Methods for site-specific modification of antibodies

Abstract

The present application relates to methods for preparing site-specific bioconjugated antibodies using metal chelators, bioconjugated antibodies and their use in nuclear medicine.

Description

[Technical field]

[0001] The present invention relates to the field of nuclear medicine, in particular for diagnosing or treating cancer. [Background technology]

[0002] For the delivery of ions or radionuclides of medical interest, suitable chelating agents that form stable complexes are of fundamental importance.In addition, chelating agents can be conjugated to monoclonal antibodies to achieve targeted delivery to cancer cells.The constructs thus generated (so-called bioconjugated antibodies, antibody payload conjugates, or antibody drug conjugates (ADCs)) find increasing interest for targeted treatment of cancer.The modified antibodies can therefore accumulate in tumor cells and concentrate the effect of radioactivity of radionuclides.

[0003] ADCs are FDA approved (Adcetris, Kadcyla, Besponsa and Mylotarg), all of which have payloads chemically linked to the antibody in a non-site-specific manner. The resulting products are therefore highly heterogeneous, both in terms of the stoichiometric relationship between the antibody and payload (payload-antibody ratio, or drug-antibody ratio, or DAR) as well as in terms of the conjugation sites on the antibody. Each of the resulting molecular species, although the same drug product, may have different properties, which can result in a wide range of different in vivo pharmacokinetic properties and activities.

[0004] Studies have shown that site-specific drug conjugation can result in significantly higher tumor uptake and reduced uptake in non-targeted tissues compared to non-site-specific ADCs. These data suggest that stoichiometrically well-defined ADCs exhibit improved pharmacokinetics and better therapeutic index compared to chemically modified ADCs.

[0005] As a site-specific technique, enzymatic conjugation has attracted great interest because these conjugation reactions are typically fast and can be carried out under physiological conditions. Among the available enzymes, microbial transglutaminase (MTG) from the fungal species Streptomyces mobaraensis has attracted increasing interest as an attractive alternative to traditional chemical protein conjugation of functional moieties, including antibodies. MTG catalyzes, under physiological conditions, the transamidation reaction between a "reactive" glutamine of a protein or peptide and a "reactive" lysine residue of a protein or peptide, although the latter can also be a simple low molecular weight primary amine, such as a 5-aminopentyl group.

[0006] Jeger et al. (Angew. Chem. Int. Ed. 2010, 49, 9995-9997) and Dennler et al. (Bioconjugate Chemistry 2014, 25, 569-578) have reported that only a few glutamines are typically targeted by MTG, making it an attractive tool for site-specific and stoichiometric protein modification. Glutamine 295 (Q295) was identified as the only reactive glutamine on the heavy chain of various IgG types that is specifically targeted by MTG with low molecular weight primary amine substrates (Jeger et al.).

[0007] It has been shown by Dennler et al. that quantitative conjugation to Q295 was only possible upon removal of the glycan moiety at asparagine residue 297 (N297) by PNGase F, whereas glycosylated antibodies could not be conjugated efficiently.

[0008] WO2017 / 025179 discloses a protein site-specific bioconjugation method for making a protein comprising at least one acylglutamine-containing amino donor sequence covalently linked to an amino donor-containing substrate via a γ-glutamyl-amide bond, wherein the at least one acylglutamine-containing amino acid donor sequence comprises at least the amino acid sequence TYFQAYG. Therefore, the method of WO2017 / 025179 requires a cumbersome manipulation process on the protein in advance to link the acylglutamine-containing amino acid tag. [Prior art documents] [Patent documents]

[0009] [Patent Document 1] WO2017 / 025179 [Non-patent literature]

[0010] [Non-Patent Document 1] Jeger et al., Angew. Chem. Int. Ed. 2010, 49, 9995~9997 [Non-Patent Document 2] Dennler et al., Bioconjugate Chemistry 2014, 25, 569~578 [Non-Patent Document 3] Wong et al., Precise characterization of intact monoclonal antibodies by the Agilent 6545XT AdvanceBio LC / Q-TF, Application Note, Agilent Technologies, 2017 Summary of the Invention [Problem to be solved by the invention]

[0011] Therefore, it remains desirable to provide a simple and efficient method to achieve antibodies with defined DAR and bioconjugated with chelators in a site-specific manner. [Means for solving the problem]

[0012] The present invention is directed to methods for site-specific conjugation of antibodies to chelators to yield defined chelator-to-antibody ratios (CARs) that are robust and easily achievable.

[0013] The method does not involve the steps of genetic engineering, propagation and purification of each antibody of interest, which are common in many bioconjugation protocols, but instead allows for the use of any antibody for bioconjugation to a chelator.

[0014] According to the object, the present invention provides a method for producing a method for manufacturing a semiconductor device comprising the steps of: Formula (I): Ab-(Linker-Chelator) n (I) (In the formula, Ab is antibody, The linker is an oligopeptide having a COOH terminus and an N-terminus, The chelator is a metal chelator, n is the chelator-antibody ratio (CAR), 0 <n≦2である) A method for preparing a site-specific bioconjugated antibody of - enzymatic deglycosylation of the antibody; - in the presence of transglutaminase, of the formula (A): Linker-Chelator (A) Coupling of the resulting deglycosylated antibody with the compound of Including, The linker is attached to the Ab at its N-terminus, * GGG), ( * KGG) and ( * AKA) (in the formula, *indicates the N-terminus of the linker that covalently binds to the Ab.

[0015] As used herein, Ab refers to an antibody, in particular a monoclonal antibody, such as a human recombinant monoclonal antibody, that is specific for a cancer antigen, such as CD38, HER2, VEGF, EGFR, and thereby targets cancer cells. Antibodies include in particular G-type immunoglobulins, such as IgG1, IgG2, IgG3, and IgG4. Suitable antibodies in the sense of the present invention include bevacizumab, cetuximab, trastuzumab, daratumumab, panitumumab, MabThera.

[0016] As used herein, "site-specific" refers to the location on the antibody where conjugation of the compound of formula (A) occurs. Typically, this location is glutamine 295 (Q295) of IgG when microbial transglutaminase (MTG) is used for bioconjugation. This corresponds to the reactive glutamine on the heavy chain of various IgG types that are specifically targeted by MTG.

[0017] By "oligopeptide" is meant a short chain of less than 15 amino acids linked by peptide (amide) bonds. Typically, the oligopeptide chain has an amino terminal group (or N-terminus) and a carboxyl (COOH) terminal group or C-terminus. Thus, according to the invention, a linker has an antibody (Ab) covalently attached at its N-terminus and a chelator covalently attached via its C-terminus.

[0018] As used herein, amino acids may be designated by their one-letter codes as follows:

[0019] [Table 1]

[0020] According to one embodiment, the linker has the formula: * GGG-(X)p-, -* KGG-(X)p-,- * AKA-(X)p- (In the formula, * indicates the N-terminus of the linker that is covalently attached to the Ab. X is an amino acid, and p is an integer such that 0≦p≦10.

[0021] According to one embodiment, the linker comprises at least the sequence GGG. Typically, the linker may be selected from -GGG- or -GGGG-.

[0022] According to a preferred embodiment, said linker is GGG.

[0023] CAR means chelator-antibody ratio. It is understood that the compound of formula (I) defined above can be a mixture of compounds (I) with different CAR. CAR is generally given as an average value. Thus, n represents the average CAR (in number) for said mixture.

[0024] CAR can be measured by intact mass analysis as described in Wong et al. "Precise characterization of intact monoclonal antibodies by the Agilent 6545XT AdvanceBio LC / Q-TF, Application Note, Agilent Technologies, 2017."

[0025] According to one embodiment, the method of the invention makes it possible to achieve a CAR comprised between 0.5 and 2, in particular between 1 and 2, typically around 1.5±0.5.

[0026] Enzymatic deglycosylation refers to the removal of glycan moieties of an antibody, typically at asparagine residue 297 (N297).

[0027] According to one embodiment, enzymatic deglycosylation is carried out using an N-glycosidase.

[0028] By "N-glycosidase" is meant an enzyme that catalyzes the cleavage of internal glycosidic bonds.

[0029] Typically, the N-glycosidase is PNGase F (protein N-glycosidase F from Flavobacterium meningosepticum).

[0030] PNGase F is commercially available and can be purchased, for example, from ROCHE or R&D SYSTEMS.

[0031] The term "chelator" is used in the sense of the present invention to denote a compound capable of chelating a metal (ie) to form two or more separate coordinate bonds with a single central metal atom. Such chelators or chelating agents are typically compounds whose structure allows the attachment of two or more of their donor atoms (or sites) simultaneously to the same metal ion and which generate one or more rings upon chelation.

[0032] According to one embodiment, the chelator is selected from chelating agents capable of chelating radioactive metal isotopes.

[0033] According to one embodiment, the chelator has formula (II):

[0034] [ka]

[0035] (In formula (II): R 5 , R 6 , and R 8 are each independently H, (C1-C6) alkyl, or (C1-C6) alkyl-C(=O)-OR 25and (C1-C6) alkyl-C(=O)-N(-R 25 )-R 26 selected from the group consisting of; R 9 , R 10 , R 11 , R 12 , R 15 , R 16 , R 17 , R 18 , R 19 , R 20 , R 21 , R 22 , R 23 , and R 24 are each independently selected from the group consisting of H, (C1-C6)alkyl; R 7 are independently H, (C1-C6) alkyl, (C1-C6) alkyl-C(=O)-N(-R 25 )-R 26 , and L 1 - selected from the group consisting of; R 13 and R 14 are each independently H, (C1-C6) alkyl, or L 1 - selected from the group consisting of; R 25 and R 26 are each independently selected from the group consisting of H, (C1-C6)alkyl, and (C1-C6)alkyl-C(=O)-OH; Chelator - L 1 - is attached to the linker - via -; However, one L 1 - is R 7 , R 13 or R 14 In any of the other R 7 , R 13 and R 14 are each H, (C1-C6) alkyl, or (C1-C6) alkyl-C(=O)-N(-R 25 )-R 26 and; L 1- is independently (C1-C6)alkyl-C(=O)-NH-(C1-C6)alkyl-C(=O)-NH-, (C1-C6)alkyl-(C6H4)-NH-C(=S)-NH-, C(-CO2H)-(C1-C6)alkyl-(C6H4)-NH-C(=S)-NH-, (C1-C6)alkyl-C(=O)-NH-, (C1-C6)alkyl-C(=O)-NH-(C1-C6)alkyl-NH-, (C1-C6)alkyl-(C6H4)-NH- and (C1-C6)alkyl-C(=O)-(O-CH2-CH2). 1-20 -C(=O)-NH- It could be.

[0036] In particular, in formula (II): R 5 , R 6 and R 8 is (C1-C6) alkyl-C(=O)-N(-R 25 )-R 26 represents; R 9 , R 10 , R 11 , R 12 , R 13 , R 14 , R 15 , R 16 , R 17 , R 18 , R 19 , R 20 , R 21 , R 22 , R 23 , R 24 , R 25 and R 26 represents H; R 7 , L 1 represents; And L 1 - represents (C1-C6)alkyl-C(=O)-NH-(C1-C6)alkyl-NH- or (C1-C6)alkyl-(C6H4)-NH-.

[0037] According to an alternative embodiment, the chelator- is derived from the following chelator precursor:

[0038] [ka] [ka] [ka] [ka]

[0039] As used above, a chelator precursor is defined as a compound that is suitable for forming a compound of formula (A) above when coupled with a linker.

[0040] Typically, the chelator precursor is DOTA, DOTAM, DOTAM-CH2-CH2-NH2, NCS-TCMC or NH2-TCMC, more preferably NH2-TCMC.

[0041] As used herein, metal typically refers to radioisotopes of metals that may be useful in nuclear medicine. Such radionuclides include: 66 Ga, 68 Ga, 86 Y, 90 Y, 111 In, 212 Pb, 212 Bi, 213 Bi, 89 Zr, 177 Lu, 225 Ac, more preferably 212 Examples include Pb (lead-212).

[0042] By "transglutaminase" is meant an enzyme capable of catalyzing the formation of an isopeptide bond between a γ-carboxamide group (-(C=O)NH2) of a glutamine residue side chain and an ε-amino group (-NH2) of a lysine residue side chain with the subsequent release of ammonia (NH3).

[0043] According to one embodiment, the transglutaminase is a microbial transglutaminase. Typically, the transglutaminase may be the transglutaminase MTGase enzyme from the fungal species Streptomyces mobaraensis.

[0044] Such transglutaminases are commercially available, for example the MTGase enzyme sold by the company ZEDIRA.

[0045] In accordance with the invention, the method comprises the step of deglycosylating the antibody with N-glycosidase F (PNGase F), which acts by cleaving N-linked oligosaccharides between the innermost GlcNAc and an asparagine residue from N-linked glycoproteins, as shown in Figure 1.

[0046] According to one embodiment, this results in the removal of the glycan moiety at asparagine residue 297 (N297).

[0047] The deglycosylation reaction may usually be carried out at a temperature of 20-40° C., typically 23° C.-37° C., for 1-24 hours, typically about 15-24 hours. The amount of N-glycosidase used may be comprised between 100 ng / mg and 1 mg / mg, typically about 500 ng / mg.

[0048] Once the antibody has been deglycosylated, the deglycosylation step may be followed by a diafiltration step to remove excess N-glycosidase F enzyme.

[0049] According to one embodiment, the method of the invention therefore comprises a diafiltration step after carrying out the deglycosylation step.

[0050] The linker-chelator (A) compound is then reacted with microbial transglutaminase (MTGase), which conjugates the compound of formula (A) to the antibody via the linker free N-terminus. This step can be accomplished by incubating the compound of formula (A) with transglutaminase.

[0051] Specifically, microbial transglutaminase acts by catalyzing the formation of a covalent bond between the γ-carbonylamide group of glutamine and the primary amine of an amino acid such as lysine, although other amide donors have also been used (Dennler 2014, Mehta 2005). In 2010, Jeger et al. showed that the conserved glutamine at position 295 of the heavy chain (constant domain) of a deglycosylated IgG1 antibody is the only position at which MTGase will react, resulting in a theoretical chelator-antibody ratio of 2 (one on each heavy chain of the antibody). The enzymatic reaction can be run using the following conditions: Temperature: 20-40°C, typically 23-37°C Amount of transglutaminase (MTGase): 1-10 U / mL, typically about 6 U / mL Time: 1-24 hours, typically 16-24 hours.

[0052] This therefore ensures that the bioconjugation of the present invention is site-specific, since coupling occurs only at this position on the antibody and the resulting bioconjugated compound or mixture of bioconjugated compounds is homogenous as far as the site at which coupling occurs.

[0053] This bioconjugation is carried out by reacting the corresponding bioconjugated antibody Ab-(linker-chelator) n This results in the formation of

[0054] A mixture of compounds may be obtained having different CARs (i.e., n). Thus, CAR(n) as used herein means an average value (in numbers).

[0055] The resulting product can be purified, typically by centrifugation-dialysis, and / or can be further exchanged into a storage buffer such as ammonium acetate.

[0056] The antibody can then be characterized by standard procedures, for example to ensure that the intermediate enzyme has been sufficiently removed, to assess antibody size, and / or to determine the chelator-antibody ratio.

[0057] Linker-chelator (A) compounds can be prepared by reacting a chelator with a linker in the form of its corresponding precursor OH-linker-Z (where Z can be a protecting group for an amine such as carboxybenzyl (Cbz)) in the presence of an ester activator such as HATU (1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate, hexafluorophosphate azabenzotriazole tetramethyluronium) in a solvent such as dimethylacetamide (DMAc or DMA) in conjunction with a base such as Hunig's base (N,N-diisopropylethylamine, DIPEA) or triethylamine. The reaction can be carried out at room temperature.

[0058] According to a further object, the present invention also provides a compound of formula (I): Ab-(Linker-Chelator) n (I) The present invention also relates to site-specific bioconjugated antibodies of the above.

[0059] As defined above, it is obtainable by the method of the invention.

[0060] The present invention also relates to a compound of formula (I): 2. A compound of formula (I) as defined above for use as an imaging agent or drug in nuclear medicine: Ab-(Linker-Chelator) n (I) The present invention also relates to site-specific bioconjugated antibodies of the above.

[0061] This can typically be used for the diagnosis and / or treatment of cancer.

[0062] Another object of the present invention are also pharmaceutical compositions comprising, as active ingredient, a compound of formula (I) according to the invention or a pharma- ceutically acceptable salt or solvate thereof. These pharmaceutical compositions contain an effective dose of at least one compound according to the invention, and at least one pharma- ceutically acceptable excipient.

[0063] The excipients may be selected from among the usual excipients known to those skilled in the art according to the desired pharmaceutical form and administration route.Typically, the compositions of the present invention are suitable for systemic administration, such as parenteral administration, for example by intraperitoneal, intramuscular or intravenous routes.

[0064] According to the usual practice, the suitable dosage for each patient is determined by the physician according to the route of administration, the weight and response of the patient. Typically, the active ingredient of the above formula (I), its salt or solvate may be mixed with a conventional pharmaceutical excipient and administered in a unit dosage form.

[0065] According to another of its aspects, the present invention also relates to a method for the diagnosis and / or treatment of cancer comprising the administration to a patient of an effective dose of a compound of formula (I) according to the invention, or a salt thereof, including a pharma- ceutically acceptable salt thereof.

[0066] Representative figures and experimental results further illustrate the invention. [Brief description of the drawings]

[0067] [Figure 1] FIG. 1 shows a schematic representation of an exemplary process of the present invention, in which (1) represents a full length antibody that is: deglycosylated in step 1, washed by diafiltration in step 2, reacted with (2): a linker-chelator and MTGase in step 3, and washed by diafiltration in step 4, thereby resulting in the corresponding bioconjugated antibody (3). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0068] Experimental Results definition PBS: Phosphate buffered saline EDTA: Ethylenediaminetetraacetic acid MWCO: Molecular Weight Cutoff CAR:chelator-antibody ratio HATU: (1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate, azabenzotriazole tetramethyluronium hexafluorophosphate) DIPEA: N,N-diisopropylethylamine DMA: dimethylacetamide (CH3C(O)N(CH3)2).

[0069] material: NH2-TCMC (compound 1) was provided by Macrocyclics, Inc. and was used as the main starting material in the synthesis of GGG-TCMC. All solvents and reagents were purchased from commercial sources and used as received.

[0070] Synthesis of GGG-TCMC:

[0071] [ka]

[0072] The following formula in DMA (64 mL):

[0073] [ka]

[0074] A solution of Z-(Gly)3-OH (3.3 g, 10.2 mmol) was prepared followed by the addition of DIPEA (5.20 mL, 29.9 mmol) and HATU (4.51 g, 11.9 mmol).

[0075] A solution of NH2-TCMC (1) (6.56 g, 10.1 mmol) in DMA (80 mL) containing 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 h. HPLC monitoring indicated the reaction was incomplete. A second equivalent of Z-(Gly)3-OH (3.35 g, 10.4 mmol) dissolved in DMA (36 mL) containing 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 h. The reaction solution was added dropwise 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 min. The precipitate was isolated by vacuum filtration and the filter cake was washed with diethyl ether (150 mL). A stock solution was prepared using acetonitrile (120 mL), HO (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 HO mobile phase. The purified fraction of 2 was concentrated by rotary evaporation and then transferred to a Parr shaker bottle. 10% palladium on carbon catalyst was added and the mixture was subjected to vacuum and then pressurized to 30 psi hydrogen. The hydrogenation batch was shaken at room temperature until HPLC monitoring showed the reaction was complete. The reaction mixture was filtered under vacuum using a 0.2 μm PVDF membrane filter and lyophilized to give 3 as a solid.

[0076] CD38-targeting IgG1 monoclonal antibody bioconjugated The antibody was diafiltered into PBS prior to bioconjugation. The antibody was deglycosylated by adding 6U / mg PNGase F enzyme and stirring overnight at 37°C. The enzyme was removed and the buffer exchanged into PBS+50mM EDTA by diafiltration through a 50kDa MWCO filter. The antibody was incubated with 80 mol equivalents of chemical linker-chelator (GGG-TCMC) and 6U / mL MTGase enzyme (Zedira) overnight at 37°C with gentle stirring. Buffer exchange and enzyme removal were performed by diafiltration into 150mM ammonium acetate, pH 4.5.

[0077] A final antibody concentration of 6.4 mg / mL was determined. The CAR was determined to be 0.8 by intact mass analysis.

[0078] [Table 2]

[0079] TEM-1 Targeted Monoclonal IgG1 Antibody Bioconjugated The antibody was diafiltered into PBS before bioconjugation began. The antibody was deglycosylated by adding 6U / mg PNGase F enzyme and stirring overnight at 37°C. The enzyme was removed and the buffer exchanged into PBS+50mM EDTA by diafiltration through a 50kDa MWCO filter. The antibody was incubated with 80 mol equivalents of chemical linker-chelator (GGG-TCMC) and 6U / mL MTGase enzyme (Zedira) overnight at 37°C with gentle stirring. Buffer exchange and enzyme removal were performed by diafiltration into 150mM ammonium acetate, pH 4.5.

[0080] A final antibody concentration of 6.5 mg / mL was determined. The CAR determined by intact mass was 1.2.

[0081] [Table 3]

[0082] IgG1 antibody bioconjugation The IgG1 antibody was diafiltered into PBS before the start of bioconjugation.

[0083] The antibody was deglycosylated by adding 6U / mg PNGase F enzyme and stirring overnight at 37°C. The enzyme was removed and the buffer exchanged into PBS+50mM EDTA by diafiltration through a 50kDa MWCO filter. The antibody was incubated with 80 mol equivalents of a chemical linker-chelator (GGG-TCMC) and 6U / mL MTGase enzyme (purchased from Zedira) overnight at 37°C with gentle stirring. Buffer exchange and enzyme removal were performed by diafiltration into 150mM ammonium acetate, pH 4.5.

[0084] A final antibody concentration of 5.1 mg / mL was determined. The CAR, as determined by intact mass analysis, was 1.1.

[0085] [Table 4]

[0086] CD20-targeting monoclonal IgG1 antibody bioconjugated The antibody was diafiltered into PBS before bioconjugation began. The antibody was deglycosylated by adding 6U / mg PNGase F enzyme and stirring overnight at 37°C. The enzyme was removed and the buffer exchanged into PBS+50mM EDTA by diafiltration through a 50kDa MWCO filter. The antibody was incubated with 80 mol equivalents of chemical linker-chelator (GGG-TCMC) and 6U / mL MTGase enzyme (Zedira) overnight at 37°C with gentle stirring. Buffer exchange and enzyme removal were performed by diafiltration into 150mM ammonium acetate, pH 4.5.

[0087] A final antibody concentration of 5.6 mg / mL was determined. The CAR was determined to be 1.0 by intact mass analysis.

[0088] [Table 5]

[0089] HER2-targeting monoclonal antibody bioconjugation The antibody was diafiltered into PBS before the start of bioconjugation.

[0090] The antibody was deglycosylated by adding 6 U / mg PNGase F (Roche) enzyme and stirring overnight at 37°C.

[0091] The enzyme was removed and the buffer exchanged into PBS+50 mM EDTA by diafiltration through a 50 kDa MWCO filter.

[0092] The antibodies were incubated with 80 mol equivalents of chemical linker-chelator (GGG-TCMC) and 6 U / mL MTGase enzyme (Zedira) overnight at 37° C. with gentle agitation.

[0093] Buffer exchange and enzyme removal was achieved by diafiltration into 150 mM ammonium acetate, pH 4.5.

[0094] A final antibody concentration of 2.5 mg / mL was determined. The CAR was determined to be 0.9 by intact mass.

[0095] [Table 6]

[0096] EGFR-targeting monoclonal IgG1 antibody bioconjugated The antibody was diafiltered into PBS before bioconjugation began. The antibody was deglycosylated by adding 6U / mg PNGase F (Roche) enzyme and stirring overnight at 37°C. The enzyme was removed and the buffer exchanged into PBS+50mM EDTA by diafiltration through a 50kDa MWCO filter. The antibody was incubated with 80 mol equivalents of a chemical linker-chelator (GGG-TCMC) and 6U / mL MTGase enzyme (Zedira) overnight at 37°C with gentle stirring. Buffer exchange and enzyme removal were performed by diafiltration into 150mM ammonium acetate, pH 4.5.

[0097] A final antibody concentration of 3.0 mg / mL was determined. The CAR was determined to be 1.1 by intact mass analysis.

[0098] HER2-Targeted Monoclonal Antibody Bioconjugation-Comparison of KGG and GGG Linkers Before the start of bioconjugation, lyophilized antibodies were resuspended and diafiltered into PBS. Antibodies were deglycosylated by adding 500ng / mg PNGase F enzyme (R&D systems) and stirring overnight at 37°C. Enzyme was removed by diafiltration through a 50kDa MWCO filter. Antibodies were incubated with 80 mol equivalents of chemical linker GGG-TCMC or KGG-TCMC and 6U / mL MTGase enzyme (Zedira) overnight at 37°C with gentle stirring. Buffer exchange and enzyme removal were performed by diafiltration into 150mM ammonium acetate, pH 4.5.

[0099] Final antibody concentrations of 5.1 mg / mL for bioconjugation with GGG-linker and 7.2 mg / mL for bioconjugation with KGG-linker were determined. CAR was determined by intact mass to be 0.8 for KGG-TCMC-antibody and 1.9 for GGG-TCMC-antibody.

[0100] Antibody bioconjugation of HER2-targeting monoclonal IgG1 antibodies, AKA-TCMC and GGG-TCMC, with and without PNGase deglycosylation This experiment aims to find out whether PNGase deglycosylation is required to conjugate a chelator to an antibody, depending on the linker used. AKA-TCMC was chosen because it should not have required PNGase. AKA-TCMC was compared to GGG-TCMC with and without PNGase deglycosylation.

[0101] procedure Before the start of bioconjugation, the lyophilized antibodies were resuspended and diafiltered into PBS.

[0102] Half of the antibodies were deglycosylated by adding 500 ng / mg PNGase F enzyme (R&D systems) and rotating overnight at 37° C. The other half did not receive PNGase F.

[0103] The enzyme was removed by diafiltration through a 50 kDa MWCO filter and diafiltered into conjugation buffer (PBS+50 mM EDTA).

[0104] Antibodies (deglycosylated and undeglycosylated) were incubated with 80 mol equivalents of chemical linker-chelator (GGG-TCMC) or (AKA-TCMC) and 6 U / mL MTGase enzyme (Zedira) overnight at 37° C. with gentle rotation.

[0105] The conjugate was sterile filtered, followed by buffer exchange and enzyme removal by diafiltration into 150 mM ammonium acetate, pH 4.5. A final sterile filtration was performed.

[0106] result

[0107] [Table 7]

[0108] The data suggest that deglycosylation is required for AKA-TCMC and GGG-TCMC in order to be conjugated to antibodies, and further that both AKA-TCMC and GGG-TCMC are capable of being conjugated to antibodies.

[0109] HER2-targeted monoclonal IgG1 antibody bioconjugation of GGG-TCMC and GGGG-TCMC procedure Before the start of bioconjugation, the lyophilized antibodies were resuspended and diafiltered into PBS.

[0110] The antibody was deglycosylated by adding 500 ng / mg PNGase F enzyme (R&D systems) and rotating overnight at 37°C.

[0111] The enzyme was removed by diafiltration through a 50 kDa MWCO filter and diafiltered into conjugation buffer (PBS+50 mM EDTA).

[0112] Antibodies were incubated with 80 mol equivalents of chemical linker-chelator (GGG-TCMC) or (GGGG-TCMC) and 2 U / mL MTGase enzyme (Zedira) overnight at 37° C. with gentle rotation.

[0113] The conjugate was sterile filtered, followed by buffer exchange and enzyme removal by diafiltration into 150 mM ammonium acetate, pH 4.5. A final sterile filtration was performed.

[0114] result

[0115] [Table 8]

Claims

1. Formula (I): Ab-(Linker-Chelator) n (I) (wherein Ab is an antibody; the linker is an oligopeptide having an N-terminus, The chelator is a metal chelator, n is the chelator-antibody ratio (CAR) and 0 <n≦2である) A method for preparing a site-specific bioconjugated antibody of - enzymatic deglycosylation of said antibody Ab; - in the presence of transglutaminase, of the formula (A): Linker-Chelator (A) Coupling of the resulting deglycosylated antibody with the compound of Including, The linker is attached to the Ab at its N-terminus, * GGG), ( * KGG) and ( * AKA) (in the formula, * represents the N-terminus of the linker that covalently binds to the Ab.

2. The linker has the formula: * GGG-(X)p-(SEQ ID NO:2), - * KGG-(X)p-(SEQ ID NO:3), * AKA-(X)p-(SEQ ID NO:4) (In the formula, * represents the N-terminus of the linker, X is an amino acid, and p is an integer such that 0≦p≦10. The method of claim 1, wherein the compound is selected from the group consisting of

3. The linker comprises at least * The method of claim 1 or 2, comprising the GGG sequence.

4. The linker is * GGG or * 3. The method of claim 1 or 2, wherein the amino acid sequence is GGGG (sequence number 5).

5. The linker * 3. The method of claim 1 or 2, wherein the amino acid sequence is GGG.

6. The method according to claim 1 or 2, wherein the enzymatic deglycosylation is carried out using an N-glycosidase.

7. 3. The method of claim 1 or 2, wherein the N-glycosidase is PNGase F (protein N-glycosidase F from Flavobacterium meningosepticum).

8. -Chelator is -NH-CH 2 -CH 2 -DOTAM or -NH 2 The method of claim 1 or 2, wherein the -TCMC.

9. The method according to claim 1 or 2, wherein the transglutaminase is a microbial transglutaminase.

10. 3. The method according to claim 1, wherein the transglutaminase is derived from the fungal species Streptomyces mobaraensis.

11. The method according to claim 1 or 2, wherein the CAR is included in 1 to 2.

12. 3. The method according to claim 1 or 2, wherein diafiltration washing is carried out between the deglycosylation step and the coupling step.

13. A compound of formula (I): obtainable by the process according to any one of claims 1 to 12: Ab-(Linker-Chelator) n (I) (wherein Ab, linker and chelator are defined as in claim 1 or 2). Site-specific bioconjugated antibodies.

14. Formula (I): Ab-(Linker-Chelator) n (I) (wherein Ab, linker and chelator are defined as in claim 1 or 2). and at least one pharma- ceutical acceptable excipient.

15. For use as an imaging agent or drug in nuclear medicine, a compound of formula (I): Ab-(Linker-Chelator) n (I) (wherein Ab, linker and chelator are defined as in claim 1 or 2). Site-specific bioconjugated antibodies.

16. 16. A site-specific bioconjugated antibody for use according to claim 15 for diagnosing and / or treating cancer.

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