Conjugates And Conjugating Reagents Comprising A Linker That Includes At Least Two (-CH2-CH2-O-) Units In A Ring

Abstract

A conjugate comprising a protein or peptide conjugated to a therapeutic, diagnostic or labelling agent via a linker, characterised in that the linker includes at least two ˜(CH.sub.2—CH.sub.2—O—)˜ units within a ring, said ring being attached via a single tethering atom within the ring to the rest of the linker, or said ring being attached via two or more tethering atoms within the ring to the rest of the linker at a single point.

Claims

1. A conjugate comprising a protein or peptide conjugated to a therapeutic, diagnostic or labelling agent via a linker, characterised in that the linker includes at least two ˜(CH.sub.2—CH.sub.2—O—)˜ units within a ring, said ring being attached via a single tethering atom within the ring to the rest of the linker, or said ring being attached via two or more tethering atoms within the ring to the rest of the linker at a single point.

2. A conjugate as claimed in claim 1, which includes within said ring a unit of the formula ˜(CH.sub.2—CH.sub.2—O—).sub.x˜ in which x is a number of at least 2, or x is a number from 2 to 50.

3. (canceled)

4. A conjugate as claimed in claim 1, in which said ring is attached via a single tethering atom in the ring to the rest of the linker at a single point.

5. A conjugate as claimed in claim 4, in which said ring has the formula: ##STR00133## ##STR00134##

6. A conjugate as claimed in claim 1, which includes a therapeutic agent.

7. A conjugate as claimed in claim 1, in which the protein or peptide is an antibody or an antibody fragment.

8. A conjugate as claimed in claim 1, which includes: i) a portion: ##STR00135## or ii a portion: ##STR00136## in which W′ represents an electron withdrawing group or a group obtained by reduction of an electron withdrawing group, each of A and B independently represents a C.sub.1-5alkylene or alkenylene chain, and Pr represents said protein or peptide bonded to A and B via nucleophiles Nu.

9. (canceled)

10. A conjugate as claimed in claim 8, which comprises an optionally substituted aryl or heteroaryl group immediately adjacent the group of formula III or Ma; and which linker also includes a —NR.sup.a.C(O)— or —C(O).NR.sup.a— group adjacent said aryl or heteroaryl group; wherein R.sup.a represents C.sub.1-4 alkyl or hydrogen.

11. A conjugate as claimed in claim 1, which includes a portion:
˜W′—(CH═CH).sub.p—(CH.sub.2).sub.2-Nu-Pr or a portion
˜NH—CO—Ar—CO—(CH.sub.2).sub.2-Nu-Pr in which W′ represents an electron withdrawing group or a group obtained by reduction of an electron withdrawing group, p is 0 or an integer from 1 to 4, and Pr represents said protein or peptide bonded to the rest of the molecule via a nucleophile Nu.

12. (canceled)

13. A conjugate as claimed in claim 1, in which each Nu represents a sulfur atom present in a cysteine residue in the protein or peptide Pr; or in which each Nu represents an imidazole group present in a polyhistidine tag attached to the protein or peptide Pr.

14. A conjugate as claimed in claim 1, which has the formula: ##STR00137## in which D represents said therapeutic, diagnostic or labelling agent, ##STR00138## represents said ring, and F′ represents said protein or peptide bonded to the remainder of the conjugate via a protein or peptide bonding portion of the linker.

15. A conjugating reagent comprising a functional group capable of reacting with a protein or peptide, which reagent also comprises a therapeutic, diagnostic or labelling agent and a linker which includes at least two ˜(CH.sub.2—CH.sub.2—O—)˜ units within a ring, said ring being attached via a single tethering atom within the ring to the rest of the linker, or said ring being attached via two or more tethering atoms within the ring to the rest of the linker at a single point.

16. A conjugating reagent as claimed in claim 15, which includes a therapeutic agent.

17. A conjugating reagent as claimed in claim 15, in which said functional group has the formula: ##STR00139## in which W represents an electron-withdrawing group; each of A and B independently represents a C.sub.1-5alkylene or alkenylene chain; and either each L independently represents a leaving group, or both Ls together represent a leaving group; or ##STR00140## in which W and A have the meanings given above, L represents a leaving group, and m is 0 to 4.

18. A conjugating reagent as claimed in claim 17, in which said functional group has the formula: ##STR00141##

19. A conjugating reagent as claimed in claim 15, in which said functional group has the formula:
˜W—(CH═CH).sub.p—(CH.sub.2).sub.2-L  (V) or
˜W—(CH═CH).sub.p—CH═CH.sub.2  (VI) in which W represents an electron withdrawing group, p represents 0 or an integer of from 1 to 4, and L represents a leaving group.

20. A conjugating reagent as claimed in claim 17, in which: i) the or each leaving group includes a portion —(CH.sub.2CH.sub.2O).sub.n— in which n is a number of two or more; or ii the or each leaving group has the formula —SP or —SO.sub.2P, in which P represents a group which includes a portion —(CH.sub.2CH.sub.2O).sub.n— in which n is a number of two or more.

21. (canceled)

22. A conjugating reagent as claimed in claim 15, which has the formula: ##STR00142## in which D represents said therapeutic, diagnostic or labelling agent, ##STR00143## represents said ring, and F represents said functional group capable of reacting with a protein or peptide.

23. A process for the preparation of a conjugate as claimed in claim 1, which comprises reacting a protein or peptide with a conjugating reagent, said conjugating reagent comprising a functional group capable of reacting with a protein or peptide, which reagent also comprises a therapeutic, diagnostic or labelling agent and a linker which includes at least two ˜(CH.sub.2—CH.sub.2—O—)˜ units within a ring, said ring being attached via a single tethering atom within the ring to the rest of the linker, or said ring being attached via two or more tethering atoms within the ring to the rest of the linker at a single point.

24. A pharmaceutical composition which comprises a conjugate as claimed in claim 1 in which the payload is a therapeutic agent, together with a pharmaceutically acceptable carrier, optionally together with a further active ingredient.

Description

BRIEF DESCRIPTION OF THF DRAWINGS

[0158] FIG. 1 shows the results of Example 8.

[0159] FIGS. 2A-2B show the results of mouse xenograft studies, with FIG. 2A showing (a) a plot of mean tumour volume±standard error over time in CB17-SCID mice following administration of vehicle or Adcetris® (brentuximab vedotin) (comparator) at 1 mg/kg on day 12 following tumour implantation; and FIG. 2B showing (b) plots of individual tumour volumes over time in CB17-SCID mice following administration of vehicle or Adcetris® (brentuximab vedotin) (comparator) at 1 mg/kg on day 12 following tumour implantation. FIGS. 3A-3B show the results of mouse xenograft studies, with FIG. 3A showing (a) a plot of mean tumour volume±standard error over time in CB17-SCID mice following administration of vehicle or conjugate 32 (comparator) at 0.8 mg/kg on day 12 following tumour implantation; and FIG. 3B showing (b) plots of individual tumour volumes over time in CB17-SCID mice following administration of vehicle or conjugate 32 (comparator) at 0.8 mg/kg on day 12 following tumour implantation.

[0160] FIGS. 4A-4B show the results of mouse xenograft studies, with FIG. 4A showing (a) a plot of mean tumour volume±standard error over time in CB17-SCID mice following administration of vehicle or conjugate 14 at 0.8 mg/kg on day 12 following tumour implantation; and FIG. 4B showing (b) plots of individual tumour volumes over time in CB17-SCID mice following administration of vehicle or conjugate 14 at 0.8 mg/kg on day 12 following tumour implantation.

[0161] FIGS. 5A-5B show the results of mouse xenograft studies, with FIG. 5A showing (a) a plot of mean tumour volume±standard error over time in CB17-SCID mice following administration of vehicle or conjugate 31 at 0.8 mg/kg on day 12 following tumour implantation; and FIG. 5B showing (b) plots of individual tumour volumes over time in CB17-SCID mice following administration of vehicle or conjugate 31 at 0.8 mg/kg on day 12 following tumour implantation.

[0162] The following Examples illustrate the invention.

Example 1: Synthesis of Conjugation Reagent 1 Comprising the Auristatin Cytotoxic Payload, MAE

[0163] ##STR00067##

Step 1: Synthesis of Compound 2

[0164] ##STR00068##

[0165] To a stirred solution of diethanolamine (2.5 g) and triethylamine (6.05 g) in dichloromethane (15 mL) was slowly added a solution of tosyl chloride (3.8 g) in dichloromethane (15 mL) at room temperature. After 2 h, water (25 mL) was added to the reaction mixture and the product was extracted with dichloromethane (5×30 mL). The combined organic extracts were dried over magnesium sulfate, the solution was then filtered and the volatiles removed in vacuo to yield compound 2 as a white solid (4.7 g). .sup.1H NMR (400 MHz, CDCl.sub.3) δ.sub.H 7.68 (d, J=8.3 Hz, 2H), 7.30 (d, J=8.3 Hz, 2H), 3.84 (t, J=5.0 Hz, 4H), 3.56 (s, 2H), 3.24 (t, J=5.0 Hz, 4H), 2.41 (s, 3H). m/z [M+Na].sup.+ (282, 95%), [M+H].sup.+ (260, 100%).

Step 2: Synthesis of Compound 3

[0166] ##STR00069##

[0167] A solution of compound 2 (176 mg) in anhydrous THF (2 mL) was added dropwise over a period of 1 h to a solution of sodium hydride (80 mg, 60% dispersion in mineral oil) in anhydrous THF (8 mL) at room temperature. After stirring for 1 h, a solution of hexaethyleneglycol di-p-toluenesulfonate (400 mg) in anhydrous THF (2 mL) was added over a period of 2 h and the reaction mixture was stirred at room temperature for 72 h. Water (30 mL) was added and the THF was removed in vacuo. The aqueous solution was extracted with chloroform (4×25 mL), the organic phases were combined and dried over magnesium sulfate before the solution was filtered and concentrated in vacuo. The residue was then purified by reverse phase C18-column chromatography eluting with buffer A (v/v): water:55% acetonitrile:0.05% trifluoroacetic acid and buffer B (v/v):acetonitrile:0.05% trifluoroacetic acid (100:0 v/v to 0:100 v/v). The organic solvent was removed in vacuo and the aqueous solvent was removed by lyophilisation to give compound 3 as a colourless oil (78 mg). .sup.1H NMR (400 MHz, CDCl.sub.3) δ.sub.H 7.68 (d, J=8.3 Hz, 2H), 7.26 (d, J=8.3 Hz, 4H), 3.67-3.58 (m, 24H), 3.58-3.53 (m, 4H), 3.38 (t, J=6.0 Hz, 4H), 2.40 (s, 3H). m/z [M+Na]*(528, 80%), [M+H].sup.+ (506, 50%).

Step 3: Synthesis of Compound 4

[0168] ##STR00070##

[0169] To a solution of compound 3 (78 mg) in anhydrous THF (6 mL) was added lithium aluminium hydride (1.13 mL, 1 M solution in THF) and the solution was heated at reflux for 16 h before the reaction mixture was cooled to 0° C. and quenched by the dropwise addition of water. The suspension was filtered and the precipitate washed with chloroform:ethanol (9:1 v/v, 5×6 mL). The filtrate and washings were combined and concentrated in vacuo to give compound 4 as a colourless oil (50 mg). m/z [M+Na].sup.+ (374, 70%), [M+H].sup.+ (352, 100%).

Step 4: Synthesis of Compound 5

[0170] ##STR00071##

[0171] To a solution of Fmoc-Glu-(OH)—OAll (48 mg) in DMF (1.0 mL) was added HATU (110 mg) and the solution was stirred for 30 min at 0° C. To this was added a solution of Val-Cit-PAB-MMAE.TFA salt (Levena Biopharma, 120 mg) and NMM (32 μL) in DMF (1 mL). The reaction mixture was stirred at 0° C. for 2.5 h. The solvent was concentrated in vacuo and the crude was dissolved in DMF (1.5 mL) before NN (32 μL) was added. Tetrakis(triphenylphosphine)palladium(0) (45 mg) was added to the reaction mixture which was then stirred at room temperature for 20 h. The reaction solution was concentrated in vacuo and the residue purified by reverse phase C18-column chromatography eluting with buffer A (v/v): water:5% acetonitrile:0.05% trifluoroacetic acid and buffer B (v/v): acetonitrile:0.05% trifluoroacetic acid (100:0 v/v to 0:100 v/v). The organic solvent was removed in vacuo and the aqueous solvent was removed by lyophilisation to give compound 5 as a white solid (98.0 mg). m/z [M+H].sup.+ (1475 Da, 100%), [M+2H].sup.2+ (738, 50%).

Step 5: Synthesis of Compound 6

[0172] ##STR00072##

[0173] To a solution of compound 5 (15 mg) in DMF (0.5 mL) at 0° C. was added HATU (4.3 mg). The solution was stirred for 20 min at 0° C. before NMM (1.3 μL) was added and the solution was stirred for a further 10 min. To a separate solution of compound 4 (5 mg) in DMF (0.3 ml) was added NMM (1.3 μL) and the solution was stirred at room temperature for 30 min. The two solutions were then combined and additional quantities of HATU (4.3 mg) and NMM (1.3 μL) were added to the combined solution which was stirred for 16 h at room temperature. The reaction solution was concentrated in vacuo to give crude Fmoc-L-Glu-[Val-Cit-PAB-MMAE]-aza-24-crown-8 (18 mg). m/z [M+Na].sup.+ (1831, 20%), [M+H].sup.+ (1809, 20%), [M+2H].sup.2+ (905, 100%). To a solution of Fmoc-L-Glu-[Val-Cit-PAB-MMAE]-aza-24-crown-8 (18 mg) in DMF (0.4 mL) was added piperidine (5 μL) and the solution was stirred for 20 min at room temperature. Additional piperidine (5 μL) was added and the solution was stirred for a further 25 min at room temperature. The reaction solution was concentrated in vacuo and the residue purified by reverse phase C18-column chromatography eluting with buffer A (v/v): water:55% acetonitrile:0.05% trifluoroacetic acid and buffer B (v/v): acetonitrile:0.05% trifluoroacetic acid (100:0 v/v to 0:100 v/v). The organic solvent was removed in vacuo and the aqueous solvent was removed by lyophilisation to give compound 6 as a colourless oil (8.5 mg). m/z [M+Na].sup.+ (1609, 10%), [M+H].sup.+ (1587, 20%), [M+2H].sup.2+ (794, 100%)

Step 6: Synthesis of Compound 2

[0174] ##STR00073##

[0175] To a stirred solution of 4-[2,2-bis[(p-tolylsulfonyl)-methyl]acetyl]benzoic acid (1.5 g, Nature Protocols, 2006, 1(54), 2241-2252) in DMF (70 mL) was added alpha-methoxy-omega-mercapto hepta(ethylene glycol) (3.2 g) and triethylamine (2.5 mL). The resulting reaction mixture was stirred under an inert nitrogen atmosphere at room temperature. After 19 h, volatiles were removed in vacuo. The resulting residue was dissolved in water (2.4 mL) and purified by reverse phase C18-column chromatography eluting with buffer A (v/v): water:55% acetonitrile:0.05% trifluoroacetic acid and buffer B (v/v): acetonitrile:0.05% trifluoroacetic acid (100:0 v/v to 0:100 v/v). The organic solvent was removed in vacuo and the aqueous solvent was removed by lyophilisation to give compound 7 as a thick clear colourless oil (1.77 g). m/z [M+H].sup.+ 901.

Step 7: Synthesis of Reagent 8

[0176] ##STR00074##

[0177] To a stirred solution of 7 (1.32 g) in methanol:water (18 mL, 9:1 v/v) at room temperature was added Oxone® (2.7 g). After 2.5 h, the volatiles were removed in vacuo and water was azeotropically removed with acetonitrile (2×15 mL). The resulting residue was dissolved in dichloromethane (3×10 mL), filtered through a column of magnesium sulfate and washed with dichloromethane (2×7 mL). The eluent and washings were combined and the volatiles were removed in vacuo to give a thick clear pale yellow oil (1.3 g). A portion of the residue (700 mg) was dissolved in water:acetonitrile (1.5 mL, 3:1 v/v), and purified by reverse phase C18-column chromatography eluting with buffer A (v/v): water:5% acetonitrile:0.05% trifluoroacetic acid and buffer B (v/v): acetonitrile:0.05% trifluoroacetic acid (100:0 v/v to 0:100 v/v). The organic solvent was removed in vacuo and the aqueous solvent was removed by lyophilisation to give reagent 8 as a thick clear colourless oil (524 mg). m/z [M+H].sup.+ 965.

Step 8: Synthesis of Reagent 1

[0178] To a solution of reagent 8 (6 mg) in DMF (0.3 mL) at 0° C. was added HATU (2.3 mg) The solution was stirred at 0° C. for 20 min before NMM (0.6 μL) was added and the solution was stirred for a further 10 min. To a separate solution of compound 6 (8.5 mg) in DMF (0.3 mL) was added NMM (0.6 μL) and the solution was stirred at room temperature for 30 min. The two solutions were then combined and additional quantities of HATU (2.3 mg) and NMM (0.6 μL) were added and the solution was stirred at room temperature for 1 h. Further quantities of HATU (1.2 mg) and NMM (0.3 μL) were then added and the solution stirred at room temperature for 1.5 h. The reaction solution was concentrated in vacuo and the residue purified by reverse phase C18-column chromatography eluting with buffer A (v/v): water:55% acetonitrile:0.05% trifluoroacetic acid and buffer B (v/v): acetonitrile:0.05% trifluoroacetic acid (100:0 v/v to 0:100 v/v). The organic solvent was removed in vacuo and the aqueous solvent was removed by lyophilisation to give reagent 1 as a colourless oil (10.7 mg). m/z [M+Na].sup.+ (2557, 5%), [M+2H].sup.2+ (1268, 40%), [M+3H].sup.3+ (845, 45%).

Example 2: Synthesis of Conjugation Reagent 9 Comprising the Auristatin Cytotoxic Payload, MMAE

[0179] ##STR00075##

Step 1: Synthesis of Compound 10

[0180] ##STR00076##

[0181] To a solution of tosyl chloride (307 mg) in dichloromethane (5 mL) was added a solution of dodecaethylene glycol (400 mg), triethylamine (255 μL) and DMAP (13 mg) in dichloromethane (5 mL) and the combined solution was stirred at room temperature for 16 h. Additional DMAP (13 mg) and triethylamine (255 μL) was added and the reaction mixture was allowed to stir at room temperature for a further 24 h. The reaction solution was concentrated in vacuo and the residue purified by reverse phase C18-column chromatography eluting with buffer A (v/v): water:5% acetonitrile:0.05% trifluoroacetic acid and buffer B (v/v): acetonitrile:0.05% trifluoroacetic acid (100:0 v/v to 0:100 v/v). The organic solvent was removed in vacuo and the aqueous solvent was removed by lyophilisation to give compound 10 as a colourless oil (287 mg). .sup.1H NMR (400 MHz, CDCl.sub.3) δ.sub.H 7.78 (d, J=8.5 Hz, 4H), 7.32 (d, J=8.5 Hz, 4H), 4.13 (t, J=4.9 Hz, 4H), 3.66 (t, J=4.9 Hz, 4H), 3.62 (br. s, 24H), 3.61-3.58 (m, 8H), 3.56 (s, 8H), 2.42 (s, 6H). m/z [M+Na].sup.+ (877, 100%), [M+H].sup.+ (855, 75%).

Step 2: Synthesis of Compound 11

[0182] ##STR00077##

[0183] A solution of compound 2 (85 mg) in anhydrous THF (2 mL) was added dropwise over a period of 1 h to a solution of sodium hydride (40 mg, 60% dispersion in mineral oil) in anhydrous THF (8 mL) at room temperature. After stirring for 1 h, a solution of compound 10 (280 mg) in anhydrous THF (20 mL) was added over a period of 2 h and the reaction mixture was stirred at room temperature for 96 h. Water (30 mL) was added and the THF was removed in vacuo. The aqueous solution was extracted with chloroform (4×25 mL) and then chloroform:isopropanol (9:1 v/v, 2×25 mL). The organic phases were combined and dried over magnesium sulfate before the solution was filtered and concentrated in vacuo. The residue was then purified by reverse phase C18-column chromatography eluting with buffer A (v/v): water:55% acetonitrile:0.05% trifluoroacetic acid and buffer B (v/v):acetonitrile:0.05% trifluoroacetic acid (100:0 v/v to 0:100 v/v). The organic solvent was removed in vacuo and the aqueous solvent was removed by lyophilisation to give compound 11 as a colourless oil (58 mg). .sup.1H NMR (400 MHz, CDCl.sub.3) δ.sub.H 7.68 (d, J=8.4 Hz, 2H), 7.27 (d, J=8.4 Hz, 4H), 3.66-3.52 (m, 52H), 3.35 (t, J=6.3 Hz, 4H), 2.40 (s, 3H). m/z [M+Na].sup.+ (792, 100%), [M+H].sup.+ (770, 55%).

Step 3: Synthesis of Compound 12

[0184] ##STR00078##

[0185] To a solution of compound 11 (54 mg) in anhydrous THF (3 mL) was added lithium aluminium hydride (513 μL, 1 M solution in THF) and the solution was heated at reflux for 4 h before the reaction mixture was cooled to 0° C. and quenched by the dropwise addition of water. The suspension was filtered and the precipitate washed with chloroform:ethanol (9:1 v/v, 3×6 mL). The filtrate and washings were combined and concentrated in vacuo to give compound 12 as a white solid (34 mg). m/z [M+Na].sup.+ (639, 10%), [M+H].sup.+ (617, 100%).

Step 4: Synthesis of Compound 13

[0186] ##STR00079##

[0187] To a solution of compound 5 (25 mg) in DMF (0.4 mL) at 0° C. was added HATU (7 mg). The solution was stirred for 20 min at 0° C. before NN (2 μL) was added and the solution was stirred for a further 10 min. To a separate solution of compound 12 (17 mg) in DMF (0.4 ml) was added NMM (2 μL) and the solution was stirred at room temperature for 30 min. The two solutions were then combined and additional quantities of HATU (7 mg) and NMM (2 μL) were added to the combined solution which was stirred for 1 h at room temperature. Further HATU (7 mg) and NMM (2 μL) were added and the solution stirred for 0.5 h before the reaction solution was concentrated in vacuo to give crude Fmoc-L-Glu-[Val-Cit-PAB-MMAE]-aza-42-crown-14 (35 mg). m/z [M+Na].sup.+ (2095, 25%), [M+H].sup.+ (2073, 15%, [M+2H].sup.2+ (1037, 100%). To a solution of crude Fmoc-L-Glu-[Val-Cit-PAB-MMAE]-aza-42-crown-14 (35 mg) in DMF (0.5 mL) was added piperidine (10 μL) and the solution was stirred for 30 min at room temperature. Additional piperidine (10 μL) was added and the solution was stirred for a further 30 min at room temperature. The reaction solution was concentrated in vacuo and the residue purified by reverse phase C18-column chromatography eluting with buffer A (v/v): water:55% acetonitrile:0.05% trifluoroacetic acid and buffer B (v/v): acetonitrile:0.05% trifluoroacetic acid (100:0 v/v to 0:100 v/v). The organic solvent was removed in vacuo and the aqueous solvent was removed by lyophilisation to give compound 13 as a colourless oil (22 mg). m/z [M+Na].sup.+ (1873, 10%), [M+H].sup.+ (1851, 50%), [M+2H].sup.2+ (926, 100%).

Step 5: Synthesis of Reagent 9

[0188] To a solution of reagent 8 (11.5 mg) in DMF (0.3 mL) at 0° C. was added HATU (3.3 mg) The solution was stirred at 0° C. for 20 min before NN (0.9 μL) was added and the solution was stirred for a further 10 min. To a separate solution of compound 13 (15 mg) in DMF (0.3 mL) was added NMM (0.9 μL) and the solution was stirred at room temperature for 30 min. The two solutions were then combined and additional quantities of HATU (3.3 mg) and NMM (0.9 μL) were added and the solution was stirred at room temperature for 1 h. Further quantities of HATU (3.3 mg) and NMM (0.9 μL) were added and the solution stirred at room temperature for 1 h. HATU (3.3 mg) and NMM (0.92 μL) were again added and the solution was stirred at room temperature for 2 h before the reaction solution was concentrated in vacuo and the residue purified by reverse phase C18-column chromatography eluting with buffer A (v/v): water:5% acetonitrile:0.05% trifluoroacetic acid and buffer B (v/v): acetonitrile:0.05% trifluoroacetic acid (100:0 v/v to 0:100 v/v). The organic solvent was removed in vacuo and the aqueous solvent was removed by lyophilisation to give reagent 9 as a colourless oil (11 mg). m/z [M+Na].sup.+ (2822, 10%), [M+H].sup.+ (2800, 20%), [M+2H].sup.2+ (1400, 80%), [M+3H].sup.3+ (933, 100%).

Example 3: Conjugation of Reagents 1 and 17 (Comparative) to Brentuximab and Anti-PSMA Antibodies to Produce Antibody Drug Conjugates (ADCs) 4 and 18 (Comparative) and 21 and 22 (Comparative), Respectively, with DAR 4

[0189] Conjugation reagents 1 and 17 were conjugated to Brentuximab or anti-PSMA antibody, giving rise to ADCs 14, 18, 21 and 22 respectively using methods analogous to those described in WO2014064423 and WO2014064424. Briefly, antibody (Brentuximab or anti-PSMA antibody) at a concentration of 5.2 mg/mL in 20 mM sodium phosphate, pH 7.5 (containing 150 mM NaCl and 20 mM EDTA) was heated to 40° C. in a heating block for 15 min. TCEP (6 eq. per mAb) was added to the mAb solution, mixed gently and incubated at 40° C. for 1 h before being allowed to cool to 22° C. Conjugation reagents were dissolved in MeCN or DMF to give a 3 mM solution. The reduced mAb solution was diluted to 4.2 mg/mL with 20 mM sodium phosphate, pH 7.5 (containing 150 mM NaCl and 20 mM EDTA). Conjugation reagent (5.6 eq. per mAb) was added to the mAb solution, the reaction was mixed gently and incubated at 22° C. for 22 h. After this the reaction was treated with 50 mM N-acetyl-L-cysteine (20 eq. over reagent) at 22° C. for 1 h. The crude conjugation mixture was analysed by hydrophobic interaction chromatography (HIC). The crude reaction was mixed with an equal volume of 50 mM sodium phosphate, pH 7 (4 M NaCl) and the resulting solution was loaded onto a ToyoPearl Phenyl-650S HIC column equilibrated with 50 mM sodium phosphate, pH 7 (2 M NaCl). The ADC was eluted from the column with a gradient of 50 mM sodium phosphate, pH 7 (20% isopropanol). Fractions containing DAR 4 ADC were pooled and concentrated (Vivaspin 20, 10 kDa PES membrane). The concentrated sample was buffer exchanged into PBS, pH 7.1-7.5, and sterile filtered (0.22 μm PVDF membranes). DAR assignments were based on A248/A280 absorption ratios. The average DAR of conjugates was calculated from the relative peak areas of individual DAR species following HIC analysis at 280 nm.

Example 4: Synthesis of Conjugation Reagent 15 (Comparative) Comprising the Auristatin Cytotoxic Payload, MMAE

[0190] Conjugation reagent 5, which contains a maleimide functional grouping, was synthesised as described within WO2015057699.

##STR00080##

Example 5: Synthesis of Conjugation Reagent 17 (Comparative) Comprising the Auristatin Cytotoxic Payload, MMAE

[0191] ##STR00081##

Step 1: Synthesis of Compound 19

[0192] ##STR00082##

[0193] A solution of Fmoc-L-Glu-(OtBu)-OH (36 mg) in DMF (2 mL) was cooled to 0° C. under an argon atmosphere and (benzotriazol-1-yloxy)tris-(dimethylamino) phosphonium hexafluorophosphate (BOP) (41 mg) was added, followed by NH.sub.2—PEG(24u)-OMe (100 mg) and DIPEA (19 μL). The solution was allowed to warm to room temperature and after 22 h the volatiles were removed in vacuo. The resulting residue was dissolved in dichloromethane (1 mL) and purified by normal phase column chromatography eluting with dichloromethane:methanol (100:0 v/v to 80:20 v/v). The organic solvent was removed in vacuo to give Fmoc-L-Glu-[OtBu]-[PEG(24u)-OMe] as a colourless oil (84 mg). Piperidine (49 μL) was added to a solution of compound Fmoc-L-Glu-[OtBu]-[PEG(24u)-OMe] (74 mg) in DMF (2 mL) under an argon atmosphere and the resulting solution stirred at room temperature for 22 h, after which the volatiles were removed in vacuo. The resulting residue was triturated with hexane (3×0.7 mL). The organic solvent was decanted each time and the resulting residue dried in vacuo to give compound 19 as a white solid (61 mg). m/z [M+H].sup.+ (1274, 70%), [M+2H].sup.2+ (638, 100%).

Step 2: Synthesis of Reagent 20

[0194] ##STR00083##

[0195] To a solution of reagent 8 (26.6 mg) in DMF (550 μL) cooled to 0° C. under an argon atmosphere was added HATU (10.5 mg) and the solution stirred for 0.5 h at 0° C. To this was added a solution of compound 19 (32 mg) in DMF (550 μL). The resulting solution was stirred for 5 min at 0° C. before the addition of NMM (2.9 μL) and HATU (10.5 mg). The reaction solution was allowed to stir at 0° C. for 2 h before being warmed to room temperature and stirred for a further 3.5 h. After this time the volatiles were removed in vacuo. The resulting residue was dissolved in water:acetonitrile (1.2 mL, 1:1 v/v), and purified by reverse phase C18-column chromatography eluting with buffer A (v/v): water:55% acetonitrile:0.1% formic acid and buffer B (v/v): acetonitrile:0.1% formic acid (100:0 v/v to 0:100 v/v). The organic solvent was removed in vacuo and the aqueous solvent removed by lyophilisation to give bis-mPEG(7u)sulfone-propanoyl-benzamide-L-Glu-[OtBu]-[PEG(24u)-OMe] as a colourless oil (30.5 mg). .sup.1H NMR (400 MHz, MeOH—O.sub.4) 8.19 (2H, d), 8.04 (2H, d), 4.83-4.71 (1H, m), 4.58 (1H, dd), 3.92-3.83 (6H, m), 3.78-3.56 (140H, m), 3.57-3.51 (6H, m), 3.40 (4H, dd), 3.36 (3H, s), 3.35 (6H, s), 2.41 (2H, t), 2.24-2.13 (1H, m), 2.10-1.98 (1H, m), 1.45 (9H, s). m/z [M+Na].sup.+ (2243, 50%), [M+H].sup.+ (2221, 40%), [M+Na+2H].sup.3+ (747, 100%). A solution of bis-mPEG(7u)sulfone-propanoyl-benzamide-L-Glu-[OtBu]-[PEG(24u)-OMe] (30 mg) in dichloromethane (2 mL) under an argon atmosphere was cooled to 0° C. to which trifluoroacetic acid (500 μL) was added and the resulting solution stirred for 1.5 h. The reaction mixture was allowed to warm to room temperature and stirred for a further 1 h. After this time the volatiles were removed in vacuo. The resulting residue was dissolved in water:acetonitrile (0.6 mL, 1:1 v/v), and purified by reverse phase C18-column chromatography eluting with buffer A (v/v): water:55% acetonitrile:0.05% trifluoroacetic acid and buffer B (v/v): acetonitrile:0.05% trifluoroacetic acid (100:0 v/v to 0:100 v/v). The organic solvent was removed in vacuo and the aqueous solvent removed by lyophilisation to give reagent 20 as a colourless oil (20 mg). .sup.1H NMR (400 MHz, MeOH-δ.sub.4) 8.19 (2H, d), 8.04 (2H, d), 4.81-4.72 (1H, m), 4.59 (1H, dd), 3.92-3.84 (6H, m), 3.67-3.50 (146H, m), 3.40 (4H, dd), 3.36 (3H, s), 3.35 (6H, s), 2.48 (2H, t), 2.26-2.15 (1H, m), 2.15-2.03 (1H, m). m/z [M+2H].sup.2+ (1083, 60%), [M+2H+Na].sup.3+ (729, 100%).

Step 3: Synthesis of Conjugation Reagent 17

[0196] A solution of compound 20 (12.4 mg) in DMF (500 μL) was cooled to 0° C. under an argon atmosphere. HATU (2.4 mg) was added and the solution stirred for 0.5 h at 0° C. To this was added a solution of Val-Cit-PAB-MMAE TFA salt (7.8 mg) and NN (0.7 μL) in DMF (500 μL), which had been stirred at room temperature for 0.5 h. After 5 min, an additional amount of HATU (1.2 mg) and NMM (0.4 μL) was added and the reaction mixture stirred at room temperature. After 2 h, an additional amount of HATU (1.2 mg) and NMM (0.4 μL) was added and the reaction mixture stirred at room temperature. After a further 1 h, the reaction solution was concentrated in vacuo and purified by reverse phase C18-column chromatography eluting with buffer A (v/v): water:55% acetonitrile:0.05% trifluoroacetic acid and buffer B (v/v): acetonitrile:0.05% trifluoroacetic acid (100:0 v/v to 0:100 v/v). The organic solvent was removed in vacuo and the aqueous solvent removed by lyophilisation to give reagent 17 as a colourless oil. m/z [M+H].sup.+ (3270, 12%), [M+2H].sup.2+ (1636, 50%), [M+3H].sup.3+ (1091, 100%).

Example 6: Conjugation of Reagent 15 (Comparative) to Brentuximab to Produce Antibody Drug Conjugate 16 (Comparative) with DAR 8

[0197] Conjugation reagent 1 was conjugated to Brentuximab, giving rise to ADC 16 using the methods described within WO2015057699, U.S. Pat. No. 7,090,843, Lyon et al., (2015) Nature Biotechnology, 33(7) p733-736 and Lyon et al., (2012), Methods in Enzymology, Volume 502, p123-137. Briefly, Brentuximab in 20 mM sodium phosphate buffer, pH 6.5 (150 mM NaCl, 20 mM EDTA) was reduced with TCEP (6 eq.) at 40° C. for 1 h. Conjugation of the reduced antibody with 2.0 molar equivalents of reagent 15 per free thiol was then performed. Reagent 15 was added to the mAb to give a final antibody concentration of 4 mg/mL. The solution was mixed gently and incubated at 22° C. for 2 h. After 2 h additional reagent 15 (0.2 molar equivalents) was added and the mixture was incubated for a further 1 h at 22° C. Excess reagent 15 was quenched with N-acetyl-L-cysteine (20 eq. over reagent) and the crude reaction was purified using a 1 mL ToyoPearl Phenyl-650S HIC column equilibrated with 50 mM sodium phosphate, pH 7 (2 M NaCl). The ADC was eluted from the column with a gradient of 50 mM sodium phosphate, pH 7 (20% isopropanol). Fractions containing ADC were pooled and concentrated (Vivaspin20, 10 kDa PES membrane) to give an average DAR 8 product. The concentrated sample was buffer exchanged into DPBS, pH 7.1-7.5 and sterile filtered.

[0198] ADC 16 was difficult to purify and characterise due to the heterogeneity of the reaction products (number of DAR variants), leading to poor resolution of the individual DAR species by preparative HIC. Results showed that the final reaction product contained significant quantities of DAR species both greater than and less than DAR 8. Purifying the DAR8 species completely from these higher and lower than DAR8 species would result in significantly lower yields of DAR8 in the final product.

Example 7: Comparison of Antibody Drug Conjugates 14 and 16 (Comparative) by Thermal Stress Test

[0199] ADC samples 14 and 16 were each prepared at 0.5 mg/mL by dilution with DPBS pH 7.1-7.5.

[0200] The two ADC samples were incubated at 65° C. for 30 min followed by incubation in an ice bath for 5 min before Size Exclusion Chromatography (SEC). SEC was performed using a TOSOH Bioscience TSK gel Super SW 3000 column. UV absorbance at 280 nm was monitored during an isocratic elution with a 0.2 M potassium phosphate buffer, pH 6.8 (0.2 M potassium chloride and 15% isopropanol).

[0201] Tables 1a and 1b below show the conformations of ADCs 14 and 16 before and after thermal stress test, as measured by the area under the curve of each peak by Abs 280 nm, following SEC.

[0202] The results in Tables 1a and 1b show that ADC 14 remains in a non-aggregated state to a much greater extent than ADC 16 following thermal stress test. In addition, the results also show that 16 dissociates into lighter molecular weight components to a greater extent than conjugate 14.

TABLE-US-00003 TABLE 1a ADC conformation before thermal stress test (% of total ADC) 14 16 Non-aggregated 98.1 97.4 Aggregated 1.9 1.8 Dissociated 0 0.7

TABLE-US-00004 TABLE 1b ADC conformation after thermal stress test (% of total ADC) 14 16 Non-aggregated 62.0 11.1 Aggregated 37.4 71 Dissociated 0.6 17.9

Example 8: Comparison of Average DARs for ADCs 14 and 16 (Comparative) Following Incubation within Human Serum

[0203] ADCs 14 and 16 were diluted to 0.1 mg/mL in human serum, 88% (v/v) serum content. Each solution was immediately sub-aliquoted into 4×0.5 mL low-bind Eppendorf tubes. Two of the Eppendorf tubes, corresponding to the ‘0’ time points were immediately transferred to the −80° C. freezer, while the remaining samples were incubated at 37° C. for 6 d. After 6 d, the samples were removed from the freezer and/or the incubator, the conjugates purified by affinity capture (CD30-coated magnetic beads), and analysed using hydrophobic interaction chromatography (HIC). CD30 affinity capture and HIC for average DAR determination were carried out as described:

[0204] CD30 affinity capture for average DAR determination. Affinity capture was performed using streptavidin coated magnetic beads (Dynabeads-Streptavidin T1, Life Technologies). CD30 (Recombinant human CD30, Sino Biological Inc.) was biotinylated and immobilized on beads through streptavidin-biotin binding and finally blocked using skimmed milk peptides. 500 μL of the plasma sample in PBS was added to the CD30-coated beads and incubated overnight at 4° C. and finally washed using PBS. Captured antibodies were eluted using acidic elution buffer for 5 min at 4° C. The eluate was subsequently neutralized to pH 7 using sodium acetate buffer, pH 8. Eluted samples were further mixed with HIC loading buffer and analysed using hydrophobic interaction chromatography with UV detection (HIC-UV).

[0205] Hydrophobic interaction chromatography for average DAR determination. Affinity captured ADCs were analysed using hydrophobic interaction chromatography in order to determine the average drug to antibody ratio (DAR). The method consisted of a linear gradient from 100% buffer A (50 mM sodium phosphate pH 7.0, 1.5 M ammonium sulfate) to 100% buffer B (50 mM sodium phosphate pH 7.0, 20% isopropanol) in 30 min using a TOSOH TSK gel Butyl-NPR HIC separation column with detection at 280 nm.

[0206] FIG. 1 shows that after 6 days at 37° C. in human serum, conjugate 16 has lost much of its cytotoxic payload, whereas conjugate 14 remains largely unchanged, as indicated by the reduction in average DAR value of the sample. This indicates that conjugates of the invention have improved stability.

Example 9: Analysis of DAR4 ADCs 14 and 18 (Comparative) and 21 and 22 (Comparative), by In Vitro Cell Viability Assay

[0207] Loss of tumour cell viability following treatment with cytotoxic drugs in vitro can be measured by growing cell lines in the presence of increasing concentrations of drug and quantifying the loss of proliferation or metabolic activity using Cell-Titer Glo® Luminescent reagent (Promega). The protocol for PSMA and CD30 overexpressing cell lines describes cell seeding, drug treatment and determination of the cell viability in reference to untreated cells based on ATP synthesis, which is directly correlated to the number of cells present in the well.

[0208] The cell lines listed in Table 2 were maintained following the suppliers' recommendations.

TABLE-US-00005 TABLE 2 Cell line Antigen Source Growth medium LNCaP clone FGC PSMA ECACC, Cat. RPMI-1640 medium 89110211 (Life Technologies ®), C4-2 PSMA 10% fetal bovine serum, Karpas-299 CD30 Dr Abraham 100 U/mL Penicillin and Karpas at the 100 ug/mL Streptomycin University of Cambridge

[0209] Adherent PSMA-positive LNCaP and C4-2 cells were detached with TrypLE and resuspended in complete medium. Cells were counted using disposable Neubauer counting chambers and cell density adjusted to 10×10.sup.4 cells/mL for LNCaP and 2×10.sup.4 cells/mL for C4-2, respectively. The cells were seeded (100 μL/well) into poly-D-Lysine coated opaque-walled 96-well white plates and incubated for 24 h at 37° C. and 5% CO.sub.2.

[0210] Human T cell lymphoma Karpas 299 cells were counted and adjusted to a cell density of 5×10.sup.4 cells/mL in complete growth medium. Cells were seeded (50 μL/well) into opaque-walled 96-well plates and incubated for 24 h at 37° C. and 5% CO.sub.2.

[0211] Eight point serial dilutions of each compound were prepared in the relevant culture medium. Cells were treated with the compounds and concentration ranges are specified in Table 3.

TABLE-US-00006 TABLE 3 Cell line Drug-conjugate Concentration range Karpas 299 14   1 nM-0.06 pM Karpas 299 18 (comparative) 0.5 nM-0.2 pM LNCaP & C4-2 21  10 nM-5 pM LNCaP & C4-2 22 (comparative)  10 nM-5 pM LNCaP & C4-2 MMAE (free drug)  10 nM-5 pM

[0212] The medium from the plate containing the adherent LNCaP or C4-2 cells was removed and replaced by 100 μL/well of the 1× serially diluted compounds. Karpas-299 cells were treated by addition of 50 μL/well of the 2× serially diluted compounds. The cells were then incubated at 37° C. and 5% CO.sub.2 for a further 96 h.

[0213] The cell viability assay was carried out using the Cell-Titer Glo® Luminescence reagent, as described by the manufacturer's instructions, (Promega Corp. Technical Bulletin TB288; Lewis Phillips G. D, Cancer Res 2008; 68:9280-9290).

[0214] Luminescence was recorded using a Molecular Devices Spectramax i3x plate reader and data subsequently analysed using GraphPad Prism four parameter non-linear regression model. Viability was expressed as % of untreated cells and calculated using the following formula:

[00001]$\%\mathrm{Viability}=100\times \frac{{\mathrm{Luminescence}}_{\mathrm{Sample}}-{\mathrm{Luminescence}}_{\mathrm{No}\mathrm{cell}\mathrm{Control}}}{{\mathrm{Luminescence}}_{\mathrm{Untreated}}-{\mathrm{Luminescence}}_{\mathrm{No}\mathrm{cell}\mathrm{Control}}}$

[0215] The % viability was plotted against the logarithm of drug concentration in nM to extrapolate the IC.sub.50 values. IC.sub.50 values indicating the anti-proliferative effects of the drug conjugates are summarised in Table 4. These indicate that conjugates of the invention have improved potency.

TABLE-US-00007 TABLE 4 Cell line Drug-conjugate IC.sub.50 (nM) Karpas 299 14 0.04 Karpas 299 18 (comparative) 0.06 LNCaP 21 0.61 LNCaP 22 (comparative) 0.76 LNCaP MMAE (free drug) 2.14 C4-2 21 0.19 C4-2 22 (comparative) 0.27 C4-2 MMAE (free drug) 0.71

Example 10: Synthesis of Conjugation Reagent 23 (Comparative) Comprising the Auristatin Cytotoxic Payload, MMAE

[0216] ##STR00084##

Step 1: Synthesis of Reagent 24

[0217] ##STR00085##

[0218] A solution of 4-[2,2-bis[(p-tolylsulfonyl)-methyl]acetyl]benzoic acid (1.0 g) was added to N-hydroxybenzotriazole hydrate (306 mg) in anhydrous THF (10 mL) under a nitrogen atmosphere. The resulting solution was cooled to 0° C. and diisopropylcarbodiimide (310 μL) was added dropwise. The reaction mixture was stirred for 20 min at 0° C. before being warmed to room temperature. Additional anhydrous THF (10 mL) was added to the reaction mixture after 1 h. After 18 h, the formed precipitate was filtered and washed with cold THF (2×5 mL) before being dried in vacuo. The solid was stirred with MeOH (10 mL) for 1 h at room temperature, collected by filtration and washed sequentially with MeOH (2×5 mL) and Et.sub.2O (5 mL). The solid was then dried in vacuo to give compound 24 as a white solid (1.1 g). m/z [M+H].sup.+ (618, 100%).

Step 2: Synthesis of Reagent 25

[0219] ##STR00086##

[0220] To a stirred suspension of L-Glutamic acid 5-tert-butyl ester (198 mg) in anhydrous DMF (20 mL) under a nitrogen atmosphere was added NMM (107 μL). The reaction mixture was cooled to 0° C. before reagent 24 (603 mg) was added. The resulting suspension was stirred at 0° C. for 1 h, after which the reaction mixture was allowed to warm to room temperature. After 19 h, the resulting solution was concentrated in vacuo and purified by reverse phase C18-column chromatography, eluting with buffer A (v/v): water:5% acetonitrile:0.1% formic acid and buffer B (v/v): acetonitrile:0.1% formic acid (100:0 v/v to 0:100 v/v). The organic solvent was removed in vacuo and the aqueous solvent removed by lyophilisation to give the reagent 25 as a white solid (198 mg). .sup.1H NMR (400 MHz, CDCl.sub.3) δ 7.98 (1H, d), 7.86 (2H), 7.71-7.65 (6H, m), 7.36 (4H, d), 4.68 (1H, ddd), 4.34 (1H, q), 3.62 (2H, ddd), 3.50 (2H, ddd), 2.69 (1H ddd), 2.55-2.45 (1H, m), 2.48 (6H, s), 2.34-2.16 (2H, m), 1.46 (9H, s); m/z [2M+H].sup.+ (1371,74%), [2M+H-tBu].sup.+ (1315, 70%), [M+H-tBu].sup.+ (630, 100%).

Step 3: Synthesis of Reagent 26

[0221] ##STR00087##

[0222] Reagent 25 (50 mg) and BOP (40 mg) were dissolved in anhydrous DMF (3 mL), cooled to 0° C. and added to a solution of NH.sub.2—PEG(24u)-OMe (99 mg) and NMM (10 μL) in anhydrous DMF (2 mL). The reaction mixture was stirred at 0° C. and after 4 h, additional amounts of BOP (10 mg) and NMM (2.5 μL) were added to the reaction mixture which was stirred for a further 15 min before being stored at −20° C. for 18 h. The reaction mixture was then concentrated in vacuo and purified by reverse phase C18-column chromatography, eluting with buffer A (v/v): water:5% acetonitrile:0.1% formic acid and buffer B (v/v): acetonitrile:0.1% formic acid (100:0 v/v to 0:100 v/v). The organic solvent was removed in vacuo and the aqueous solvent removed by lyophilisation to give bis-tolylsulfonyl-propanoyl-benzamide-L-Glu-[OtBu]-[PEG(24u)-OMe] as a colourless oil (128 mg). m/z [M+H].sup.+ (1757, 100%), [M+2H].sup.2+ (879, 100%). Bis-tolylsulfonyl-propanoyl-benzamide-L-Glu-[OtBu]-[PEG(24u)-OMe] (126.5 mg) was dissolved in formic acid (2.5 mL) and stirred under a nitrogen atmosphere at room temperature. After 20 h, the reaction mixture was concentrated in vacuo and dried under high vacuum for 18 h to give reagent 26 as a colourless oil (122 mg, assumed quantitative yield). m/z [M+Na].sup.+ (1723, 15%), [M+H].sup.+ (1700, 100%).

Step 4: Synthesis of Reagent 23

[0223] A solution of reagent 26 (13 mg), HATU (4.1 mg) and Val-Cit-PAB-MMAE.TFA salt (9 mg) in anhydrous DMF (1 mL) under an argon atmosphere was cooled to 0° C. To this was added NMM (2 μL). After 1 h, an additional amount of HATU (4.1 mg) and NMM (2 μL) was added, and after a further 1.5 h the solution was stored at −20° C. for 72 h. The reaction solution was concentrated in vacuo, dissolved in acetonitrile (1 ml) and purified by reverse phase C18-column chromatography eluting with buffer A (v/v): water:5% acetonitrile:0.05% trifluoroacetic acid and buffer B (v/v): acetonitrile:0.05% trifluoroacetic acid (100:0 v/v to 0:100 v/v). The organic solvent was removed in vacuo and the aqueous solvent removed by lyophilisation to give reagent 23 as a thick clear colourless oil (11.4 mg). m/z [M+H].sup.+ (2805, 20%), [M+2H].sup.2+ (1403, 75%), [M+3H].sup.3+ (936, 100%).

Example 11: Synthesis of Conjugation Reagent 27 (Comparative) Comprising the Auristatin Cytotoxic Payload, MMAE

[0224] ##STR00088##

Step 1: Synthesis of Compound 28

[0225] ##STR00089##

[0226] To a solution of NH.sub.2—PEG(7u)-OMe (300 mg) in ethanol (2.5 mL) was added tert-butyl acrylate (194 μL) and the reaction mixture was stirred at room temperature for 72 h. The solution was concentrated in vacuo and purified by normal phase chromatography eluting with dichloromethane:methanol (100:0 v/v to 70:30 v/v). The solvent was removed in vacuo to give compound 28 as a pale yellow oil (324 mg). m/z [M+H].sup.+ (468, 100%).

[0227] Step 2: Synthesis of reagent 29.

##STR00090##

[0228] To a solution of 4-[2,2-bis[(p-tolylsulfonyl)-methyl]acetyl]benzoic acid (257 mg) in anhydrous DMF (2.5 mL) at 0° C. was added HATU (204 mg) and the reaction mixture was stirred at 0° C. for 10 min. NMM (57 μL) was added and the reaction solution was stirred for a further 20 min at 0° C. Additional HATU (102 mg) and NN (29 μL) were added and the solution was warmed to room temperature and left to stir for 30 min. After this time, a solution of compound 28 (200 mg) in anhydrous DMF (2.5 mL) was added. The reaction mixture was stirred for 5 h. The solution was then concentrated in vacuo and purified by reverse phase C18-column chromatography eluting with buffer A (v/v): water:55% acetonitrile:0.05% trifluoroacetic acid and buffer B (v/v): acetonitrile:0.05% trifluoroacetic acid (100:0 v/v to 0:100 v/v). The organic solvent was removed in vacuo and the aqueous solvent removed by lyophilisation to give reagent 2 as a colourless solid (173 mg). m/z [M+Na].sup.+ (973, 40%) [M+H].sup.+ (951, 100%), [M+H-tBu]+ (895, 90%).

Step 3: Synthesis of Reagent 30

[0229] ##STR00091##

[0230] To a solution of reagent 29 (160 mg) in dichloromethane (1.6 mL) at 0° C. was added trifluoroacetic acid (0.5 mL) and the solution was stirred at 0° C. for 1 h and then stored at 4° C. for 16 h. The volatiles were then removed in vacuo, the residue was dissolved in acetonitrile (1 mL) and diluted with aqueous buffer (v/v): water:5% acetonitrile:0.05% trifluoroacetic acid (5 mL). The solvent was then removed by lyophilisation to give reagent 30 as an amber oil (quantitative yield). m/z [M+Na].sup.+ (916, 50%), [M+H].sup.+ (894, 100%).

Step 4: Synthesis of Reagent 27

[0231] To a solution of reagent 30 (15 mg) in anhydrous DMF (0.6 mL) at 0° C. was added HATU (7.3 mg) and the reaction mixture was stirred at 0° C. for 20 min. NMM (2 μL) was added and the solution was stirred for a further 20 min at 0° C. Additional HATU (7.3 mg) and NMM (2 μL) were then added, the solution was warmed to room temperature and stirred for 30 min. A separate solution of Val-Cit-PAB-MMAE.TFA salt (21.8 mg) and NN (2 μL) in anhydrous DMF (0.6 mL) which had previously been stirred at room temperature for 10 min, was then added to the reaction solution. After stirring at room temperature for 3 h, the reaction solution was concentrated in vacuo and purified by reverse phase C18-column chromatography eluting with buffer A (v/v): water:55% acetonitrile:0.05% trifluoroacetic acid and buffer B (v/v): acetonitrile:0.05% trifluoroacetic acid (100:0 v/v to 0:100 v/v). The organic solvent was removed in vacuo and the aqueous solvent removed by lyophilisation to give reagent 2 as a white solid (23.7 mg). m/z [M+H].sup.+ (2000, 10%), [M+2H].sup.2+ (1000, 100%).

Example 12: Conjugation of Reagents 9 and 27 (Comparative) to Brentuximab to Produce Antibody Drug Conjugates (ADCs) 31 and 32 (Comparative) Respectively, with DAR 4

[0232] Conjugation reagents 9 and 27 (comparative) were conjugated to Brentuximab giving rise to ADCs 31 and 32 (comparative) respectively, using a method analogous to that described in Example 3. Briefly, Brentuximab (5.0 mg/mL in 20 mM sodium phosphate, 150 mM NaCl, 20 mM EDTA, pH 7.5) was heated to 40° C. in a heating block for 15 min. TCEP (6 eq. per mAb) was added to the mAb solution, mixed gently and incubated at 40° C. for 1 h before being allowed to cool to 22° C. Conjugation reagent 9 was dissolved in acetonitrile and conjugation reagent 2 was dissolved in DMF to give 3 mM and 1.6 mM stock solutions respectively. The reduced mAb solutions were diluted to approximately 4.3 mg/mL with 20 mM sodium phosphate, 150 mM NaCl, 20 mM EDTA, pH 7.5. Conjugation reagents (5.6 to 6.0 eq. per mAb) were added to the reduced mAb solutions to give a final antibody concentration of 4 mg/mL. The reaction solutions were mixed gently and incubated at 22° C. for 16 to 24 h. After this time, each reaction solution was treated with 50 mM N-acetyl-L-cysteine (20 eq. over reagent) at 22° C. for 1 h. Each crude conjugation mixture was analysed by hydrophobic interaction chromatography (HIC). The crude reactions were mixed with an equal volume of 50 mM sodium phosphate, 4 M NaCl, pH 7 and the resulting solutions loaded onto a ToyoPearl Phenyl-650S HIC column equilibrated with 50 mM sodium phosphate, 2 M NaCl, pH 7. The ADCs were eluted from the column with a gradient of 50 mM sodium phosphate, pH 7 (20% isopropanol). Fractions containing DAR 4 ADC were pooled and concentrated (Vivaspin 20, 10 kDa PES membrane). The concentrated samples were buffer exchanged into PBS, pH 7.1-7.5, and sterile filtered (0.22 μm PVDF membranes).

Example 13: Synthesis of Conjugation Reagent 33 Comprising a Duocarmycin Cytotoxic Payload

[0233] ##STR00092##

Step 1: Synthesis of Compound 34

[0234] ##STR00093##

[0235] To a solution of Fmoc-Glu(OtBu)-OH (78 mg) in anhydrous DMF (500 μL) at 0° C. was added HATU (108 mg) and NMM (34 μL) and the mixture was stirred at 0° C. for 10 min. To this was added a solution of compound 4 (44 mg) in anhydrous DMF (500 μL) and the mixture was stirred at 0° C. under an argon atmosphere for 15 min. The reaction mixture was then concentrated in vacuo and the residue dissolved in anhydrous DMF (500 μL). Piperidine (70 μL) was added and the solution stirred for 90 min at room temperature. The reaction solution was concentrated in vacuo and purified by reverse phase C18-column chromatography, eluting with buffer A (v/v): water:5% acetonitrile:0.05% trifluoroacetic acid and buffer B (v/v): acetonitrile:0.05% trifluoroacetic acid (100:0 v/v to 0:100 v/v). The organic solvent was removed in vacuo and the aqueous solvent removed by lyophilisation to give compound 34 as an orange oil (44 mg). m/z [M+H].sup.+ (537, 45%).

Step 2: Synthesis of Reagent 35

[0236] ##STR00094##

[0237] To a solution of 4-[2,2-bis[(p-tolylsulfonyl)-methyl]acetyl]benzoic acid (37.5 mg) in anhydrous DMF (500 μL) at 0° C. was added HATU (65 mg) and NMM (20 μL) and the mixture was stirred at 0° C. for 10 min. To this was added a solution of compound 34 (44.3 mg) in anhydrous DMF (500 μL) and the mixture was stirred at 0° C. under an argon atmosphere for 1 h. The reaction mixture was then concentrated in vacuo, the residue dissolved in DMF (1 mL) and purified by reverse phase C18-column chromatography, eluting with buffer A (v/v): water:0.05% trifluoroacetic acid and buffer B (v/v): acetonitrile:0.05% trifluoroacetic acid (60:40 v/v to 0:100 v/v). The solvent was removed by lyophilisation to give bis-tolylsulfonyl-propanoyl-benzamide-L-Glu-(OtBu)-aza-24-crown-8 as a white solid (28.5 mg). m/z [M+Na].sup.+ (1041, 20%), [M+H].sup.+ (1019, 5%). To a solution of bis-tolylsulfonyl-propanoyl-benzamide-L-Glu-(OtBu)-aza-24-crown-8 (26.5 mg) in anhydrous dichloromethane (1 mL) was added trifluoroacetic acid (500 μL) and the solution stirred at room temperature under an argon atmosphere for 1 h. The volatiles were removed in vacuo to give reagent 35 as a white solid (assumed quantitative yield). m/z [M+Na].sup.+ (985, 35%), [M+H].sup.+ (963, 30%).

Step 3: Synthesis of Compound 3

[0238] ##STR00095##

[0239] To a suspension of Boc-Val-Cit-PAB-Duocarmycin (Abzena, TCRS, 17 mg) in anhydrous dichloromethane (2 mL) at 0° C. was added trifluoroacetic acid (1 mL) and the resulting solution stirred at 0° C. for 75 min. The volatiles were then removed in vacuo to give compound 36 as a yellow solid (assumed quantitative yield). m/z [M+H].sup.+ (798, 100%).

Step 3: Synthesis of Reagent 33

[0240] To a solution of reagent 35 (13.4 mg) in anhydrous DMF (500 μL) at 0° C. was added HATU (13.2 mg) and NN (4 μL) and the mixture stirred at 0° C. for 10 min. To this was added a solution of compound 36 (12.7 mg) in anhydrous DMF (500 μL) and the mixture was stirred at 0° C. under an argon atmosphere for 20 min. Additional quantities of HATU (7.9 mg) and NMM (2.6 μL) were added and the reaction mixture stirred for a further 80 min. The solution was then concentrated in vacuo and purified by reverse phase C18-column chromatography, eluting with buffer A (v/v): water:55% acetonitrile:0.05% trifluoroacetic acid and buffer B (v/v): acetonitrile:0.05% trifluoroacetic acid (100:0 v/v to 0:100 v/v). The solvent was removed by lyophilisation to give reagent 33 as a yellow solid (5.1 mg). m/z [M+Na].sup.+ (1766, 60%), [M+H].sup.+ (1744, 70%), [M+Na+H].sup.2+ (884, 90%), [M+2H].sup.2+ (872, 100%).

Example 14: Synthesis of Conjugation Reagent 37 (Comparative) Comprising a Duocarmycin Cytotoxic Payload

[0241] ##STR00096##

[0242] To a solution of reagent 30 (14.1 mg) in anhydrous DMF (500 μL) at 0° C. was added HATU (24 mg) and NMM (7.6 μL) and the mixture stirred at 0° C. for 5 min. To this was added a solution of compound 36 (14.4 mg) in anhydrous DMF (500 μL) and the mixture stirred at 0° C. under an argon atmosphere for 40 min. The solution was then concentrated in vacuo, the residue dissolved in water:acetonitrile (3:7 v/v, 1 mL) and purified by reverse phase C18-column chromatography eluting with buffer A (v/v): water:55% acetonitrile:0.05% trifluoroacetic acid and buffer B (v/v): acetonitrile:0.05% trifluoroacetic acid (100:0 v/v to 0:100 v/v). The solvent was removed by lyophilisation to give reagent 37 as a yellow solid (8.9 mg). m/z [M+Na].sup.+ (1696, 10%), [M+H].sup.+ (1675, 50%), [M+Na+H].sup.2+ (848, 40%), [M+2H].sup.2+ (838, 85%).

Example 15: Conjugation of Reagents 33 and 37 (Comparative) to Brentuximab to Produce Antibody Drug Conjugates (ADCs) 38 and 39 (Comparative) Respectively, with DAR 4

[0243] Conjugation reagents 3 and 37 (comparative) were conjugated to Brentuximab, giving rise to ADCs 38 and 39 (comparative) respectively, using a method analogous to that described in Example 3. Briefly, Brentuximab (8.5 mg/mL in 20 mM sodium phosphate, 150 mM NaCl, 20 mM EDTA, pH 7.5) was heated to 40° C. in a heating block for 15 min. TCEP (6 eq. per mAb) was added to the mAb solution, mixed gently and incubated at 40° C. for 1 h before being allowed to cool to 22° C. Conjugation reagents were dissolved in propylene glycol to give 0.3 mM solutions. Conjugation reagents (5.6 eq. per mAb) were then added to the mAb solutions and the reactions mixed gently and incubated at 22° C. for 42 to 78 h, during which time additional reagents (up to 1.2 eq. per mAb) were added to the reactions as required. The crude reaction solutions were then mixed with an equal volume of 50 mM sodium phosphate, 4 M NaCl, pH 7, and 5 times the volume of the crude reaction mixture of 50 mM sodium phosphate, 2 M NaCl, pH 7. The resulting solutions were loaded onto a ToyoPearl Phenyl-650S HIC column equilibrated with 50 mM sodium phosphate, 2 M NaCl, pH 7. The ADCs were eluted from the column with a gradient of 50 mM sodium phosphate, pH 7 (20% isopropanol). Fractions containing DAR 4 ADC were pooled and concentrated (Vivaspin 20, 30 kDa PES membrane), prior to being buffer exchanged into PBS, pH 7.1-7.5, and sterile filtered (0.22 μm PVDF membranes). The samples were further purified using a Superdex 200 pg SEC column by isocratic elution with PBS, pH 7.1-7.5 (10 or 20% isopropanol). Fractions containing DAR 4 ADC were pooled and concentrated (Vivaspin 20, 30 kDa PES membrane) prior to being buffer exchanged into PBS, pH 7.1-7.5, and sterile filtered (0.22 μm PVDF membranes).

Example 16: Synthesis of Conjugation Reagent 40 Comprising the Auristatin Cytotoxic Payload, MMAE

[0244] ##STR00097##

Step 1: Synthesis of Compound 4

[0245] ##STR00098##

[0246] To a solution of Fmoc-Glu(OtBu)-OH (469 mg) in anhydrous DMF (8 mL) at 0° C. was added HATU (419 mg) and the mixture was stirred at 0° C. for 20 min. NMM (121 μL) was then added and the solution stirred at 0° C. for a further 15 min. To a separate solution of 2-aminomethyl-15-crown-5 (250 mg) in anhydrous DMF (2 mL) was added NN (121 μL) and the solution stirred at 0° C. for 20 min. The two solutions were then combined, additional quantities of HATU (419 mg) and NMM (121 μL) were added and the mixture stirred at room temperature for 3 h. The reaction solution was then concentrated in vacuo and purified by reverse phase C18-column chromatography, eluting with buffer A (v/v): water:0.05% trifluoroacetic acid and buffer B (v/v): acetonitrile:0.05% trifluoroacetic acid (95:5 v/v to 0:100 v/v). The organic solvent was removed in vacuo and the aqueous solvent removed by lyophilisation to give Fmoc-L-Glu(OtBu)-amidomethyl-15-crown-5 as a white solid (440 mg). m/z [M+Na].sup.+ (679, 25%), [M+H].sup.+ (657, 30%), [M+H-tBu].sup.+ (601, 100%). To a solution of Fmoc-L-Glu(OtBu)-amidomethyl-15-crown-5 (440 mg) in dichloromethane (20 mL) was added piperidine (463 μL) and the solution stirred at room temperature for 3.5 h. Additional piperidine (198 μL) was added and the mixture stirred at room temperature for a further 1.5 h. The reaction mixture was then concentrated in vacuo and the residue suspended in acetonitrile (4 mL). The acetonitrile mixture was extracted with hexane (50 mL) before the acetonitrile layer was reduced in vacuo to give an oily residue. The residue was then purified by reverse phase C18-column chromatography, eluting with buffer A (v/v): water:0.05% trifluoroacetic acid and buffer B (v/v): acetonitrile:0.05% trifluoroacetic acid (95:5 v/v to 0:100 v/v). The organic solvent was removed in vacuo and the aqueous solvent removed by lyophilisation to give reagent 41. m/z [M+Na].sup.+ (457, 10%), [M+H].sup.+ (435, 100%), [M+H-tBu].sup.+ (379, 30%).

Step 2: Synthesis of Reagent 42

[0247] ##STR00099##

[0248] To a solution of compound 24 (82 mg) in anhydrous DMF (2 mL) was added compound 41 (50 mg) and NMM (12.7 μL) and the mixture stirred at room temperature under an argon atmosphere for 2 h. The reaction solution was then concentrated in vacuo, the residue dissolved in acetonitrile (500 μL) and purified by reverse phase C18-column chromatography, eluting with buffer A (v/v): water:0.05% trifluoroacetic acid and buffer B (v/v): acetonitrile:0.05% trifluoroacetic acid (70:30 v/v to 0:100 v/v). The organic solvent was removed in vacuo and the aqueous solvent removed by lyophilisation to give reagent 42 as a white solid (37.4 mg). m/z [M+Na].sup.+ (939, 80%), [M+H].sup.+ (917, 100%), [M+H-tBu].sup.+ (861, 50%).

Step 3: Synthesis of Reagent 43

[0249] ##STR00100##

[0250] To a solution of reagent 42 (36 mg) in anhydrous dichloromethane (2 mL) was added trifluoroacetic acid (1 mL). The solution was stirred at room temperature for 80 min before the reaction mixture was concentrated in vacuo. The residue was then dissolved in acetonitrile:water (1:2 v/v, 1.5 mL) and the solvent removed by lyophilisation to give reagent 43 as a white solid (assumed quantitative yield). m/z [M+Na].sup.+ (883, 75%), [M+H].sup.+ (861, 100%).

Step 4: Synthesis of Reagent 40

[0251] To a solution of reagent 43 (5.6 mg) in anhydrous DMF (300 μL) at 0° C. was added HATU (2.4 mg) and the mixture stirred at 0° C. for 20 min. NN (1 μL) was added and the solution stirred at 0° C. for a further 10 min. To a separate solution of Val-Cit-PAB-MMAE.TFA salt (6.6 mg) in anhydrous DMF (300 μL) at 0° C. was added NMM (1 μL) and the solution stirred at 0° C. for 30 min. The two solutions were then combined, additional quantities of HATU (2.4 mg) and NMM (1 μL) were added and the mixture allowed to warm to room temperature under stirring for 3.25 h. The reaction solution was then concentrated in vacuo, the residue dissolved in acetonitrile:DMSO (6:1 v:v, 350 μL) and purified by reverse phase C18-column chromatography, eluting with buffer A (v/v): water:0.05% trifluoroacetic acid and buffer B (v/v): acetonitrile:0.05% trifluoroacetic acid (70:30 v/v to 0:100 v/v). The organic solvent was removed in vacuo and the aqueous solvent removed by lyophilisation to give reagent 40 as a white solid (7 mg). m/z [M+Na].sup.+ (1989, 20%), [M+H].sup.+ (1967, 30%), [M+2Na].sup.2+ (1006, 30%), [M+H+Na].sup.2+ (995, 90%), [M+2H].sup.2+ (984, 100%).

Example 17: Synthesis of Conjugation Reagent 44 Comprising the Auristatin Cytotoxic Payload, MMAE

[0252] ##STR00101##

Step 1: Synthesis of Compound 45

[0253] ##STR00102##

[0254] To a solution of Fmoc-Glu(OtBu)-OH (216 mg) in anhydrous DMF (8 mL) at 0° C. was added HATU (193 mg) and the mixture stirred at 0° C. for 20 min. NMM (56 μL) was added and the solution stirred at room temperature for a further 15 min. To a separate solution of compound 12 (284 mg) in anhydrous DMF (2 mL) was added NMM (56 μL) and the solution was stirred at 0° C. for 20 min. The two solutions were then combined, additional quantities of HATU (192 mg) and NMM (56 μL) were added and the mixture stirred at room temperature for 3.5 h. The reaction solution was then concentrated in vacuo before the residue was dissolved in ethyl acetate (50 mL). The solution was then washed with saturated sodium hydrogen carbonate solution (2×10 mL), followed by saturated brine solution (10 mL). The organic phase was then concentrated in vacuo to give crude Fmoc-L-Glu(OtBu)-aza-42-crown-14. m/z [M+Na].sup.+ (1046, 20%), [M+H].sup.+ (1023, 100%). To a solution of crude Fmoc-L-Glu(OtBu)-aza-42-crown-14 in DMF (7 mL) was added piperidine (320 μL) and the solution stirred at room temperature for 2 h. The reaction mixture was then concentrated in vacuo and purified by reverse phase C18-column chromatography, eluting with buffer A (v/v): water: 0.05% trifluoroacetic acid and buffer B (v/v): acetonitrile:0.05% trifluoroacetic acid (95:5 v/v to 0:100 v/v). The organic solvent was removed in vacuo and the aqueous solvent removed by lyophilisation. The solid residue was then triturated with hexane (3×10 mL), the solid isolated by filtration and then dried in vacuo to give compound 45 as an off-white solid (201 mg). m/z [M+H].sup.+ (801, 100%).

Step 2: Synthesis of Reagent 46

[0255] ##STR00103##

[0256] To a solution of compound 45 (86 mg) in anhydrous dichloromethane (3 mL) was added N-succinimidyl 6-maleimidohexanoate (47 mg) and the solution stirred at room temperature. DIPEA (3×19 μL) was added to the reaction solution after 17.5, 22.5 and 24 h. The solution was then stirred for a further 18 h at room temperature before the reaction mixture was concentrated in vacuo, the residue dissolved in acetonitrile:DMSO (2:1 v/v) and purified by reverse phase C18-column chromatography, eluting with buffer A (v/v): water:0.05% trifluoroacetic acid and buffer B (v/v): acetonitrile:0.05% trifluoroacetic acid (90:10 v/v to 0:100 v/v). The organic solvent was removed in vacuo and the aqueous solvent removed by lyophilisation to give reagent 46 as a white solid (38 mg). m/z [M+Na].sup.+ (1016, 30%), [M+H].sup.+ (994, 20%).

Step 3: Synthesis of Reagent 47

[0257] ##STR00104##

[0258] To a solution of reagent 46 (32 mg) in anhydrous dichloromethane (1.4 mL) was added trifluoroacetic acid (375 μL) and the solution stirred at room temperature for 2.5 h. Additional trifluoroacetic acid (375 μL) was then added and the solution stirred at room temperature for a further 2 h before the solution was concentrated in vacuo and the residue dried under high vacuum for 18 h. The residue was then dissolved in water (1 mL) and the solvent removed by lyophilisation to give reagent 47 as a colourless solid (assumed quantitative yield). m/z [M+Na].sup.+ (960, 20%), [M+H].sup.+ (938, 20%).

Step 4: Synthesis of Reagent 44

[0259] To a solution of reagent 47 (33.3 mg) in anhydrous DMF (750 μL) at 0° C. was added HATU (14.8 mg) and the mixture stirred at 0° C. for 20 min. NN (4.3 μL) was added and the solution stirred at 0° C. for a further 15 min. To a separate solution of Val-Cit-PAB-MMAE.TFA salt (45.7 mg) in anhydrous DMF (500 μL) at 0° C. was added NMM (4.3 μL) and the solution stirred at 0° C. for 25 min. The two solutions were then combined, additional quantities of HATU (14.8 mg) and NMM (4.3 μL) were added and the mixture allowed to warm to room temperature under stirring for 3.5 h. The solution was then concentrated in vacuo and purified by reverse phase C18-column chromatography, eluting with buffer A (v/v): water:5% acetonitrile:0.05% trifluoroacetic acid and buffer B (v/v): acetonitrile:0.05% trifluoroacetic acid (100:0 v/v to 0:100 v/v). The organic solvent was removed in vacuo and the aqueous solvent removed by lyophilisation to give reagent 44 as a white solid (48.3 mg). m/z [M+Na].sup.+ (2066, 20%), [M+H].sup.+ (2044, 25%), [M+H+Na].sup.2+ (1033, 50%), [M+2H].sup.2+ (1022, 100%).

Example 18: Synthesis of Conjugation Reagent 48 Comprising the Auristatin Cytotoxic Payload, MMAE

[0260] ##STR00105##

[0261] To a stirred solution of compound 13 (21.8 mg) in anhydrous DMF (500 μL) was added NN (7.8 μL). This solution was then added dropwise over a period of 20 min to a stirred solution of suberic acid bis(N-hydroxysuccinimide ester) (43.8 mg) in anhydrous DMF (1.5 mL) at room temperature under an argon atmosphere. The reaction mixture was then stirred at room temperature for 20 h before the solution was concentrated in vacuo and purified by reverse phase C18-column chromatography, eluting with buffer A (v/v): water:0.05% trifluoroacetic acid and buffer B (v/v): acetonitrile:0.05% trifluoroacetic acid (90:10 v/v to 0:100 v/v). The organic solvent was removed in vacuo and the aqueous solvent removed by lyophilisation to give reagent 48 as a white solid (15.2 mg). m/z [M+Na].sup.+ (2126, 10%), [M+H].sup.+ (2104, 20%), [M+2Na].sup.2+ (1074, 50%), [M+Na+H].sup.2+ (1063, 50%) [M+2H].sup.2+ (1052, 100%).

Example 19: Conjugation of Reagent 44 to Brentuximab to Produce Antibody Drug Conjugate (ADC) 49 with DAR 4.6

[0262] Conjugation reagent 44 was conjugated to Brentuximab, giving rise to ADC 49, using a method analogous to that described within Example 6. Briefly, Brentuximab in 20 mM sodium phosphate buffer, 150 mM NaCl, 20 mM EDTA, pH 7.5, was reduced with TCEP (2 eq. per mAb) at 40° C. for 1 h. Conjugation of the reduced antibody with reagent 44 (6 eq. per mAb) was then performed. Reagent 44 was dissolved in acetonitrile to give a 4.8 mM stock solution. The reduced mAb solution was diluted to 4.2 mg/mL with 20 mM sodium phosphate buffer, 150 mM NaCl, 20 mM EDTA, pH 7.5. Reagent 44 and additional acetonitrile were added to the mAb solution to give a final antibody concentration of 4 mg/mL. The solution was mixed gently and incubated at 22° C. for 1 h. Excess reagent 44 was quenched with N-acetyl-L-cysteine (20 eq. over reagent) and the crude reaction mixture purified using an hydroxyapatite column equilibrated with 10 mM sodium phosphate, pH 6.7. The ADC was eluted from the column with a gradient of 10 mM sodium phosphate, 2 M NaCl, pH 6.7. Fractions containing ADC were pooled and concentrated (Vivaspin 20, 30 kDa PES membrane) and the concentrated sample was buffer exchanged into DPBS, pH 7.1-7.5 and sterile filtered. An average DAR of 4.6 was assigned to the conjugate using the method described in Example 3.

Example 20: Conjugation of Reagent 48 to Brentuximab to Produce Antibody Drug Conjugate (ADC) 50

[0263] Conjugation reagent 4 was conjugated to Brentuximab, giving rise to ADC 50. Briefly, reagent 48 was dissolved in DMF to give a 5.3 mM stock solution. To a Brentuximab solution (4.4 mg/mL in 20 mM sodium phosphate buffer, 150 mM NaCl, 20 mM EDTA, pH 7.5) was added reagent 48 (10 eq. per mAb) and additional DMF to give a final antibody concentration of 4 mg/mL. The solution was mixed gently and incubated at 22° C. for 1 h. The crude reaction solution was purified using an hydroxyapatite column equilibrated with 10 mM sodium phosphate, pH 6.7. The ADC was eluted from the column with a gradient of 10 mM sodium phosphate, 2 M NaCl, pH 6.7. Fractions containing ADC were pooled and concentrated (Vivaspin 20, 30 kDa PES membrane) and the concentrated sample was buffer exchanged into DPBS, pH 7.1-7.5 and sterile filtered. An average DAR of 4.1 for the conjugate was calculated from the relative peak intensities of the individual DAR species following mass spectrometry.

Example 21: Conjugation of Reagent 40 to Brentuximab to Produce Antibody Drug Conjugate (ADC) 51 with DAR 4

[0264] Conjugation reagent 40 was conjugated to Brentuximab giving rise to ADC 51 using a method analogous to that described in Example 3. Briefly, Brentuximab (5.2 mg/mL in 20 mM sodium phosphate, 150 mM NaCl, 20 mM EDTA, pH 7.5) was heated to 40° C. in a heating block for 15 min. TCEP (6 eq. per mAb) was added to the mAb solution, mixed gently and incubated at 40° C. for 1 h before being allowed to cool to 22° C. Conjugation reagent 40 was dissolved in DMF to give a 1.5 mM solution. Reagent 40 (5.6 eq. per mAb) was added to the mAb solution and the reaction was mixed gently and incubated at 22° C. for 16 h. The crude reaction mixture was mixed with an equal volume of 50 mM sodium phosphate, 4 M NaCl, pH 7 buffer and the resulting solution loaded onto a ToyoPearl Phenyl-650S HIC column equilibrated with 50 mM sodium phosphate, 2 M NaCl, pH 7. The ADC was eluted from the column with a gradient of 50 mM sodium phosphate, pH 7 (20% isopropanol). Fractions containing DAR 4 ADC were pooled and concentrated (Vivaspin 20, 30 kDa PES membrane) before the concentrated sample was buffer exchanged into PBS, pH 7.1-7.5, and sterile filtered (0.22 μm PVDF membranes).

Example 22: Analysis of ADC 31 by In Vitro Cell Viability Assay

[0265] Brentuximab conjugate 31, containing the payload MMAE, was prepared as described within Example 12. The cell viability assay using Karpas-299 cells was performed as described within Example 9. Concentration ranges used are described in Table 5. IC50 values obtained are listed in Table 6.

TABLE-US-00008 TABLE 5 Cell line Drug-conjugate Concentration range Karpas 299 31 0.4 nM-0.7 pM Karpas 299 MMAE (Free Drug) 2.5 nM-1 pM  

TABLE-US-00009 TABLE 6 Cell line Drug-conjugate IC.sub.50 (nM) Karpas 299 31 0.02 Karpas 299 MMAE (Free Drug) 0.15

[0266] The IC50 value obtained for conjugate 31 shows that the ADC of the invention has potent cell killing properties in vitro.

Example 23: Analysis of ADC 38 by In Vitro Cell Viability Assay

[0267] Brentuximab conjugate 38, containing a duocarmycin payload, was prepared as described within Example 15. The cell viability assay using Karpas-299 cells was performed as described within Example 9. The concentration range used for the conjugate was 50 nM-0.6 pM and the IC50 value obtained was 0.14 nM.

[0268] The IC50 value obtained for conjugate 38 shows that the ADC of the invention has potent cell killing properties in vitro.

Example 24: Analysis of ADCs 49, 50 and 51 by In Vitro Cell Viability Assay

[0269] Brentuximab conjugates 49, 50 and 51, containing the payload MMAE, were prepared as described within Examples 19, 20 and 21 respectively. The cell viability assay using Karpas-299 cells was performed as described within Example 9. Concentration ranges used for each conjugate are described in Table 7. IC50 values obtained for each conjugate are listed in Table 8.

TABLE-US-00010 TABLE 7 Cell line Drug-conjugate Concentration range Karpas 299 49 1.0 nM-0.5 pM Karpas 299 50 1.0 nM-0.5 pM Karpas 299 51 1.0 nM-0.5 pM Karpas 299 MMAE (Free Drug) 2.5 nM-1 pM

TABLE-US-00011 TABLE 8 Cell line Drug-conjugate IC.sub.50 (nM) Karpas 299 49 0.02 Karpas 299 50 0.04 Karpas 299 51 0.03 Karpas 299 MMAE (Free Drug) 0.20

[0270] IC50 values obtained show that ADCs of the invention have potent cell killing properties in vitro.

Example 25: Karpas-299 Mouse Xenograft Studies Comparing Brentuximab-Drug Conjugates 14, 31, 32 (Comparative) and Adcetris© (Comparative)

[0271] Healthy female CB17-SCID mice (CBySmn.CB17-Prkdcscid/J, Charles River Laboratories) with an average body weight of 18.1 g were used for cell inoculation (Day 0). 24 to 72 h prior to tumour cell injection, the mice were γ-irradiated (1.44 Gy, .sup.60Co). The animals were maintained in SPF health status according to the FELASA guidelines in housing rooms under controlled environmental conditions.

[0272] Tumours were induced by subcutaneous injection of 10.sup.7 Karpas-299 cells (T-anaplastic large cell lymphoma, ALCL) in 200 μL of RPMI 1640 into the right flank. Tumours were measured twice a week with calipers, and the volume was estimated using the formula:

[00002]$\mathrm{Tumour}\mathrm{Volume}\left({\mathrm{mm}}^3\right)=\frac{{\mathrm{width}}^2\times \mathrm{length}}{2}$

[0273] Twelve days after tumour implantation (Day 12), the animals were randomised into groups of eight mice using Vivo manager® software (211 mm.sup.3 mean tumour volume) and treatment was initiated. The animals from the vehicle group received a single intravenous (i.v.) injection of PBS. The treated groups were dosed with a single i.v. injection of ADC at 0.8 mg/kg, or Adcetris© (brentuximab vedotin) at 1 mg/kg.

[0274] Treatment tolerability was assessed by bi-weekly body weight measurement and daily observation for clinical signs of treatment-related side effects. Mice were euthanized when a humane endpoint was reached (e.g. 1,600 mm.sup.3 tumour volume) or after a maximum of 9 weeks post-dosing. Asterisks within the graphs indicate where animals were euthanized.

[0275] The mean tumour volumes±standard error for each treatment are represented in FIGS. 2a, 3a, 4a and 5a, whereas individual tumour volumes for each treatment are represented in FIGS. 2b, 3b, 4b and 5b. All compounds were well tolerated.

[0276] Mice were dosed with 0.8 mg/kg of ADC 14, 31 or 32 or 1 mg/kg Adcetris© at day 12 post-tumour induction. Adcetris© was used at a higher dose to ensure that a reduction in tumour volume could be observed for this group. Each cohort dosed showed an initial reduction in tumour volume up to approximately day 20. However, after day 20, animals dosed with Adcetris© (comparative), displayed tumour re-growth in 7/8 animals, as shown by the increased average tumour volume and the individual tumour volumes in FIGS. 2a and 2b, respectively. The graph showing the mean tumour volume for this cohort (FIG. 2a) was no longer plotted when half of the animals had been euthanized due to tumour volume being greater than 1600 mm.sup.3, in accordance with accepted practice. In this group, only 1/8 animals had no measureable tumour by the end of the study at day 71.

[0277] For ADC 32 (comparative), 4/8 animals had tumour re-growth after day 22 post-tumour induction. This is shown in FIGS. 3a and 3b, which display an increase in average and individual tumour volumes from day 22 to day 71. Within this group, two mice were euthanized at days 41 and 68 (as indicated by an asterisk), as their tumour volumes had reached the maximum allowable size for this model. Only 4/8 animals displayed no measureable tumour at day 71.

[0278] For ADC 14, the response rate was markedly better than ADC 32 (comparative), with 7/8 animals showing no measureable tumour volume at the end of the study at day 71 (see FIGS. 4a and 4b). Only one mouse showed any tumour re-growth, which was euthanized at day 48 post-tumour induction (as indicated by an asterisk). Even more impressively, for the cohort dosed with ADC 31, no animals showed any tumour re-growth at all after dosing. 8/8 animals survived to the end of the study at day 71 with no measureable tumour (see FIGS. 5a and 5b).

[0279] From these studies it is clear that ADCs of the invention are more efficacious than comparative ADCs of the prior art, with both 14 and 31 displaying better tumour-killing potencies than 32 and Adcetris©.

[0280] Further Inventive Aspects

[0281] While working in this area, the inventors have found that particularly advantageous results can be obtained when using a particular form of conjugation technology for conjugating a therapeutic, diagnostic and labelling agent to a peptide or protein. Accordingly, disclosed herein is an invention described by the following clauses.

[0282] 1. A conjugate comprising a protein or peptide conjugated to a therapeutic, diagnostic or labelling agent via a linker, characterised in that the linker includes at least two ˜(CH.sub.2—CH.sub.2—O—)˜ units within a ring, and also includes a portion:

##STR00106##

in which W′ represents an electron withdrawing group or a group obtained by reduction of an electron withdrawing group, each of A and B independently represents a C.sub.1-5alkylene or alkenylene chain, and Pr represents said protein or peptide bonded to A and B via nucleophiles Nu.

[0283] 2. A conjugate as described in clause 1, which includes a portion:

##STR00107##

[0284] 3. A conjugate as described in either clause 1 or clause 2, in which each Nu represents a sulfur atom present in a cysteine residue in the protein or peptide Pr; or in which each Nu represents an imidazole group present in a polyhistidine tag attached to the protein or peptide Pr.

[0285] 4. A conjugate as described in any one of the preceding clauses, in which W′ represents a —CO— or —CH(OH)— group.

[0286] 5. A conjugate as described in any one of the preceding clauses, which includes within said ring a unit of the formula ˜(CH.sub.2—CH.sub.2—O—).sub.x˜ in which x is a number of at least 2.

[0287] 6. A conjugate as described in clause 5, in which x is from 2 to 50.

[0288] 7. A conjugate as described in any one of the preceding clauses, in which the ring is attached via a single tethering atom within the ring to the rest of the linker at a single point or at two or more points; or in which the ring is attached via two or more tethering atoms within the ring to the rest of the linker at a single point or at two or more points.

[0289] 8. A conjugate as claimed in claim 7, in which said ring has the formula:

##STR00108## ##STR00109##

[0290] 9. A conjugate as described in clause 7, in which the ring is attached via two or more tethering atoms within the ring to the rest of the linker at two or more points.

[0291] 10. A conjugate as described in clause 9, in which said ring has the formula:

##STR00110##

[0292] 11. A conjugate as described in any one of the preceding clauses, which comprises an optionally substituted aryl or heteroaryl group immediately adjacent the group of formula III or IIIa; and which linker also includes a —NR.sup.a.C(O)— or —C(O).NR.sup.a— group adjacent said aryl or heteroaryl group; wherein R.sup.a represents C.sub.1-4 alkyl or hydrogen.

[0293] 12. A conjugate as described in any one of the preceding clauses, which includes a therapeutic agent.

[0294] 13. A conjugate as described in any one of the preceding clauses, in which the protein or peptide is an antibody or an antibody fragment.

[0295] 14. A conjugating reagent comprising a functional group capable of reacting with a protein or peptide, which reagent also comprises a therapeutic, diagnostic or labelling agent and a linker which includes at least two ˜(CH.sub.2—CH.sub.2—O—)˜ units within a ring, and in which said functional group has the formula:

##STR00111##

in which W represents an electron-withdrawing group; each of A and B independently represents a C.sub.1-5alkylene or alkenylene chain; and either each L independently represents a leaving group, or both Ls together represent a leaving group; or

##STR00112##

in which W and A have the meanings given above, L represents a leaving group, and m is 0 to 4.

[0296] 15. A conjugating reagent as described in clause 14, in which said functional group has the formula:

##STR00113##

[0297] 16. A conjugating reagent as described in either clause 14 or clause 15, in which W represents a —CO— group.

[0298] 17. A conjugating reagent as described in any one of clauses 14 to 16, which includes a feature according to any one of clauses 2 to 11.

[0299] 18. A conjugating reagent as described in any one of clauses 14 to 17, in which the or each leaving group includes a portion —(CH.sub.2CH.sub.2O).sub.n— in which n is a number of two or more.

[0300] 19. A conjugating reagent as described in clause 18, in which the or each leaving group has the formula —SP or —SO.sub.2P, in which P represents a group which includes a portion —(CH.sub.2CH.sub.2O).sub.n— in which n is a number of two or more.

[0301] 20. A process for the preparation of a conjugate as described in any one of clauses 1 to 13, which comprises reacting a protein or peptide with a conjugating reagent as described in any one of clauses 14 to 19.

[0302] 21. A pharmaceutical composition which comprises a conjugate as described in any one of clauses 1 to 13, in which the payload is a therapeutic agent, together with a pharmaceutically acceptable carrier, and optionally together with a further active ingredient.

DETAILED DESCRIPTION OF THIS INVENTIVE ASPECT

[0303] The reagent of the invention may be represented schematically by the formula:

##STR00114##

in which D represents the therapeutic, diagnostic or labelling agent, F represents a functional grouping capable of bonding to a protein or peptide, and

##STR00115##

represents a ring which includes at least two ethylene glycol, ˜(CH.sub.2—CH.sub.2—O—)˜, units. The functional grouping F is capable of reacting with a protein or peptide as explained in more detail below.

[0304] The conjugate of the invention may be represented schematically by the formula:

##STR00116##

in which D represents the therapeutic, diagnostic or labelling agent, F′ represents the protein or peptide bonded to the remainder of the conjugate via a protein or peptide bonding portion of the linker, and

##STR00117##

represents a ring which includes at least two ethylene glycol, ˜(CH.sub.2—CH.sub.2—O—)˜, units.

[0305] The Polyethylene Glycol Ring

[0306] Throughout, “polyethylene glycol ring” should be understood to mean a ring which includes at least two ethylene glycol, ˜(CH.sub.2—CH.sub.2—O—)˜, units. The ring may include two or more separate ˜(CH.sub.2—CH.sub.2—O—)˜ units, or it may include one or more units of the formula ˜(CH.sub.2—CH.sub.2—O—).sub.x˜ in which x is a number of at least 2. The ring may contain one or more additional atoms to complete the cyclic structure. Additional atoms may for example be nitrogen, carbon, oxygen, sulfur, silicon and/or phosphorus atoms.

[0307] The ring may be attached via a single tethering atom within the ring to the rest of the linker at a single point, or it may be attached at two or more points. Alternatively, the ring may be attached via two or more tethering atoms within the ring to the rest of the linker at a single point, or it may be attached at two or more points. Tethering atoms may for example be nitrogen, carbon, phosphorus or silicon atoms, especially nitrogen and/or carbon atoms, and the atoms present at the point of attachment to the rest of the linker may for example be nitrogen or carbon atoms.

[0308] The following are schematic drawings of possible forms of attachment of the ring to the rest of the linker in conjugates or reagents of the invention, T representing a tethering atom in the ring, and PEG representing at least two ˜(CH.sub.2—CH.sub.2—O—)˜ units:

##STR00118##

Specific examples of suitable rings include the following, where the symbol ˜ indicates a point of incorporation of the ring into the linker:

##STR00119##

[0309] Preferably the ring is attached via a single tethering atom in the ring to the rest of the linker at a single point. In another preferred embodiment, the ring is attached via two or more tethering atoms within the ring to the rest of the linker at two or more points.

[0310] The ring may for example consist of ˜(CH.sub.2—CH.sub.2—O—).sub.x˜ units in which x is at least 2, preferably from 2 to 20. Alternatively, the ring may contain ˜(CH.sub.2—CH.sub.2—O—).sub.x˜ units in which x is at least 2, preferably from 2 to 50, especially from 2 to 20, but may also include one or more additional atoms as mentioned above, or may be derivatised in some other way.

[0311] Conjugates and reagents may be readily synthesised from crown ethers. Crown ethers are cyclic oligomers of ethylene glycol, and many different crown ethers are known, some of which consist entirely of ethylene glycol units, and some of which contain additional atoms within the ring. For example, aza-crown ethers contain a nitrogen atom, while diaza-crown ethers contain two nitrogen atoms. Many crown ethers are commercially available, and these provide convenient starting points for synthesis of the conjugates and reagents according to the invention. Crown ethers carrying functional groups through which they may be reacted with other compounds are known, for example crown ethers carrying carboxy, hydroxy, amino, or aldehyde groups are known, as are crown ethers fused to a benzene ring optionally carrying a functional group such as a carboxy, hydroxy, amino, isocyanate, nitro or aldehyde group.

[0312] Crown ethers are known to chelate cations, and perfluoro crown ethers have been described within U.S. Pat. No. 4,838,274 for use in MRI. Therefore the conjugates of the invention may be used in applications within imaging techniques such as MRI or PET.

[0313] Typical crown ethers which can be incorporated into the conjugates and reagents according to the invention include the structures shown below.

##STR00120## ##STR00121## ##STR00122##

[0314] These may be incorporated into the linker of the conjugates and reagents of the invention by reaction through atoms, especially nitrogen atoms, present within the ring, or via groups, for example hydroxy, amino, carboxy, aldehyde, isocyanate or nitro groups, present on a side-chain. Rings having two functional groups or atoms can be attached to the rest of the linker of conjugates and reagents of the invention at two separate attachment points, hence being incorporated into the backbone of the linker. Typical linkages are as shown below:

##STR00123## ##STR00124##

[0315] In addition to rings derived from crown ethers, rings derived from cryptands may be used in the present invention. Such rings are described for example in US 2014/0072900, and include the following:

##STR00125##

[0316] As an alternative to synthesising the conjugates and reagents of the invention starting from crown ethers or cryptands, it is possible to prepare a conjugate or reagent having a PEG chain attached at one end to the rest of the linker, and then to react the free end of the chain with a functional group present elsewhere on the linker, using conventional chemistry. In yet another alternative synthesis method, it is possible to prepare a conjugate or reagent containing two pendant PEG chains, and then to create a loop by reaction of appropriate reactive groups on each of the chains. Alkene and alkyne ring-closing metathesis may for example be used. All such methods of synthesis would be known to the skilled person, and permit the preparation of a very wide range of conjugates and reagents according to the invention, including ones in which the ring is incorporated into the backbone of the linker.

[0317] The conjugates and reagents of the invention may contain one ring including at least two ˜(CH.sub.2—CH.sub.2—O—)˜ units, or they may contain two or more such rings. The ring may be monocyclic, or it may be bi- or multi-cyclic. Two or more rings may be attached to or incorporated into the backbone of the linker, or they may be attached to each other, thus:

##STR00126##

[0318] It will be understood that many different sizes and structures of rings are possible. The important feature of the invention is that a PEG chain forms part of a cyclic structure: this chain is not a linear PEG chain which forms part of the backbone of the linker, neither is it a pendant PEG chain which is tethered at one end to the linker but which has a free untethered end.

[0319] In one preferred embodiment, all of the PEG in the conjugate or reagent according to the invention is present within one or more rings. In another embodiment, PEG may also be present elsewhere in the linker, specifically in the backbone of the linker or in a group linking the ring to the backbone of the linker, and this is discussed in more detail below.

[0320] The total number of ˜(CH.sub.2—CH.sub.2—O—)˜ units present in the conjugates and reagents of the invention will of course depend on the intended application. For some applications, high molecular weight PEGs may be used, for example the number average molecular weight may be up to around 75,000, for example up to 50,000, 40,000 or 30,000 g/mole. For example, the number average molecular weight may be in the range of from 500 g/mole to around 75,000. However, smaller PEG portions may be preferred for some applications.

[0321] As with the total quantity of PEG present in the conjugates or reagents of the invention, the number of ˜(CH.sub.2—CH.sub.2—O—)˜ units present in the ring will depend on the intended application. For example the cyclic PEG portion may have a molecular weight up to 3,000 g/mole. However, cyclic groups containing as few as 2 ethylene glycol units, for example from 2 to 50 ethylene glycol units, are useful for some applications, and are present as a cyclic PEG group in one preferred embodiment of the invention. PEG-containing rings with 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 repeat units, or 24, 36, 40 or 48 repeat units, may for example be used.

[0322] Conjugating Reagents and Processes

[0323] The conjugating reagent according to the invention is capable of reacting with two nucleophiles. If two or more leaving groups are present, these may be the same or different. Alternatively, a conjugating reagent may contain a single group which is chemically equivalent to two leaving groups and which single group is capable of reacting with two nucleophiles.

[0324] Nucleophilic groups include sulfur atoms and amine groups, and nucleophilic groups in proteins are for example provided by cysteine, lysine or histidine residues. In one preferred embodiment of the invention, a nucleophilic group is a sulfur atom present in a cysteine residue present in the protein. Such structures may be obtained by reduction of a disulfide bond present in the protein. In another embodiment, a nucleophilic group may be an imidazole group present in a histidine residue present in a polyhistidine tag attached to the protein.

[0325] The conjugating reagent contains the functional grouping F:

##STR00127##

in which W represents an electron-withdrawing group, for example a keto group, an ester group —O—CO—, or a sulfone group —SO.sub.2—; each of A and B independently represents a C.sub.1-5alkylene or alkenylene chain; and either each L independently represents a leaving group, or both Ls together represent a leaving group. When reagents containing such groups react with proteins, a first leaving group L is lost to form in situ a conjugating reagent containing a functional grouping of formula:

##STR00128##

in which m is 0 to 4, which reacts with a first nucleophile. The second leaving group L is then lost, and reaction with a second nucleophile occurs. As an alternative to using a reagent containing the functional grouping I as starting material, reagents containing the functional grouping II may be used, as the functional groupings I and II are chemical equivalents of each other.

[0326] These conjugating reagents of the invention are of the general type disclosed in WO 2005/007197 and WO 2010/100430. Such reagents may for example be used to target two sulfur atoms obtained by reduction of a disulfide bond in a protein, or imidazole groups present in histidine residues present in a polyhistidine tag attached to a protein. It has been found that the incorporation of a cyclic PEG group according to the present invention into reagents of this type gives particularly good results, with conjugation reactions occurring efficiently to produce stable conjugates with a high degree of homogeneity.

[0327] A leaving group L may for example be —SP, —OP, —SO.sub.2P, —OSO.sub.2P, —N.sup.+PR.sup.2R.sup.3, halogen, or —OØ, in which P represents a hydrogen atom or an alkyl (preferably C.sub.1-6alkyl), aryl (preferably phenyl), or alkyl-aryl (preferably C.sub.1-6alkyl-phenyl) group, or is a group which includes a portion —(CH.sub.2CH.sub.2O).sub.n— in which n is a number of two or more, and each of R.sup.2 and R.sup.3 independently represents a hydrogen atom, a C.sub.1-4alkyl group, or a group P, and Ø represents a substituted aryl, especially phenyl, group, containing at least one substituent, for example —CN, CF.sub.3, —NO.sub.2, —CO.sub.2R.sup.a, —COH, —CH.sub.2OH, —COR.sup.a, —OR.sup.a, —OCOR.sup.a, —OCO.sub.2R.sup.a, —SR.sup.a, —SOR.sup.a, —SO.sub.2R.sup.a, —NHCOR.sup.a, —NR.sup.aCOR.sup.a, —NHCO.sub.2R.sup.a, —NR.sup.aCO.sub.2R.sup.a, —NO, —NHOH, —NR.sup.a OH, —CH═N—NR.sup.a COR.sup.a, —N.sup.+R.sup.a.sub.3, —, halogen, especially chlorine or, especially, fluorine, —C≡CR.sup.a, and —CH═CR.sup.a.sub.2, in which each R.sup.a independently represents a hydrogen atom or an alkyl (preferably C.sub.1-6alkyl), aryl (preferably phenyl), or alkyl-aryl (preferably C.sub.1-6alkyl-phenyl) group. The presence of electron withdrawing substituents is preferred.

[0328] Conjugating reagents in which P represents a group which includes a portion —(CH.sub.2CH.sub.2O).sub.n— in which n is a number of two or more are the subject of our copending application GB 1418186, published as WO 2016/059377, and are described above.

[0329] An especially preferred leaving group L present in a novel conjugating reagent according to the present invention is —SP or —SO.sub.2P, especially —SO.sub.2P. Within this group, one preferred embodiment is where P represents a phenyl or, especially, a tolyl group. Another preferred embodiment is where P represents a group which includes a portion —(CH.sub.2CH.sub.2O).sub.n—, especially one in which n has one of the values mentioned above, especially 7. An especially preferred leaving group L is —SO.sub.2—(CH.sub.2CH.sub.2O).sub.n—H/Me, especially —SO.sub.2—(CH.sub.2CH.sub.2O).sub.7—HMe.

[0330] Throughout this Specification, any reference to a leaving group L should be understood to include a specific reference to these preferred groups, especially —SO.sub.2—(CH.sub.2CH.sub.2O).sub.n—H/Me, and more especially —SO.sub.2—(CH.sub.2CH.sub.2O).sub.7—H/Me.

[0331] Preferably W represents a keto group. Preferably each of A and B represents —CH.sub.2—.

[0332] Reagents of the formula I and II above form conjugates which include the grouping F′:

##STR00129##

in which W′ represents an electron withdrawing group or a group obtained by reduction of an electron withdrawing group, and Pr represents a protein or peptide bonded to A and B via nucleophiles Nu. The immediate product of the conjugation process (as described in more detail below) is a conjugate which contains an electron-withdrawing group W. However, the conjugation process is reversible under suitable conditions. This may be desirable for some applications, for example where rapid release of the protein is required, but for other applications, rapid release of the protein may be undesirable. It may therefore be desirable to stabilise the conjugates by reduction of the electron-withdrawing moiety to give a moiety which prevents release of the protein. Accordingly, the conjugation process may comprise an additional optional step of reducing the electron withdrawing group in the conjugate. The use of a borohydride, for example sodium borohydride, sodium cyanoborohydride, potassium borohydride or sodium triacetoxyborohydride, as reducing agent is particularly preferred. Other reducing agents which may be used include for example tin (II) chloride, alkoxides such as aluminium alkoxide, and lithium aluminium hydride.

[0333] Thus, for example, a moiety W containing a keto group may be reduced to a moiety containing a CH(OH) group; an ether group CH.OR.sup.a may be obtained by the reaction of a hydroxy group with an etherifying agent; an ester group CH.O.C(O)R.sup.a may be obtained by the reaction of a hydroxy group with an acylating agent; an amine group CH.NH.sub.2, CH.NHR.sup.a or CH.NR.sup.a.sub.2 may be prepared from a ketone by reductive amination; or an amide CH.NHC(O)R.sup.a or CH.N(C(O)R.sup.a).sub.2 may be formed by acylation of an amine. A sulfone may be reduced to a sulfoxide, sulfide or thiol ether.

[0334] Preferably the groupings F′ and F have the formula:

##STR00130##

especially

##STR00131##

##STR00132##

[0335] In the above formulae, preferred leaving groups are as described above. Preferably each Nu is a sulfur atom.

[0336] Conjugating reagents according to the invention may contain more than one functional grouping for reaction with a protein. For example, a reagent may contain a functional grouping, preferably of formula I or II, at one end of the molecule, and one or more additional functional groupings, elsewhere in the molecule. Such structures are described in for example Belcheva et al, J. Biomater. Sci Polymer Edn. 9(3), 207-226 and are useful in the synthesis of conjugates containing multiple proteins.

[0337] The novel conjugating reagents of the present invention may be prepared by methods analogous to known methods. Specific reactions are illustrated in the Examples.

[0338] Conjugating reagents according to the invention may be reacted with a protein or peptide to form a conjugate according to the invention, and such a reaction forms a further aspect of the invention.

[0339] A key feature of using conjugating reagents of the formulae I or II is that an α-methylene leaving group and a double bond are cross-conjugated with an electron withdrawing function that serves as a Michael activating moiety. If the leaving group is prone to elimination in the cross-functional reagent rather than to direct displacement and the electron-withdrawing group is a suitable activating moiety for the Michael reaction then sequential intramolecular bis-alkylation can occur by consecutive Michael and retro Michael reactions. In reagents containing the functional grouping I, a leaving group serves to mask a latent conjugated double bond that is not exposed until after the first alkylation has occurred to give a reagent including the functional grouping II and bis-alkylation results from sequential and interactive Michael and retro-Michael reactions. The cross-functional alkylating agents may contain multiple bonds conjugated to the double bond or between the leaving group and the electron withdrawing group.

[0340] Where bonding to the protein is via two sulfur atoms derived from a disulfide bond in the protein, the process may be carried out by reducing the disulfide bond following which the reduced product reacts with the reagent according to the invention. Preferably the disulfide bond is reduced and any excess reducing agent is removed, for example by buffer exchange, before the conjugating reagent is introduced. The disulfide bond can be reduced, for example, with dithiothreitol, mercaptoethanol, or tris-carboxyethylphosphine using conventional methods.

[0341] Conjugation reactions may be carried out under similar conditions to known conjugation processes, including the conditions disclosed in WO 2005/007197, WO 2009/047500, WO 2014/064423, WO 2014/064424, and WO 2015/057699. The process may for example be carried out in a solvent or solvent mixture in which all reactants are soluble. For example, the protein may be allowed to react directly with the polymer conjugating reagent in an aqueous reaction medium. This reaction medium may also be buffered, depending on the pH requirements of the nucleophile. The optimum pH for the reaction will generally be at least 4.5, typically between about 5.0 and about 8.5, preferably about 6.0 to 7.5. The optimal reaction conditions will of course depend upon the specific reactants employed.

[0342] Reaction temperatures between 3-40° C. are generally suitable when using an aqueous reaction medium. Reactions conducted in organic media (for example THF, ethyl acetate, acetone, DMSO, DMF, MeCN) are typically conducted at temperatures up to ambient. In one preferred embodiment, the reaction is carried out in aqueous buffer which may contain a proportion of organic solvent, for example up to 20% by volume of organic solvent, typically from 5 to 20% by volume of organic solvent.

[0343] The protein can be effectively conjugated using a stoichiometric equivalent or a slight excess of conjugating reagent. However, it is also possible to conduct the conjugation reaction with an excess stoichiometry of conjugating reagent, and this may be desirable for some proteins. The excess reagent can easily be removed, for example by ion exchange chromatography or HPLC, during subsequent purification of the conjugate.

[0344] Of course, it is possible for more than one conjugating reagent to be conjugated to a protein, where the protein contains sufficient suitable attachment points. For example, in a protein which contains two different disulfide bonds, or in a protein which contains one disulfide bond and also carries a polyhistidine tag, it is possible to conjugate two molecules of reagent per molecule of protein, and such conjugates form part of the present invention.

[0345] The Payload, the Protein, the Linker, and Pharmaceutical Compositions and Utility

[0346] All material present in the sections “The payload”, “The protein”, “The Linker”, and “Pharmaceutical compositions and utility” above applies to this aspect of the invention.