PROCESS FOR SITE-SPECIFIC MODIFICATION OF AN ANTIBODY

Abstract

A process is for preparing a site-specific bioconjugated antibody of a formula (I): Ab-(Linker-Chelator)n (I). The Linker is an oligopeptide with an N-terminal end. The Chelator is a metal chelating agent. n is a Chelator-to antibody ratio (CAR), wherein 0<n≤2. The process includes enzymatic deglycosylation of the antibody; coupling of the obtained deglycosylated antibody with a compound of a formula (A): Linker-Chelator (A) in the presence of a transglutaminase. The Linker is bound to the Ab at its N-terminal end, and comprising a sequence chosen among (*G-G-G), (*K-G-G) and (*A-K-A), where * denotes the N-terminal end of the Linker which is covalently bound to the Ab.

Claims

1-16. (canceled)

17. A process for preparing a site-specific bioconjugated antibody of a formula (I):
Ab-(Linker-Chelator).sub.n  (I), the Linker being an oligopeptide with an N-terminal end, the Chelator being a metal chelating agent, n being a Chelator-to antibody ratio (CAR), wherein 0<n≤2; the process comprising: enzymatic deglycosylation of the antibody; coupling of the obtained deglycosylated antibody with a compound of a formula (A):
Linker-Chelator  (A) in the presence of a transglutaminase, the Linker being bound to the Ab at its N-terminal end, and comprising a sequence chosen among (*G-G-G), (*K-G-G) and (*A-K-A), where * denotes the N-terminal end of the Linker which is covalently bound to the Ab.

18. The process according to claim 17, wherein the Linker is chosen from a group consisting of a formula: -*G-G-G-(X)p- (SEQ ID No 2), -*K-G-G-(X)p- (SEQ ID No 3), and -*A-K -A-(X)p- (SEQ ID No 4), where * denotes the N-terminal end of the Linker, X is an amino-acid and p is an integer such that 0≤p≤10.

19. The process according to claim 17, wherein said Linker comprises at least the *G-G-G sequence.

20. The process according to claim 17, wherein the Linker is *G-G-G or *G-G-G-G (SEQ ID No 5).

21. The process according to claim 17, wherein the Linker is *G-G-G.

22. The process according to claim 17, wherein the enzymatic deglycosylation is carried out with a N-glycosidase.

23. The process according to claim 22, wherein the N-glycosidase is PNGase F (protein N-glycosidase F from Flavobacterium meningosepticum).

24. The process according to claim 17, wherein the Chelator is —NH—CH.sub.2—CH.sub.2-DOTAM or —NH-TCMC.

25. The process according to claim 17, wherein the transglutaminase is a microbial transglutaminase.

26. The process according to claim 17, wherein the transglutaminase is transglutaminase from the species Streptomyces mobaraensis.

27. The process according to claim 17, wherein the CAR is comprised between 1 and 2.

28. The process according to claim 17, wherein a diafiltration wash is carried out between the deglycosylation step and the coupling step.

29. A site-specific bioconjugated antibody formed by the process according to claim 17.

30. A pharmaceutical composition comprising: the site-specific bioconjugated antibody according to claim 29; and at least one pharmaceutically acceptable excipient.

31. A contrast agent or a drug in nuclear medicine comprising: the site-specific bioconjugated antibody according to claim 29.

32. A method for diagnosing and/or treating cancer comprising: administering the contrast agent or drug according to claim 31 to a patient.

33. A site-specific bioconjugated antibody comprising:
Ab-(Linker-Chelator).sub.n  (I), the Linker being an oligopeptide with an N-terminal end, the Chelator being a metal chelating agent, n being a Chelator-to antibody ratio (CAR), wherein 0<n≤2, the Linker being bound to the Ab at its N-terminal end, and comprising a sequence chosen among (*G-G-G), (*K-G-G) and (*A-K-A), where * denotes the N-terminal end of the Linker which is covalently bound to the Ab.

34. A pharmaceutical composition comprising: the a site-specific bioconjugated antibody according to claim 33; and at least one pharmaceutically acceptable excipient.

35. A contrast agent or a drug in nuclear medicine comprising: the site-specific bioconjugated antibody according to claim 33.

36. A method for diagnosing and/or treating cancer comprising: administering the contrast agent or drug according to claim 35 to a patient.

Description

DETAILED DESCRIPTION

[0083] FIG. 1 is a schematic representation of an illustrative process of the present disclosure, where (1) represents a full length antibody, which is: [0084] in step 1 deglycosylated, [0085] in step 2: washed by diafiltration, [0086] in step 3: reacted with (2): Linker-Chelator and MTGase [0087] in step 4: washed by diafiltration, [0088] to lead to the corresponding bioconjugated antibody (3).

Experimental Results

[0089] Definitions

[0090] PBS—phosphate buffered saline

[0091] EDTA—ethylenediaminetetraacetic acid

[0092] MWCO—molecular weight cut off

[0093] CAR—Chelator-to-antibody ratio

[0094] HATU: (1-[Bis(dimethyamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate, Hexafluorophosphate Azabenzotriazole Tetramethyl Uronium)

[0095] DIPEA: N,N-diisopropylethylamine

[0096] DMA: Dimethylacetamide (CH.sub.3C(O)N(CH.sub.3).sub.2).

[0097] Materials:

NH.sub.2-TCMC (compound 1), was provided by Macrocyclics and used as the principal starting material in the synthesis of GGG-TCMC. All solvents and reagents were purchased from commercial sources and used as received.

[0098] Synthesis of GGG-TCMC:

##STR00006##

[0099] A solution of Z-(Gly).sub.3—OH (3.3 g, 10.2 mmol) of formula:

##STR00007##

in DMA (64 mL) was prepared followed by the addition of DIPEA (5.20 mL, 29.9 mmol) and HATU (4.51 g, 11.9 mmol).

[0100] A solution of NH.sub.2-TCMC (1) (6.56 g, 10.1 mmol) in DMA (80 mL) with DIPEA (5.80 mL, 33.3 mmol) was prepared and added to the prepared peptide solution. The reaction was stirred at room temperature for 17 hours. HPLC monitoring indicated the reaction was incomplete. A second equivalent of Z-(Gly).sub.3—OH (3.35 g, 10.4 mmol) dissolved in DMA (36 mL) with DIPEA (5.20 mL, 29.9 mmol) and HATU (4.53 g, 11.9 mmol) was added to the reactor and stirred for an additional 22 hours. The reaction solution was added drop-wise to diethyl ether (1.50 L) to precipitate the crude material. Additional diethyl ether (1.0 L) was added to the vessel and stirred for 20 minutes. The precipitate was isolated by vacuum filtration and the filter cake washed with diethyl ether (150 mL). A stock solution was prepared with acetonitrile (120 mL), H.sub.2O (480 mL), and TFA (6 mL). The crude material was purified by reverse phase chromatography using 0.1% TFA in acetonitrile and 0.1% TFA in H.sub.2O mobile phase. Purified fractions of 2 were concentrated by rotary evaporation then transferred to a Parr shaker bottle. 10% Palladium on Carbon catalyst was added and the mixture subjected to vacuum then pressurized to 30 psi hydrogen. Hydrogenation batches were shaken at room temperature until HPLC monitoring indicated reaction completion. Reaction mixture was filtered using 0.2 μm PVDF membrane filter under vacuum and lyophilized to obtain 3 as a solid.

IgG1 Monoclonal Antibody Targeting CD38 Bioconjugation

[0101] This antibody was diafiltered into PBS prior to bioconjugation. Antibody was deglycosylated by adding 6 U/mg of PNGase F enzyme and stirring at 37° C. overnight. Enzyme was removed and buffer exchanged to PBS+50 mM EDTA by diafiltration through a 50 kDa MWCO filter. Antibody was incubated with 80 mol equivalent of chemical Linker-Chelator (GGG-TCMC) and 6U/ml of MTGase enzyme (Zedira) overnight at 37° C. with gently stirring. Buffer exchange and enzyme removal were performed by diafiltration into 150 mM ammonium acetate, pH 4.5.

[0102] A final antibody concentration of 6.4 mg/ml was determined. CAR was determined by intact mass analysis to be 0.8.

TABLE-US-00002 TABLE 1 Chelators Per Antibody Abundance 0 292,483 1 825,940 2 61,925 TOTAL 1180348 CAR 0.8 CAR = ((825,940) + (2*61.925))/(1180348) Monoclonal IgG1 antibody targeting TEM-1 Bioconjugation

[0103] This antibody was diafiltered into PBS before starting bioconjugation. Antibody was deglycosylated by adding 6 U/mg of PNGase F enzyme and stirring at 37° C. overnight. Enzyme was removed and buffer exchanged to PBS+50 mM EDTA by diafiltration through a 50 kDa MWCO filter. Antibody was incubated with 80 mol equivalent of chemical Linker-Chelator (GGG-TCMC) and 6 U/ml of MTGase enzyme (Zedira) overnight at 37° C. with gently stirring. Buffer exchange and enzyme removal were performed by diafiltration into 150 mM ammonium acetate, pH 4.5.

[0104] A final antibody concentration of 6.5 mg/ml was determined. CAR determined by intact mass is 1.2

TABLE-US-00003 TABLE 2 Chelators Per Antibody Abundance 0 161,429 1 1,752,176 2 591,348 TOTAL 2,504,953 CAR 1.2 IgG1 Antibody Bioconjugation IgG1 antibody was diafiltered into PBS before starting bioconjugation.

[0105] Antibody was deglycosylated by adding 6 U/mg of PNGase F enzyme and stirring at 37° C. overnight. Enzyme was removed and buffer exchanged to PBS+50 mM EDTA by diafiltration through a 50 kDa MWCO filter. Antibody was incubated with 80 mol equivalent of chemical Linker-Chelator (GGG-TCMC) and 6 U/ml of MTGase enzyme (purchased from Zedira) overnight at 37° C. with gently stirring. Buffer exchange and enzyme removal were performed by diafiltration into 150 mM ammonium acetate, pH 4.5.

[0106] A final antibody concentration of 5.1 mg/ml was determined. CAR determined by intact mass analysis is 1.1.

TABLE-US-00004 TABLE 3 Chelators Per Antibody Abundance 0 672,786 1 4,854,318 2 1,258,650 TOTAL 6,785,754 CAR 1.1 Monoclonal IgG1 antibody targeting CD20 Bioconjugation

[0107] This antibody was diafiltered into PBS before starting bioconjugation. Antibody was deglycosylated by adding 6 U/mg of PNGase F enzyme and stirring at 37° C. overnight. Enzyme was removed and buffer exchanged to PBS+50 mM EDTA by diafiltration through a 50 kDa MWCO filter. Antibody was incubated with 80 mol equivalent of chemical Linker-Chelator (GGG-TCMC) and 6 U/ml of MTGase enzyme (Zedira) overnight at 37° C. with gently stirring. Buffer exchange and enzyme removal were performed by diafiltration into 150 mM ammonium acetate, pH 4.5.

[0108] A final antibody concentration of 5.6 mg/ml was determined. CAR was determined by intact mass to be 1.0.

TABLE-US-00005 TABLE 4 Chelators Per Antibody Abundance 0 266,076 1 2,257,476 2 233,893 TOTAL 2,757,445 CAR 1.0 Monoclonal antibody targeting HER2 Bioconjugation

[0109] This antibody was diafiltered into PBS before starting bioconjugation.

[0110] Antibody was deglycosylated by adding 6 U/mg of PNGase F (Roche) enzyme and stirring at 37° C. overnight.

[0111] Enzyme was removed and buffer exchanged to PBS+50 mM EDTA by diafiltration through a 50 kDa MWCO filter.

[0112] Antibody was incubated with 80 mol equivalent of chemical Linker-Chelator (GGG-TCMC) and 6 U/ml of MTGase enzyme (Zedira) overnight at 37° C. with gently stirring.

[0113] Buffer exchange and enzyme removal were performed by diafiltration into 150 mM ammonium acetate, pH 4.5.

[0114] A final antibody concentration of 2.5 mg/ml was determined. CAR was determined by intact mass to be 0.9.

TABLE-US-00006 TABLE 5 Chelators Per Antibody Abundance 0 200,359 1 731,247 2 96,722 TOTAL 1,028,328 CAR 0.9 Monoclonal IgG1 Antibody targeting EGFR Bioconjugation

[0115] Antibody was diafiltered into PBS before starting bioconjugation. Antibody was deglycosylated by adding 6 U/mg of PNGase F (Roche) enzyme and stirring at 37° C. overnight. Enzyme was removed and buffer exchanged to PBS+50 mM EDTA by diafiltration through a 50 kDa MWCO filter. Antibody was incubated with 80 mol equivalent of chemical Linker-Chelator (GGG-TCMC) and 6 U/ml of MTGase enzyme (Zedira) overnight at 37° C. with gently stirring. Buffer exchange and enzyme removal performed by diafiltration into 150 mM ammonium acetate, pH 4.5.

[0116] A final antibody concentration of 3.0 mg/ml was determined. CAR was determined by intact mass analysis to be 1.1.

Monoclonal Antibody Targeting HER2 Bioconjugation—Comparison KGG and GGG Linkers

[0117] Lyophilized antibody was resuspended and diafiltered into PBS before starting bioconjugation. Antibody was deglycosylated by adding 500 ng/mg of PNGase F enzyme (R&D systems) and stirring at 37° C. overnight. Enzyme was removed by diafiltration through a 50 kDa MWCO filter. Antibody was incubated with 80 mol equivalent of chemical linker GGG-TCMC or KGG-TCMC and 6 U/ml of MTGase enzyme (Zedira) overnight at 37° C. with gently stirring. Buffer exchange and enzyme removal were performed by diafiltration into 150 mM ammonium acetate, pH 4.5.

[0118] A final antibody concentration of 5.1 mg/ml was determined for the bioconjugation with GGG- Linker and 7.2 mg/ml for the bioconjugation with KGG-linker. CAR was determined by intact mass to be 0.8 for KGG-TCMC-Antibody and 1.9 with GGG-TCMC-Antibody.

Monoclonal IgG1 Antibody Targeting HER2 Antibody Bioconjugation of AKA-TCMC and GGG-TCMC with and without PNGase Deglycosylation

[0119] This experiment aims at finding whether a PNGase deglycosylation is required to conjugate Chelators to antibodies depending on the linker used. AKA-TCMC was selected as it should not have required PNGase. AKA-TCMC was compared to GGG-TCMC with and without PNGase deglycosylation.

Procedure

[0120] Lyophilized antibody is resuspended and diafiltered into PBS before starting bioconjugation.

[0121] Half of the antibody was deglycosylated by adding 500 ng/mg of PNGase F enzyme (R&D systems) and rotating at 37° C. overnight. The other half did not receive PNGase F.

[0122] Enzyme was removed by diafiltration through a 50 kDa MWCO filter and diafiltered into conjugation buffer (PBS+50 mM EDTA).

[0123] Antibody (deglycosylated and non-deglycosylated) was incubated with 80 mol equivalent of chemical Linker-Chelator (GGG-TCMC) or (AKA-TCMC) and 6 U/ml of MTGase enzyme (Zedira) overnight at 37° C. with gentle rotation.

[0124] Conjugates were sterile filtered followed by buffer exchange and enzyme removal by diafiltration into 150 mM ammonium acetate, pH 4.5. Final sterile filtration performed.

Results

[0125]

TABLE-US-00007 TABLE 7 Final Protein Concentration CAR Value Deglycosylated Ab with 5.7 mg/ml 1.1 GGG-TCMC Deglycosylated Ab with 6.7 mg/ml 0.6 AKA-TCMC Non-deglycosylated Ab 6.7 mg/ml 0 with GGG-TCMC Non-deglycosylated Ab 7.1 mg/ml 0 with AKA-TCMC

[0126] The data suggests that deglycosylation is required for AKA-TCMC and GGG-TCMC to be conjugated to an antibody and furthermore, that both AKA- and GGG-TCMC are capable to conjugate to the antibody.

Monoclonal IgG1 Antibody Targeting HER2Bioconjugation of GGG-TCMC and GGGG-TCMC

Procedure

[0127] Lyophilized antibody resuspended and diafiltered into PBS before starting bioconjugation.

[0128] Antibody was deglycosylated by adding 500 ng/mg of PNGase F enzyme (R&D systems) and rotating at 37° C. overnight.

[0129] Enzyme was removed by diafiltration through a 50 kDa MWCO filter and diafiltered into conjugation buffer (PBS+50 mM EDTA).

[0130] Antibody was incubated with 80 mol equivalent of chemical Linker-Chelator (GGG-TCMC) or (GGGG-TCMC) and 2 U/ml of MTGase enzyme (Zedira) overnight at 37° C. with gentle rotation.

[0131] Conjugates were sterile filtered followed by buffer exchange and enzyme removal by diafiltration into 150 mM ammonium acetate, pH 4.5. Final sterile filtration performed.

Results

[0132]

TABLE-US-00008 TABLE 8 Final Protein CAR Concentration Value TCMC-GGG-Antibody 1.3 mg/ml 0.5 TCMC-GGGG-Antibody 0.9 mg/ml 0.8