount of MMAE released after incubation in human plasma at 37° C. Comparison between McSAF-pyridine and an ADC produced with a maleimide attachment head (a single bond)=McSAF-maleimidel of the formula:

##STR00053##

[0286] Materials and method

[0287] MMAE calibration curve

[0288] 25 μL of RP424 (H.sub.2O+0.1% FA) were added to 25 μL of samples (MMAE-ds standard or MMAE concentration range) and then the mixture was agitated. 75 μL of a standard solution “MMAE-d8” at 0.04 μg/mL were added before agitating for 30 seconds. The mixture was centrifuged at 20 000 g at 4° C. for 10 minutes.

[0289] The supernatant was removed and transferred to polypropylene flasks. A fresh centrifugation was carried out at 2500 g at 4° C. for 5 min before injection in LC-MS/MS. The samples were injected onto an Acquity BEH UPLC C18, 50×2.1 mm, 1.7 μm, column connected to an LC-20AD and LC-20ADXR system (Shimadzu) coupled to a Shimadzu mass detector (8060) with an ESI+ source.

[0290] Elution was performed with a gradient from 75% buffer D (10 mM ammonium acetate) in 25% buffer E (acetonitrile) to 5% buffer D in 95% buffer E over 5 minutes followed by an increase to 75% buffer D in 25% buffer E over 5.1 minutes. The results were processed with the Labsolution 6.60 software.

[0291] Human plasma incubation

[0292] The samples were incubated in sterile human EDTA-2K plasma (BiolVT) at an initial concentration of 100 μg/mL. 3 samples were collected just after having agitated the mixture (T0), then after 6 h, 12 h, 24 h, 48 h and 96 h of incubation at 37° C. The samples were stored at −80° C. prior to LC-MS/MS analysis thereof as described above.

[0293] Results

[0294] The results, presented in FIG. 8, show that McSAF-pyridine releases less MMAE into the plasma compared to McSAF-maleimide1. McSAF-pyridine is therefore more stable than McSAF-maleimidel under physiological conditions.

[0295] Stability at 37° C. in the presence or absence of HSA (human serum albumin)

[0296] Materials and method

[0297] The concentration of McSAF-pyridine and of McSAF-maleimide1, in a PBS buffer (1 mM monobasic sodium phosphate, 3 mM dibasic sodium phosphate, 155 mM sodium chloride, 1 mM sodium azide, pH 7.4), was adjusted to 2 mg/mL. Twelve (12) 20 μL samples of McSAF-pyridine and of McSAF-maleimidel were placed in twelve polypropylene flasks (Fisher Scientific, 0.6 mL) and then 20 μL of PBS buffer (1 mM monobasic sodium phosphate, 3 mM dibasic sodium phosphate, 155 mM sodium chloride, 1 mM sodium azide, pH 7.4) or 20 μL of a 20 mg/mL solution of HSA in PBS buffer (1 mM monobasic sodium phosphate, 3 mM dibasic sodium phosphate, 155 mM sodium chloride, 1 mM sodium azide, pH 7.4) were added to each flask. After agitation, each of the 12 flasks is centrifuged for 30 seconds at 5000 g before incubation at 37° C. in an incubator (VWR INCU-line IL23). The flasks were removed three by three from the incubator and stored at −80° C. after 1 minute (T0), 24 h, 48 h and 120 h.

[0298] After dilution by a factor of 2 with PBS buffer (1 mM monobasic sodium phosphate, 3 mM dibasic sodium phosphate, 155 mM sodium chloride, 1 mM sodium azide, pH 7.4), the contents of each of the 12 flasks are filtered through 0.22 μm and analyzed by HIC. For McSAF-pyridine, 50 μg of products were injected onto an MAbPac HIC-Butyl, 5 μm, 4.6×100 mm, column (ThermoScientific), connected to a Waters Alliance HPLC system (e2695) equipped with a PDA (e2998) set for detection at 280 nm. The samples were eluted at a flow rate of 1 mL/min with a gradient from 100% buffer A (1.5 M ammonium sulfate, 50 mM monobasic sodium phosphate, 5% isopropanol (v/v), pH 7.0) to 100% buffer B (50 mM monobasic sodium phosphate, 20% isopropanol (v/v), pH 7.0) in 50 minutes. The temperature was maintained at 25° C. throughout the separation.

[0299] For McSAF-maleimide1, 50 pg of products were injected onto a TSKgel Butyl NPR, 2.5 μm, 4.6×100 mm, column (Tosoh), connected to a Waters Alliance HPLC system (e2695) equipped with a PDA (e2998) set for detection at 280 nm. The samples were eluted at a flow rate of 0.6 mL/min with a gradient from 100% buffer A (1.5 M ammonium sulfate, 50 mM monobasic sodium phosphate, pH 7.0) to 20% buffer B (50 mM monobasic sodium phosphate, 20% isopropanol (v/v), pH 7.0) in 60 minutes and then this gradient was maintained for 6 min. The temperature was maintained at 30° C. throughout the separation.

[0300] Results

[0301] The monitoring of the change of the average DAR by the HIC method after incubation at 37° C. is presented in FIG. 9. The results show perfect stability at 37° C. for McSAF-pyridine, which is not the case for McSAF-maleimide1. This is explained by the increased stability of the antibody-drug conjugate according to the invention.

[0302] The monitoring of the change of the average DAR by the HIC method after incubation with an excess of HSA at 37° C. is presented in FIG. 10. The results show perfect stability in the presence of HSA for McSAF-pyridine, which is not the case for McSAF-maleimide1. This is explained by the increased stability of the antibody-drug conjugate according to the invention.

[0303] Stability at 40° C.

[0304] Materials and method

[0305] The concentration of McSAF-pyridine and McSAF-maleimide1, in a PBS buffer (1 mM monobasic sodium phosphate, 3 mM dibasic sodium phosphate, 155 mM sodium chloride, pH 7.4), was adjusted to 1 mg/mL. Six (6) 150 pL samples of McSAF-pyridine and of McSAF-maleimidel were placed in six polypropylene flasks (Eppendorf Protein LoBind, 0.5 mL). After agitation, the samples were incubated in an incubator (VWR INCU-line IL23) at 40° C. The flasks were removed three by three from the incubator, centrifuged for 2 minutes at 5000 g and stored at −80° C. after 1 minute and 4 weeks.

[0306] The contents of each of the 6 flasks were filtered through 0.22 μm and analyzed by HIC and SEC.

[0307] HIC

[0308] The McSAF-pyridine ADC was analyzed by carrying out the protocol described in example 3A.

[0309] For McSAF-maleimide1, 50 ═g of products were injected onto a TSKgel Butyl NPR, 2.5 μm, 4.6×100 mm, column (Tosoh), connected to a Waters Alliance HPLC system (e2695) equipped with a PDA (e2998) set for detection at 280 nm. The samples were eluted at a flow rate of 0.6 mL/min with a gradient from 100% buffer A (1.5 M ammonium sulfate, 50 mM monobasic sodium phosphate, pH 7.0) to 80% buffer B (50 mM monobasic sodium phosphate, 20% isopropanol (v/v), pH 7.0) in buffer A in 45 minutes and then this gradient was maintained for 6 min. The temperature was maintained at 30° C. throughout the separation.

[0310] SEC

[0311] 50 μg of products were injected onto an AdvanceBio SEC, 2.7 pm, 7.8×300 mm, column (Agilent Technologies), connected to a Waters Alliance HPLC system (e2695) equipped with a PDA (2998) set for detection at 280 nm. The ADCs were eluted at a flow rate of 1 mL/min with an isocratic buffer C (1 mM monobasic sodium phosphate, 155 mM sodium chloride, 3 mM dibasic sodium phosphate, 3 mM sodium azide, pH 7.4) in 24 minutes. The temperature was maintained at 25° C. throughout the separation.

[0312] Results

[0313] The monitoring of the change of the average DAR by the HIC method after incubation at 40° C. for 28 days is presented in FIG. 11 (N=3). The results show that the average DAR of McSAF-maleimidel varies from 4.00 (at t0) to 2.61 (at t28), attesting to a lack of stability under the stressful conditions simulated, while that of McSAF-pyridine varies little (3.93 (at t0) to 3.98 (at t28)), demonstrating its improved stability compared to McSAF-maleimidel using maleimide technology.

[0314] The monitoring of the change in the DAR4 percentage by the HIC method after incubation at 40° C. for 28 days is presented in FIG. 12 (N=3). The results show that the DAR4 percentage decreases over the course of time for McSAF-maleimidel (30% at t0 against 20% at t28) while that of McSAF-pyridine varies little (67% at t0 against 63% at t28). This demonstrates the increased stability and better preservation of the DAR4 percentage of McSAF-pyridine compared to McSAF-maleimidel using maleimide technology.

[0315] The monitoring of the change in the monomer percentage by the SEC method after incubation at 40° C. for 28 days is presented in FIG. 13 (N=3). The results show that the monomer percentage decreases in the same way for McSAF-maleimidel (77% at t28) and for McSAF-pyridine (80% at t28).

Example 6: In vivo evaluation of the antibody-drug conjugate (McSAF-pyridine)

[0316] Materials and method

[0317] All experiments were carried out in an environment complying with the recommendations of the French authorities (Authorization No. B 21 231 011 EA) and of FELASA. The survival study was performed on a BT-474 xenograft model. A suspension of tumor cells was implanted subcutaneously in immunosuppressed BALB/c nude mice (Charles River) 24 to 72 h after complete irradiation with a γ source (2 Gy, 60Co, BioMep, Bretenieres, France). After the tumor graft had taken, the mice were randomized into groups (N=8) once the mean volume had reached 150-200 mm.sup.3. The ADCs (McSAF-pyridine or the T-DM1 reference) were administered on days 1 and 26 by the intravenous route (IV, bolus) into the caudal vein of the mice at an amount of 1 or 5 mg/kg. The tumor volume as a function of time was calculated twice a week using the following formula: (Length x thickness.sup.2)/2. The animals were euthanized when the tumor volumes reached 10% of their weight, or approximately 2000 mm.sup.3.

[0318] Results

[0319] The results of administering a 5 mg/kg dose are presented in FIG. 14 and FIG. 15. The results of administering a 1 mg/kg dose are presented in FIG. 16 and FIG. 17. The results show that at both doses, 1 and 5 mg/kg, McSAF-pyridine is more effective than T-DM1. At 5 mg/kg of McSAF-pyridine, complete and lasting tumor regression was observed with 8 mice cured out of 8 mice treated.

[0320] A complementary immunohistochemistry (IHC) analysis on mice, at the end of the study, was performed. It made it possible to confirm the complete eradication of the HER2+ cells in the zone corresponding to the xenograft for the McSAF-pyridine ADC, for all the mice treated with 5 mg/kg, thus confirming complete tumor regression.

TABLE-US-00008 Sequence listing Sequence number Sequence type Amino acid sequence SEQ ID NO: 1 Trastuzumab light DIQMTQSPSSLSASVGDRVTITCRASQ chain DVNTAVAWYQQKPGKAPKLLIYSASFL YSGVPSRFSGSRSGTDFTLTISSLQPE DFATYYCQQHYTTPPTFGQGTKVEIKR TVAAPSVFIFPPSDEQLKSGTASVVCLL NNFYPREAKVQWKVDNALQSGNSQES VTEQDSKDSTYSLSSTLTLSKADYEKH KVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 2 Trastuzumab heavy EVQLVESGGGLVQPGGSLRLSCAASG chain FNIKDTYIHWVRQAPGKGLEWVARIYP TNGYTRYADSVKGRFTISADTSKNTAY LQMNSLRAEDTAVYYCSRWGGDGFYA MDYWGQGTLVTVSSASTKGPSVFPLA PSSKSTSGGTAALGCLVKDYFPEPVTV SWNSGALTSGVHTFPAVLQSSGLYSLS SVVTVPSSSLGTQTYICNVNHKPSNTK VDKKVEPKSCDKTHTCPPCPAPELLGG PSVFLFPPKPKDTLMISRTPEVTCVVVD VSHEDPEVKFNWYVDGVEVHNAKTKP REEQYNSTYRVVSVLTVLHQDWLNGK EYKCKVSNKALPAPIEKTISKAKGQPRE PQVYTLPPSREEMTKNQVSLTCLVKGF YPSDIAVEWESNGQPENNYKTTPPVLD SDGSFFLYSKLTVDKSRWQQGNVFSC SVMHEALHNHYTQKSLSLSPGK
References cited in the format “[reference number]”: [0321] 1. Fekete, S. et al., J. Pharm. Biomed. Anal., 130, 3-18, 2016 [0322] 2. Barran, P. et al., EuPA Open Proteomics, 11, 23-27, 2016 [0323] 3. Goyon, A et al., J. Chromatogr. B, 1065-1066, 35-43, 2017