Patent Application
20240191272
The present invention relates to a method for producing an antibody-drug conjugate. The present invention also relates to a mutant enzyme of endo-β-N-acetylglucosaminidase used in the production method.
Antibody drug conjugates (henceforth referred to as ADCs) are drugs conjugated to antibodies that specifically bind to a target. For example, an ADC consisting of an antibody targeting cancer cells conjugated with a payload having potent cytotoxic activity can selectively deliver a drug (payload) to cancer cells, and thereby effectively kill cancer cells with greatly reduced systemic toxicity as a side effect compared with the use of the payload alone. A typical ADC is T-DM1 (Kadcyla (registered trademark)), which is the anti-HER2 antibody trastuzumab conjugated with a tubulin inhibitor, and it is approved as a drug (Non-patent documents 1 to 3). Concerning T-DM1, payloads are randomly bound to Lys residues on the antibody, and therefore it is produced as a mixture of conjugates heterogeneous for binding positions and binding numbers of the payloads on the antibody. Similar ADCs include gemtuzumab ozogamicin (generic name Mylotarg, Non-patent document 4), brentuximab vedotin (generic name Adcetris, Non-patent document 5), and so forth.
Recently, it has been reported that the homogeneity of ADCs affects pharmacokinetics, drug release rate, and drug efficacy (Non-patent document 6). In addition, from the viewpoint of ADC quality control, regioselective payload binding methods have been actively developed to produce ADCs with high homogeneity. One of such methods is a method for binding payloads to antibody glycans using an enzyme. The ADC production processes using this method reported so far comprise: (1) homogenization of glycans by enzymatic cleavage of heterogeneous antibody glycans (hydrolysis step), (2) enzymatic binding of glycan-homogenized antibodies and another glycan (glycosyltransfer step), and (3) binding of azide group on the other glycan and a payload (chemical reaction step).
In the above method, a family of enzymes called endo-β-N-acetylglucosaminidases is used in the hydrolysis and glycosyltransfer steps in order to convert glycans (Non-patent document 7). Endo-β-N-acetylglucosaminidases are isolated from a variety of species, and are used in different ways depending on the type of antibody glycan. Typical endo-β-N-acetylglucosaminidases used in the hydrolysis step include EndoA, EndoD, EndoM, EndoH, EndoF2, EndoF3, EndoE, EndoS, EndoS2, and so forth.
It has been reported that, among these, hydrolysis abilities of EndoS D233Q, i.e., EndoS in which the 233rd Asp is replaced with Gln (Patent document 1 and Non-patent document 8), EndoS2 D184M or EndoS2 D184Q, i.e., EndoS in which the 184th Asp is replaced with Met or Gln (Patent document 2 and Non-patent document 9), and EndoF3 D165A or EndoF3 D165Q, i.e., EndoF3 in which the 165th Asp is replaced with Ala or Gln (Patent document 3 and Non-patent document 10), are suppressed. It has also been reported that these mutant enzymes promote glycosyltransfer between glycan-homogenized antibodies (GlcNAc antibodies) and a substrate with oxazolinated glycan reducing end.
Furthermore, it has been reported that a homogeneous ADC was produced by transferring a substrate glycan having oxazolinated reducing end and azide group to a GlcNAc antibody using EndoS D233Q, and subsequent click reaction of the azide group of the resulting conjugate with MMAE, which is one type of payload (Non-patent documents 11 and 12).
The ADC production processes using sugar chain-homogenized antibody and enzyme reported so far require three steps: the steps of hydrolysis of antibody glycan, glycosyltransfer to glycan-homogenized antibody, and linkage of payload to antibody-glycan. If ADC purification in each step is also taken into consideration, increase in ADC production cost and decrease in the performance and function of the antibody due to purification are anticipated, and therefore a simpler production method is desired.
In general, enzymatic reactions are substrate-specific. Therefore, if it is attempted to conjugate a substrate glycan modified with a payload with a glycan-homogenized antibody in a single step using a glycosyltransferase, a significant decrease in the reaction efficiency of the enzyme is expected. In fact, when the inventors of the present invention attempted to conjugate peptide- or drug-modified glycans to antibodies with various glycosyltransferases, the reaction efficiency was significantly reduced compared with that for linking simple glycans to antibodies.
An object of the present invention is to provide an enzyme that can efficiently conjugate a glycan modified with a payload to a glycan-homogenized antibody, and to provide a novel production method of ADC that allows such conjugation of an antibody and a payload, which conventionally required two steps, in a single step by using such an enzyme.
The present invention provides the followings.
the step of conjugating a glycan-homogenized antibody and a glycosylated drug, wherein:
the glycosylated drug consists of a drug bound to a glycan having an oxazolinated reducing end via a linker.
A, B, C, and E are each independently selected from the group consisting of an alkyl group, ester group, carbamoyl group, alkoxyalkyl group, imine group, hydrazone group, azo group, sulfone group, aromatic group, and single bond; and
D is a peptide consisting of 2 to 6 amino acid residues].
A is —(CH2 CH2O)m1—(CH2)m2-, —(CH2)m2-, or a single bond;
B is selected from the group consisting of
C is —C(O)—(CH2)n1—C(O)—, —C(O)—(CH2)n1—C(O)—NH—(CH2CH2O)n2—(CH2)n3—C(O)—, —C(O)—(CH2)n1—C(O)—NH—(CH2)n3—C(O)—, or a single bond,
E is -aminobenzylalcohol-C(O)—, NH—(CH2)o-, or a single bond,
m1 is an integer of from 1 to 12
m2 is an integer of from 1 to 6,
n1 is an integer of from 1 to 6,
n2 is an integer of from 1 to 12,
n3 is an integer of from 1 to 6, and
o is an integer of from 1 to 6].
FIG. is an HPLC chromatogram of ADC obtained by conjugation of antibody-GlcNAc and an MMAE-attached glycan using an EndoF3 mutant enzyme.
The present invention relates to a method for producing an antibody drug conjugate (hereinafter referred to as ADC) comprising the following step. The production method of the present invention comprises the step of conjugating a glycan-homogenized antibody and a glycosylated drug. The step of conjugating a glycan-homogenized antibody and a glycosylated drug can be performed by a synthetic method or an enzymatic method.
When the conjugation step is performed by an enzymatic method, an endo-β-N-acetylglucosaminidase mutant enzyme can be used. Concerning the present invention, the term endo-β-N-acetylglucosaminidase mutant enzyme refers to an endo-β-N-acetylglucosaminidase mutant enzyme showing suppressed hydrolysis ability. Examples of typical endo-β-N-acetylglucosaminidases include EndoA, EndoD, EndoM, EndoH, EndoF2, EndoF3, EndoE, EndoS, and EndoS2.
In a preferred embodiment, among the endo-β-N-acetylglucosaminidase mutant enzymes, a mutant enzyme that is EndoS of which 233rd asparagine (D) is replaced, a mutant enzyme that is EndoS2 of which 184th asparagine (D) is replaced, or a mutant enzyme that is EndoF3 of which 165th asparagine (D) is replaced, preferably a mutant enzyme that is EndoS of which 233rd asparagine (D) is replaced, or a mutant enzyme that is EndoF3 of which 165th asparagine (D) is replaced is used.
An example of more preferred endo-β-N-acetylglucosaminidase mutant enzyme is a mutant enzyme comprising any one of the following proteins.
Another example of the endo-β-N-acetylglucosaminidase mutant enzyme is a mutant enzyme comprising any one of the following proteins:
In the sequence listing, the amino acid sequence of EndoF3 D165Q, corresponding to EndoF3 in which the 165th amino acid is replaced with glutamine (Q), is shown as SEQ ID NO: 1. The amino acid sequence of EndoS D233Q, corresponding to EndoS in which the 233rd amino acid is replaced with glutamine (Q) is also shown as SEQ ID NO: 11. In the following descriptions, the present invention may be explained by referring to the cases using EndoF3 D165Q or EndoS D233Q as the endo-β-N-acetylglucosaminidase mutant enzyme, but those skilled in the art can also understand the cases using other mutant enzymes by appropriately applying the explanations.
The endo-β-N-acetylglucosaminidase mutant enzyme used for the conjugation may be the mutant enzyme described above into which a point mutation is further introduced. Examples of such an enzyme include a mutant enzyme consisting of a protein consisting of the amino acid sequence of SEQ ID NO: 1, or the amino acid sequence of SEQ ID NO: 1 in which the 165th glutamine (Q) is replaced with alanine (A), and having a mutation corresponding to at least one selected from the group consisting of K81T, M97W, Y, F, V, G, S, or Q, V129G, Q250I, N270R, L or T, and Y282W. Particularly preferred examples include EndoF3 D165Q/M97G, which is a protein consisting of the amino acid sequence of SEQ ID NO: 1, or the amino acid sequence of SEQ ID NO: 1 in which the 165th glutamine (Q) is replaced with alanine (A), wherein the 97th M is replaced with G.
Concerning the present invention, the term conjugation activity used for the endo-β-N-acetylglucosaminidase mutant enzyme means such an activity that when the conjugation reaction between a glycan-homogenized antibody and a glycosylated drug is performed with the enzyme, at least 20% of drug-added antibodies (adducts) can be generated, unless otherwise specified (100 (%)—Remaining ratio (%) is at least 20%). The ratio (%) mentioned here can be calculated on the basis of the peak area of the HPLC chromatogram for the conjugation reaction solution. If more than one type of adducts are formed, for example, if one drug adduct (1-adduct) and two drugs adduct (2-adduct) are formed, all adducts should be considered. Specifically, the remaining ratio can be calculated in accordance with the following equation.
Remaining ratio (%)=[Peak area value of unreacted antibody]/[(Peak area value of unreacted antibody)+(Peak area value of adduct*)]×100
1-Adduct conversion rate (%)=[Peak area value of 1-adduct]/[(Peak area value of unreacted antibody)+(Peak area value of 1-adduct)+(Peak area value of 2-adduct)]×100
2-Adduct conversion rate (%)=[Peak area value of 2-adduct]/[(Peak area value of unreacted antibody)+(Peak area value of 1-adduct)+(Peak area value of 2-adduct)]×100
The present invention also provides a polynucleotide encoding an endo-β-N-acetylglucosaminidase mutant enzyme that can be used in the method for producing an antibody-drug conjugate. Namely, the present invention provides:
a polynucleotide encoding any one of the following proteins:
Another example of the polynucleotide encoding an endo-β-N-acetylglucosaminidase mutant enzyme that can be used in the method for producing an antibody-drug conjugate is a polynucleotide that encodes any one of the following proteins:
The antibody used in the present invention may be any of a mouse antibody, chimeric antibody, humanized antibody, and human antibody, but a humanized or human antibody is preferred. The antibody used may also be modified or created by the Potelligent technology (removal of fucose from the Fc region of IgG).
An example of the antibody is an anti-HER2 antibody. Anti-HER2 antibodies are recombinant anti-HER2 monoclonal antibodies, and include a chimeric antibody with a human constant region, humanized antibody in which the CDR (complementarity determining region) of a human antibody is replaced into the CDR of an anti-HER2 antibody of a non-human animal (CDR transplanted antibody), and human antibody that is an expression product of a human-derived antibody gene, but a humanized antibody and human antibody are preferred. Fully human monoclonal antibodies can be produced by immunizing mice into which most of the human immunoglobulin heavy and light chain loci have been introduced by transgenesis. A preferred anti-HER2 antibody is trastuzumab (Herceptin (registered trademark)), which is a humanized IgG1 antibody.
The present invention can be applied to antibodies used as antibody drugs. Specific examples include ibritumomabtiuxetan, iodine131, catumaxomab, blinatumomab, muromonab-CD3, abciximab, rituximab, basiliximab, infliximab, cetuximab, brentuximab, siltuximab, dinutuximab, obiltoxaximab, daclizumab, palivizumab, gemtuzumab, alemtuzumab, omalizumab, efalizumab, bevacizumab, natalizumab, tocilizumab, ranibizumab, eculizumab, certolizumab pegol, mogamulizumab, pertuzumab, obinutuzumab, vedolizumab, pembrolizumab, idarucizumab, mepolizumab, elotuzumab, daratumumab, ixekizumab, reslizumab, adalimumab, panitumumab, golimumab, ustekinumab, canakinumab, ofatumumab, denosumab, ipilimumab, belimumab, raxibacumab, ramucirumab, nivolumab, secukinumab, evolocumab, alirocumab, and necitumumab.
An example of the class of antibodies used in the production method of the present invention is IgG, and an example of the subclass is IgG1.
In the present invention, anticancer agents can be used as the drug. Examples of anticancer agents include known anticancer agents such as tubulin synthesis inhibitors (anticancer plant alkaloids), topoisomerase inhibitors, alkylating agents that are inhibitors of nucleic acid synthesis, antimetabolites, antibiotic anticancer agents, hormonal agents, platinum agents, and non-specific anti-malignant tumor agents, as well as anticancer agents to be newly developed. Specifically, there can be mentioned tubulin synthesis inhibitors (anticancer plant alkaloids) such as auristatin, vincristine sulfate, vinblastine sulfate, vindesine sulfate, docetaxel hydrate, paclitaxel, vinorelbine tartrate, and maytansinoids; topoisomerase inhibitors such as camptothecin, topotecan, etoposide, irinotecan hydrochloride and nogitecan hydrochloride; alkylating agents such as cyclophosphamide, ifosfamide, melphalan, thiotepa, busulfan, carboquone, dacarbazine, nimustine hydrochloride and ranimustine; antimetabolites such as methotrexate, mercaptopurine, 6-mercaptopurine riboside, fluorouracil (5-FU), tegafur, tegafur-uracil, carmofur, doxyfluoridine, cytarabine ocfosfate, hydroxycarbamide, cytarabine, gemcitabine hydrochloride, fludarabine phosphate, enocitabine, and leucovorin; anticancer antibiotics such as doxorubicin hydrochloride, idarubicin hydrochloride, epirubicin hydrochloride, pirarubicin hydrochloride, daunorubicin hydrochloride, aclarubicin hydrochloride, mitomycin C, actinomycin D, bleomycin, peplomycin sulfate, neocartinostatin, and zinostatin stimalamar; hormonal agents such as estramustine sodium phosphate, flutamide, bicalutamide, goserelin acetate, leuprorelin acetate, tamoxifen citrate, fadrozole hydrochloride hydrate, anastrozole, mepithiostan, epithiostanol, and medroxyprogesterone acetate; platinum preparations such as cisplatin, carboplatin and nedaplatin; nonspecific anti-malignant tumor agents such as Krestin, lentinan, schizophyllan, and ubenimex; mitoxantrone hydrochloride, procarbazine hydrochloride, pentostatin, sobuzoxane, tretinoin, L-asparaginase, asegratone, mitotane, sodium porphymer, and so forth.
In a preferred embodiment, the drug is any selected from the group consisting of anthracyclines, auristatins, maytansines, camptothecins, pyrrolobenzodiazepine dimers, calicheamicins, and duocarmycins.
Examples of anthracyclines include daunorubicin (daunomycin), doxorubicin (adriamycin), PNU-159682, and pirarubicin.
Examples of auristatins include auristatin, which is a tubulin synthesis inhibitor and an extremely potent antitumor agent. Auristatins include monomethyl auristatin E (MMAE), monomethyl auristatin D (MMAD), monomethyl auristatin F (MMAF), and so forth. Among these, monomethyl auristatin E (MMAE) is preferred.
Examples of maytansines include ansamitosin P3 (AP3), DM1, and DM4.
Examples of camptothecins include exatecan, SN-38, and DXd.
Other specific examples include PBD-dimer, calicheamicin, and duocarmycin.
In the glycosylated drug used in the present invention, the drug is bound to a glycan having an oxazolinated reducing end via a linker. In a preferred embodiment, the linker contains a spacer (-C-B-A- described below) and a cleavable portion (-E-D-).
The spacer is a moiety that connects the glycan and a binding product of the linker (in a narrow sense, -E-D- as described below) and the drug. Examples of the binding group include an amide, ester, alkoxyalkyl, carbamoyl, sulfonamide, sulfonate, phosphonamide, phosphonate, aldoimine, ketoimine, oxime, click binding site formed from alkyne and azide etc., and so forth.
Typical click binding sites are listed in the following references: Chem, Rev., 113, 4905-4979 (2013); Method Mol. Biol., 2078, 83-97 (2020); Chem. Soc. Rev., 48, 4361-4374 (2019); and Protein Cell, Jan., 9(1), 33-46 (2018)). Preferred examples of such binding sites include 1,2,3-triazole moiety formed from alkyne and azide, and tetracyclic moiety containing 1,2,3-triazole formed from DBCO and azide. An even more preferred example is tetracyclic moiety containing 1,2,3-triazole formed from azide.
A common technique for improving water solubility of the linker for the purpose of reducing ADC aggregation is to introduce PEG groups. In the production method of the present invention, for example, PEG groups are introduced into the spacer. Such PEG can have a linear or branched chain, and is preferably a linear PEG. The PEG chain length is not particularly limited, and examples of PEG include PEG2 (the number represents the number of repeats), PEG3, PEG4, and PEGS to PEG12.
The cleavable portion is a moiety cleavable under specific conditions that do not cause denaturation or degradation of the protein. In other words, it contains a structure that can be cleaved in vivo. The specific conditions that do not cause denaturation and degradation of the protein are conditions of degradation caused by one or more substances selected from the group consisting of acidic substances, basic substances, reducing agents, oxidizing agents, and enzymes (References: Bioorg. Med. Chem., 20 571-582 (2012); J. Control. Release, 99, 423-434 (2004); Bioconjugate Chem., 28, 1906-1915 (2017)). Examples of such a cleavable portion include disulfide bond, acetal bond, ketal bond, aldoimine bond, ketoimine bond, oxime bond, ester bond, amide bond, carbamoyl bond, carbonate bond, alkoxyalkyl bond, sulfone-containing bond, and phosphonic acid-containing bond. Preferred examples of these cleavable portions include peptides consisting of 2 to 6 amino acid residues. Examples thereof include Val-Cit, Gly-Gly-Phe-Gly (GGFG), Val-Ala, Lys-Phe (KF), and Gly-Gly-Val-Gly (GGVG).
In a preferred embodiment, the linker has a structure represented by the general formula -E-D-C-B-A-, where A is bound to the glycan and E is bound to the drug.
In the formula,
A, B, C, and E are each independently selected from the group consisting of an alkyl group, ester group, carbamoyl group, alkoxyalkyl group, imine group, hydrazone group, azo group, sulfone group, aromatic group, and single bond; and
D is a peptide consisting of 2 to 6 amino acid residues.
In the formula,
A, B, C, and E may also be independently selected from the group consisting of —(CH2)1-12-, —(CH2)1-12—O—C(O)—(CH2)0-12, —O—C(O)—NH—, —(CH2CH2O)1-12—(CH2)1-12-, —C(═NR1)— or —(CH2)1-12—N(R1)— (R1 is hydrogen atom or —(CH2)1-6), —R2C═N—NR3— or —R2C═N—OR3— (R2 and R3 are independently hydrogen atom or —(CH2)1-6), —N═N—, —SO3—, a divalent aromatic hydrocarbon group (group formed by removing two hydrogen atoms from an aromatic hydrocarbon ring, such as phenylene, indenylene, naphthylene, fluorenylene, phenanthrenylene, anthrylene, and pyrenylene), and a single bond.
A can be —(CH2CH2O)m1—(CH2)m2-, —(CH2)m2-, or a single bond.
B may be any selected from the group consisting of the following groups:
In the formulas, R is independently hydrogen atom, alkyl having 1 to 4 carbon atoms, alkoxy having 1 to 4 carbon atoms, —CN, —OH, —CF3, or NRR.
C is —C(O)—(CH2)n1—C(O)—, —C(O)—(CH2)n1—C(O)—NH—(CH2CH2O)n2—(CH2)n3—C(O)—, —C(O)—(CH2)n1—C(O)—NH—(CH2)n3—C(O)—, or a single bond,
E is -aminobenzylalcohol-C(O)—, NH—(CH2)o-,or a single bond
m1 is an integer of from 1 to 12,
m2 is an integer of from 1 to 6,
n1 is an integer of from 1 to 6,
n2 is an integer of from 1 to 12,
n3 is an integer of from 1 to 6, and
o is an integer of from 1 to 6.
B may be any of the following groups:
In a particularly preferred embodiment, the linker consists of the following structure.
In a particularly preferred embodiment, the production method of the present invention can be carried out as follows.
Concerning the present invention, in the expression of “an amino acid sequence derived by substitution, deletion, insertion, and/or addition of one or more amino acids” used for a protein or an amino acid sequence, the number of amino acids modified by substitution or the like is not particularly limited for any proteins so long as the proteins consisting of the amino acid sequence have the desired function, unless especially specified, but it may be about 1 to 750, 1 to 500, 1 to 250, 1 to 200, 1 to 150, 1 to 100, 1 to 50, 1 to 40, 1 to 30, 1 to 20, 1 to 15, 1 to 9, or 1 to 4. However, in the case of substitutions of amino acids having similar properties, substitutions of a further larger number of amino acids may be possible. The means for preparing polynucleotides for such amino acid sequences or proteins are well known to those skilled in the art.
In the explanations of the present invention, as for amino acids or amino acid residues, A stands for alanine, C for cysteine, D for aspartic acid, E for glutamic acid, F for phenylalanine, G for glycine, H for histidine, I for isoleucine, K for lysine, L for leucine, M for methionine, N for asparagine, P for proline, Q for glutamine, R for arginine, S for serine, T for threonine, U for selenocysteine, V for valine, W for tryptophan, and Y for tyrosine, unless especially noted,.
Concerning the present invention, the term “identity” used for nucleotide sequences (also referred to as “base sequences”) or amino acid sequences means percentage of number of matching nucleotides or amino acids between two optimally aligned sequences for any nucleotide sequences and amino acid sequences, unless otherwise noted. In other words, it can be calculated in accordance with the equation: Identity=(Number of matched positions/Total number of positions)×100, and can be calculated by using commercially available algorithms. Such algorithms are also incorporated in the NBLAST and XBLAST programs described in Altschul et al., J. Mol. Biol., 215(1990) 403-410. In more detail, searches and analyses for nucleotide or amino acid sequence identities can be performed with algorithms or programs well known to those skilled in the art (e.g., BLASTN, BLASTP, BLASTX, ClustalW). When using programs, the parameters can be appropriately set by those skilled in the art, or the default parameters of each program can also be used. The specific methods of these analyses are also well known to those skilled in the art. The genetic information processing software Genetyx (registered trademark, Genetyx Corporation) may be used to calculate the identity. If the target sequence for which the % identity is to be calculated has an additional sequence at the end, such as a tag sequence, that is not present in the sequence to be compared, the additional sequence is not included in the calculation of the % identity.
Concerning the present invention, the term identity used for nucleotide sequences or amino acid sequences means sequence identity of at least 50%, for example, 60% or higher, 70% or higher, preferably 80% or higher, more preferably 85% or higher, even more preferably 90% or higher, even more preferably 95% or higher, even more preferably 97.5% or higher, even more preferably 99% or higher, in any case, unless otherwise stated.
The polynucleotides or genes and proteins or enzymes used in the present invention can be prepared by those skilled in the art using conventional techniques.
Hereafter, the present invention will be specifically explained with reference to the following examples. However, the present invention is not limited to these examples. The reagents, solvents and starting materials mentioned without any particular explanations are commercially available or can be obtained from well-known supply sources.
The protein concentrations mentioned in this description were measured by the Bradford method (measurement wavelength: 595 nm).
The following abbreviations are used in the following examples.
The nucleotide sequence of EndoF3 (NZ_CP067018, locus_tag I6H88_03475; WP_034868774.1 for the amino acid sequence) was modified with the D165Q mutation (EndoF3 D165Q amino acid sequence is shown as SEQ ID NO: 1), a codon-optimized sequence thereof for expression in E. coli (SEQ ID NO: 2) was artificially synthesized (ThermoFisher Scientific), and this sequence was amplified by PCR using primers FEndoF3-NtermMBP and REndoF3-NtermMBP (30 cycles of 98° C. for 10 seconds, 60° C. for 5 seconds, and 68° C. for 8 seconds).
Similarly, the maltose binding protein (amino acid sequence is shown as SEQ ID NO: 5) and Factor Xa cleavage sequence ATCGAGGGAAGG (SEQ ID NO: 6; amino acid sequence is IEGR (SEQ ID NO: 7)) were cloned into the pRSF-MBP vector (pRSDuet-1,
The two fragments obtained were cloned by using the In-Fusion kit (TAKARA) so that the Factor Xa sequence should be placed at the C-terminus of MBP and the EndoF3 sequence of which N-terminal signal sequence is removed should be placed downstream thereof E. coli JM109 (TAKARA) cells were transformed with the resulting In-Fusion solution and cultured overnight at 37° C. Colony PCR was performed on the obtained colonies, and pRSFDuet-1-MBP-EndoF3 D165Q (SEQ ID NO: 10) was obtained as a plasmid from a positive clone.
PCR was performed to introduce the target point mutation into pRSFDuet-1-MBP-EndoF3 D165Q. DpnI was added in a volume of 0.001 mL to the fragment obtained by PCR, the reaction was allowed at 37° C. for 1 hour, and the DNA fragment was purified. The obtained fragment was introduced into E. coli JM109 cells by the heat shock method, and then the cells were applied to an LK plate, and incubated overnight at 37° C. to obtain colonies. The colonies were inoculated into the LB medium containing kanamycin (3 mL), and incubated overnight, and plasmid extraction was performed with Wizard Plus SV Minipreps DNA Purification Systems to obtain 0.05 mL of a plasmid DNA solution from the culture.
The above plasmid (0.001 mL) was added to BL21(DE3) competent cells (Novagen, 0.05 mL), and the cells were left standing at 0° C. for 20 minutes, and left standing on an aluminum block at 42° C. for 45 seconds for heat shock treatment. The SOC medium (0.45 mL) warmed to 37° C. was added, and the mixture was incubated at 37° C. for 1 hour. The culture was centrifuged (room temperature, 6000 g, 3 minutes) to precipitate the cells. After removing the supernatant (0.4 mL), the cells were resuspended, inoculated on LB-Agar plates (containing 0.05 mg/mL of kanamycin), and cultured overnight at 37° C. as standing culture.
One colony obtained by the above transformation was added to Superbroth (10 mL, containing Bacto Trypton, (32 g), Bacto Yeast Extract (5 g), NaCl (5 g), 1 M aqueous sodium hydroxide (5 mL), and pure water (950 mL), and containing 0.05 mg/mL of kanamycin), and cultured overnight at 37° C. The culture (2 mL) was added to Superbroth (200 mL, containing 0.05 mg/mL kanamycin) in a 500 mL baffled flask. The cells were cultured at 37° C. for 6 hours, IPTG was added at a final concentration of 1 mM, and the cells were further cultured overnight at 37° C. The culture was centrifuged (4° C., 10000 rpm, 5 minutes), the supernatant was removed, and the cells were collected.
BugBuster (40 mL) and Lysonase (0.04 mL) were added to the cells, and the cells were suspended by pipetting, and the suspension was made homogenous by inversion for 20 minutes. This cell suspension was centrifuged (room temperature, 10000 rpm, 20 minutes), the supernatant was removed, and the precipitates were obtained as the insoluble fraction. To this insoluble fraction, a 0.15% CHAPS/20% acetic acid solution (40 mL) was added, they were mixed by inversion at 37° C. for 2.5 hours, and the mixture was centrifuged (room temperature, 10000 rpm, 10 minutes) to obtain a supernatant. This supernatant was added to Slide-A-Lyzer Dialysis Flask (20 kDa MWCO, Thermo Fischer Scientific) and dialyzed against ultrapure water at 4° C. for 2 days (the outer dialysis solution was changed 3 times with intervals of 4 hours or longer). The dialysis was further continued overnight at 4° C. against 10 mM Tris-HCl buffer (pH 8.0). This solution was subjected to buffer replacement with a 50 mM Tris-HCl buffer, pH7.4 by ultrafiltration (Amicon Ultra 30 kDa) to obtain an EndoF3 mutant enzyme.
The nucleotide sequence of EndoS (CP043530, locus_ag MGAS2221_1552, WP 011285695.1 for the amino acid sequence) was modified with the D233Q mutation (amino acid sequence of EndoS_D233Q is shown as SEQ ID NO: 11), and a codon-optimized sequence thereof for expression in E. coli (SEQ ID NO: 12) was artificially synthesized as two parts, those for the N-terminus portion and the C-terminus portion (ThermoFisher Scientific). By using the above sequence as a template, the N-terminus side sequence and the C-terminus side sequence were amplified by PCR (30 cycles of 98° C. for 10 seconds, 60° C. for 5 seconds, and 68° C. for 8 seconds) using primers FEndoS_D233Q_His_pRSF and RendoS_D233Q_front, and FendoS_D233Q_back and REndoS_D233Q_His_pRSF, respectively. Similarly, pRSFDuet-1 was amplified by PCR using primers FpRSF_InF_His_BamHI and RpRSF_InF_His_NcoI (30 cycles of 98° C. for 10 seconds, 60° C. for 5 seconds, and 68° C. for 8 seconds).
The resulting three fragments were ligated using the In-Fusion kit (TAKARA). The start codon Met of EndoS sequence was removed and the fragment was placed downstream of the His tag. E. coli JM109 cells (TAKARA) were transformed with the resulting In-Fusion solution, and cultured overnight at 37° C. Colony PCR was performed on the obtained colonies, and pRSFDuet-1-EndoS D233Q (SEQ ID NO: 19) was obtained as a plasmid from a positive clone.
The above plasmid (0.001 mL) was added to BL21(DE3) competent cells (0.05 mL, Novagen), and the cells were left standing at 0° C. for 20 minutes, and then left standing on an aluminum block at 42° C. for 45 seconds for heat shock treatment. The SOC medium (0.45 mL) warmed to 37° C. was added, and the mixture was incubated at 37° C. for 1 hour. The culture was centrifuged (room temperature, 6000 g, 3 minutes) to precipitate the cells. After removing the supernatant (0.4 mL), the cells were resuspended, inoculated on LB-Agar plates (containing 0.05 mg/mL of kanamycin), and cultured overnight at 37° C. as standing culture.
One colony obtained by the above transformation was added to Superbroth (10 mL, containing Bacto Trypton, (32 g), Bacto Yeast Extract (5 g), NaCl (5 g), 1 M aqueous sodium hydroxide (5 mL), and pure water (950 mL), and containing 0.05 mg/mL of kanamycin), and cultured overnight at 37° C. The culture (2 mL) was added to Superbroth (200 mL, containing 0.05 mg/mL kanamycin) in a 500 mL baffled flask. The cells were cultured at 37° C. for 6 hours, IPTG was added at a final concentration of 1 mM, and the cells were further cultured overnight at 37° C. The culture was centrifuged (4° C., 10000 rpm, 5 minutes), the supernatant was removed, and the cells were collected.
BugBuster (5 mL) and Lysonase (0.5 mL) were added to the cells, the cells were suspended by pipetting, and the suspension was made homogenous by inversion for 20 minutes. This cell suspension was centrifuged (4° C., 10000 rpm, 20 minutes), and the supernatant was collected. Streptomycin sulfate was added to the supernatant at a final concentration of 1%, and the mixture was left standing overnight at 4° C. This suspension was centrifuged (4° C., 10000 rpm, 20 minutes), and the supernatant was collected. To this supernatant, ammonium sulfate was added to a final concentration of 40%, and the mixture was left standing at 0° C. for 1 hour. This suspension was centrifuged (4° C., 10000 rpm, 20 minutes), and the supernatant was collected. To this supernatant, ammonium sulfate was added to a final concentration of 60%, and the mixture was left standing at 0° C. for 1 hour. This suspension was centrifuged (4° C., 10000 rpm, 20 minutes), and the supernatant was collected. To this supernatant, 1 M Tris-HCl buffer, pH 7.5 was added to a final concentration of 20%, and purification was performed by column chromatography (His Trap HP) to obtain EndoS D233Q (SEQ ID NO: 11).
A schematic diagram of the preparation of antibody-GlcNAc is shown in
By using the EndoF3 mutant enzymes prepared in Example 1 or the EndoS mutant enzyme prepared in Example 2, the antibody-GlcNAc prepared in Example 3, and the payload-attached glycan (the daunorubicin-attached glycan of Example 6 or MMAE-attached glycan of Example 11 described later) were conjugated (see Examples 5 and 6 described later). A schematic diagram of this conjugation is shown as
Column used: AdvanceBio HIC
Column size: 30×4.6 mm
Packing material particle size: 3.5 μm
Mobile phase A: 50 mM Aqueous sodium phosphate, pH 7.4
Mobile phase B: 2 M Aqueous ammonium sulfate, pH 7.0
Mobile phase C: 2-Propanol
The gradient was as follows.
The HPLC chromatogram for the resultant of the conjugation with daunorubicin-attached glycan by the EndoF3 mutant enzyme is shown in
Antibody-GlcNAc remaining ratio (%)=[Peak area value of antibody-GlcNAc]/[(Peak area value of antibody-GlcNAc)+(Peak area value of 1-adduct)+(Peak area value of 2-adduct)]×100
1-Adduct conversion ratio (%)=[Peak area value of 1-adduct]/[(Peak area value of antibody-GlcNAc)+(Peak value area of 1-adduct)+(Peak area value of 2-adduct)]×100
2-Adduct conversion ratio (%)=[Peak area value of 2-adduct]/[(Peak area value of antibody-GlcNAc)+(Peak area value of 1-adduct)+(Peak area value of 2-adduct)]×100
Intact mass analysis of each conjugate was also performed. The obtained MS chromatograms were deconvoluted. The result of conjugation with daunorubicin-attached glycan attained with the EndoF3 mutant enzyme is shown in
The activity of EndoS mutant enzyme was also measured in the same manner as that for the EndoF3 mutant enzyme.
The activities of the mutant enzymes for daunorubicin-attached glycan are shown in the following table.
Among the EndoF3 mutant enzymes, D165Q/M97G showed the lowest remaining ratio. This mutant enzyme is expected to show similarly high activity even for different glycan structures.
The activities of the mutant enzymes for the MMAE-attached glycan are shown in the following table.
The EndoS mutant enzyme showed high conversion ratio.
Although the conversion can also be attained with the EndoS2 mutant enzyme at a certain degree, the EndoF3 mutant enzyme and EndoS mutant enzyme showed higher conversion ratios under the experimental conditions. In addition, it had been expected that the conversion ratio would be reduced in the case of conjugation of glycans to which a payload was added compared with the case of conjugation of glycans having a linker, but contrary to the expectation, higher conversion ratios were obtained in the case of conjugation of glycans with daunorubicin or MMAE.
To a solution of the compound 2 (Fmoc-Val-Cit-PAB-PNP, 0.99 g) synthesized by the method of WO2004/010957 in DMF (50 mL), the compound 1 (daunomycin, 0.72 g) and diisopropylethylamine (0.45 mL) were added, and the mixture was stirred at room temperature for 19 hours. The reaction solution was concentrated under reduced pressure, and the resulting residue was washed with chloroform and filtered. The filtration residue was dissolved in DMF, and the solution was filtered and concentrated to obtain the compound 3 (1.43 g).
To a solution of the compound 3 (1.43 g) in DMF (20 mL), diethylamine (0.5 mL) was added, and the resulting mixture was stirred at room temperature for 1.5 hours. After concentrating the reaction solution under reduced pressure, the resulting residue was washed with ethyl acetate and filtered to obtain a filtration residue (0.90 g). Of the filtration residue obtained, 0.40 g was dissolved in DMF (10 mL). To this solution, the compound 4 (Fmoc-PEG2-NHS ester, 0.38 g) and diisopropylethylamine (0.15 mL) were added, and the resulting mixture was stirred at room temperature for 17 hours. After concentrating the reaction solution under reduced pressure, the resulting residue was purified by gel filtration chromatography (LH-20, chloroform/methanol=1/1) to obtain the compound 5 (0.45 g).
To a solution of the compound 5 (0.45 g) in DMF (15 mL), diethylamine (1.0 mL) was added, and the resulting mixture was stirred at room temperature for 30 minutes. The reaction solution was concentrated under reduced pressure, and the resulting residue was washed with ethyl acetate, and filtered to obtain a filtration residue (0.30 g). Of the filtration residue obtained, 0.15 g was dissolved in DMF (15 mL). To this solution, the compound 6 (DBCO-NHS ester, 0.06 g) and diisopropylethylamine (0.05 mL) were added, and the resulting mixture was stirred at room temperature for 4 hours. After concentrating the reaction solution under reduced pressure, the resulting residue was purified by gel filtration chromatography (LH-20, chloroform/methanol =1/1) to obtain the compound 7 (0.05 g).
To a solution of the compound 8 (a2,6-sialylglycan, 0.10 g) in DMF (12 mL), the compound 9 (azido-PEG2-amineTos-OH, 0.88 g, Tokyo Chemical Industry) and DMT-MM (0.72 g) were added, and the resulting mixture was stirred overnight at 37° C. The reaction solution was purified by gel filtration chromatography (LH-20; methanol). The obtained purified fraction was further purified by silica gel column chromatography (water/acetonitrile) using Wakosil C-18 to obtain the compound 10 (0.11 g).
To a solution of the compound 7 (0.05 g) in DMF (10 mL), the compound 10 (0.02 g) was added, and the resulting mixture was stirred overnight at room temperature. The reaction solution was concentrated under reduced pressure, and the resulting residue was purified by preparative HPLC (water/acetonitrile) to obtain the compound 11 (0.03 g).
To a solution of the compound 11 (0.02 g) in a mixture of DMF/water (1.5 mL/1.5 mL), CDMBI (0.02 g) and triethylamine (0.02 mL) were added, and the resulting mixture was stirred at 0° C. for 30 minutes. The reaction solution was purified by gel filtration column chromatography (LH-20, DMF) to obtain the compound 12 (0.01 g).
To an Eppendorf tube, the mutant enzyme (one of the EndoF3 mutant enzymes prepared in Example 1, 0.003 mg) or the EndoS mutant enzyme prepared in Example 2 (0.003 mg)), the antibody-GlcNAc prepared in Example 3 (0.03 mg), and a solution of the compound 12 in DMSO (40 mg/mL, 0.001 mL) were added, and a 50 mM Tris-HCl buffer, pH 7.4 was further added to the mixture to adjust the antibody-GlcNAc concentration to 5.0 mg/mL. The reaction solution was incubated at 37° C. for 3 hours. The reaction solution was subjected to buffer replacement with a 50 mM phosphate buffer, pH7.0 by ultrafiltration (Amicon Ultra 100 kDa), and the obtained compound 13 was analyzed (
A solution A was prepared by adding HOBt (0.11 g) and diisopropylethylamine (0.08 mL) to DMF (10 mL). The compound 14 (0.22 g, Asta Tech, Inc.) and the compound 2 (0.22 g) were dissolved in this solution A (6.3 mL). This reaction solution was stirred at room temperature for 3 hours. The reaction solution was concentrated under reduced pressure, and the resulting residue was purified by reprecipitation (chloroform/ethyl acetate) to obtain the compound 15 (0.31 g).
To a solution of the compound 15 (0.31 g) in DMF (4.0 mL), diethylamine (1.0 mL) was added, and the resulting mixture was stirred overnight at room temperature. The reaction solution was concentrated under reduced pressure, and the resulting residue was purified by reprecipitation (chloroform/ethyl acetate) to obtain a yellow solid (0.17 g). Of the solid obtained, 0.11 g was dissolved in the solution A described above (2.5 mL), the compound 4 (0.11 g) was added, and the resulting mixture was stirred at 30° C. for 3 hours. The reaction solution was concentrated under reduced pressure, and the resulting residue was purified by reprecipitation (DMF/ethyl acetate) to obtain the compound 16 (0.11 g).
A solution B was prepared with DMF (10 mL), HOAt (0.11 g), and diisopropylethylamine (0.08 mL). The compound 9 (0.09 g) and the compound 6 (0.03 g) were dissolved in this solution B (2.5 mL). This reaction solution was stirred at 30° C. for 3 hours. The reaction solution was concentrated under reduced pressure, and the resulting residue was purified by reprecipitation (chloroform/methanol/ether) to obtain the compound 17 (0.11 g).
To a solution of the compound 17 (0.07 g) in DMF (1.5 mL), the compound 10 (0.02 g) was added, and the resulting mixture was stirred at 30° C. for 3 hours. The reaction solution was concentrated under reduced pressure, and purified by preparative HPLC (methanol/water) to obtain the compound 18 (0.01 g).
To a solution of the compound 18 (0.004 g) in a mixture of DMF/water (2.0 mL/2.0 mL), CDMBI (0.01 g) and triethylamine (0.1 mL) were added, and the resulting mixture was stirred at 0° C. for 2 hours. This reaction solution was purified by gel filtration chromatography (LH-20, DMF) to obtain the compound 19 (0.005 g).
To an Eppendorf tube, the antibody-GlcNAc obtained in Example 2 (0.03 mg), one of the EndoF3 mutant enzymes prepared in Example 1 (0.003 mg) or EndoS mutant enzyme prepared in Example 2 (0.003 mg), and a solution of the compound 19 in DMSO (40 mg/mL, 0.002 mL) were added, and a 50 mM Tris-HCl buffer, pH 7.4 was further added to the mixture to adjust the antibody-GlcNAc concentration to 5.0 mg/mL. The reaction solution was incubated at 37° C. for 1.5 hours. The reaction solution was subjected to buffer replacement with a 50 mM phosphate buffer, pH 7.0 by ultrafiltration (Amicon Ultra 100 kDa), and the compound 20 was analyzed (
To a solution of the compound 21 (0.1 g) prepared from maytansinol as the starting material according to the descriptions of WO2014/145090 in a mixture of acetonitrile/water (6.0 mL/2.0 mL), the compound 22 (0.3 g) prepared according to the descriptions of WO2014/145090 and 1 M aqueous sodium hydrogencarbonate (0.5 mL) were added, and the resulting mixture was stirred at room temperature for 23 hours. This reaction solution was extracted with ethyl acetate, and the organic layer was dried over magnesium sulfate. The organic layer was concentrated under reduced pressure, and the resulting residue was purified by silica gel column chromatography (ethyl acetate/methanol) to obtain the compound 23 (0.08 g).
To a solution of the compound 23 (0.04 g) in a mixture of acetonitrile/water (3.0 mL/1.0 mL), TFA (1.0 mL) was added, and the resulting mixture was stirred at room temperature for 30 minutes. To this reaction solution, saturated aqueous sodium hydrogencarbonate was added, the mixture was extracted with a mixture of chloroform/methanol (7/1), and the organic layer was dried over magnesium sulfate. The organic layer was concentrated under reduced pressure to obtain the compound 24 (0.04 g).
To a solution of the compound 25 (0.07 g, MedChemExpress) in DMF (5.0 mL), the compound 4 (0.07 g) and diisopropylethylamine (0.04 mL) were added, and the resulting mixture was stirred at room temperature for 3 hours. This reaction solution was concentrated under reduced pressure, and the resulting residue was purified by reprecipitation (DMF/ether) to obtain the compound 26 (0.07 g).
To a solution of the compound 26 (0.07 g) in DMF (5.0 mL), diethylamine (0.5 mL) was added, and the resulting mixture was stirred at room temperature for 1 hour. The reaction solution was concentrated under reduced pressure, the resulting residue was dissolved in DMF (5.0 mL), DBCO-NHS (0.04 g) and diisopropylethylamine (0.04 mL) were added, and the resulting mixture was stirred overnight at room temperature. The reaction solution was concentrated under reduced pressure, and the resulting residue was purified by reprecipitation (DMF/ether) to obtain the compound 27 (0.07 g).
To a solution of the compound 27 (0.07 g) in DMF (5.0 mL), bis(4-nitrophenyl) carbonate (0.03 g) and diisopropylethylamine (0.04 mL) were added, and the resulting mixture was stirred at room temperature for 3 hours. The reaction solution was concentrated under reduced pressure, and the resulting residue was purified by reprecipitation (DMF/ether) to obtain the compound 28 (0.07 g).
To a solution of the compound 24 (0.008 g) in DMF (0.9 mL), the compound 28 (0.01 g) and triethylamine (0.008 mL) were added, and the resulting mixture was stirred at room temperature for 3 hours. The reaction solution was concentrated under reduced pressure, and the resulting residue was purified by preparative HPLC (acetonitrile/water) to obtain the compound 29 (0.011 g).
To a solution of the compound 29 (0.008 g) in DMF (1.6 mL), the compound 10 (0.002 g) was added, and the resulting mixture was stirred overnight at 50° C. The reaction solution was purified by gel filtration chromatography (LH-20, DMF) to obtain the compound 30 (0.004 g).
To a solution of the compound 30 (0.002 g) in a mixture of DMF/water (0.5 mL/0.5 mL), CDMBI (0.04 g) and triethylamine (0.002 mL) were added, and the resulting mixture was stirred at 0° C. for 1 hour. This reaction solution was purified by gel filtration chromatography (LH-20, DMF) to obtain the compound 31 (0.003 g).
To an Eppendorf tube, the antibody-GlcNAc obtained in Example 2 (0.03 mg), the EndoF3 mutant enzymes prepared in Example 1 (0.003 mg), or EndoS mutant enzyme prepared in Example 2 (0.003 mg), or WP1328 enzyme prepared in Example 12 (0.003 mg), and a solution of the compound 31 in DMSO (40 mg/mL, 0.002 mL) were added, and a 50 mM Tris-HCl buffer, pH 7.4 was further added to the mixture to adjust the antibody-GlcNAc concentration to 5.0 mg/mL. The reaction solution was incubated at 30° C. or 37° C. for 1.5 to 3.0 hours. The reaction solution was subjected to buffer replacement with a 50 mM phosphate buffer, pH 7.0 by ultrafiltration (Amicon Ultra 100 kDa), and the compound 32 was analyzed (
To a solution of the compound 33 (0.07 g, MedChemExpress) in DMF (5.0 mL), the compound 2 (0.04 g), diisopropylethylamine (0.04 mL), and HOBt (0.02 g) were added, and the resulting mixture was stirred at 30° C. for 3 hours. The reaction solution was concentrated under reduced pressure, and the resulting residue was purified by reprecipitation (DMF/ether). To the resulting purple solid, DMF (5.0 mL) and diethylamine (0.1 mL) were added, and the resulting mixture was stirred at room temperature for 1 hour. The reaction solution was concentrated under reduced pressure, and the resulting residue was purified by reprecipitation (DMF/ether) and further purified by gel filtration chromatography (LH-20, DMF) to obtain the compound 34 (0.09 g).
To a solution of the compound 34 (0.09 g) in DMF (5.0 mL), the compound 4 (0.03 g), diisopropylethylamine (0.02 mL) and HOBt (0.01 g) were added, and the resulting mixture was stirred at 30° C. for 3 hours. The reaction solution was concentrated under reduced pressure, and the resulting residue was purified by reprecipitation (DMF/ether). To the resulting purple solid, DMF (5.0 mL) and diethylamine (0.1 mL) were added, and the resulting mixture was stirred at room temperature for 1 hour. The reaction solution was concentrated under reduced pressure, and the resulting residue was purified by reprecipitation (DMF/ether) and further purified by gel filtration chromatography (LH-20, DMF) to obtain the compound 35 (0.07 g).
To a solution of the compound 35 (0.07 g) in DMF (5.0 mL), the compound 6 (DBCO-NHS ester, 0.03 g), diisopropylethylamine (0.01 mL), and HOBt (0.01 g) were added, and the resulting mixture was stirred at 30° C. for 1 hour. The reaction solution was concentrated under reduced pressure, and the resulting residue was purified by reprecipitation (DMF/ether) to obtain the compound 36 (0.05 g).
To a solution of the compound 36 (0.009 g) in DMF (3.0 mL), the compound 10 (0.005 g) was added, and the resulting mixture was stirred at 40° C. for 2 hours. The reaction solution was concentrated under reduced pressure, and the resulting residue was purified by preparative HPLC (acetonitrile/water) to obtain the compound 37 (0.003 g).
To a solution of the compound 37 (0.003 g) in a mixture of DMF/water (1.0 mL/1.0 mL), CDMBI (0.007 g) and triethylamine (0.001 mL) were added, and the resulting mixture was stirred at 0° C. for 30 minutes. This reaction solution was purified by gel filtration chromatography (LH-20, DMF) to obtain the compound 38 (0.003 g).
To an Eppendorf tube, the antibody-GlcNAc obtained in Example 2 (0.03 mg), one of the EndoF3 mutant enzymes prepared in Example 1 (0.003 mg), EndoS mutant enzyme prepared in Example 2 (0.003 mg), or WP1328 enzyme prepared in Example 12 (0.003 mg), and a solution of the compound 38 in DMSO (40 mg/mL, 0.002 mL) were added, and a 50 mM Tris-HCl buffer, pH 7.4 was further added to the mixture to adjust the antibody-GlcNAc concentration to 5.0 mg/mL. The reaction solution was incubated at 30° C. or 37° C. for 1.5 to 3.0 hours. The reaction solution was subjected to buffer replacement with a 50 mM phosphate buffer, pH 7.0 by ultrafiltration (Amicon Ultra 100 kDa), and the compound 39 was analyzed (
To a solution of the compound 8 (0.05 g) in DMF (6.0 mL), the compound 40 (azido-PEG3-amine, 0.25 g, Combi-Blocks) and DMT-MM (0.34 g) were added, and the resulting mixture was stirred overnight at 37° C. The reaction solution was purified by gel filtration chromatography (LH-20, methanol). The obtained purified fraction was further purified by silica gel column chromatography (water/acetonitrile) using Wakosil C-18 to obtain the compound 41 (0.02 g).
To a solution of the compound 17 (0.02 g) in DMF (3.0 mL), the compound 41 (0.006 g) was added, and the resulting mixture was stirred at room temperature for 22 hours. The reaction solution was concentrated under reduced pressure, and the resulting residue was purified by preparative HPLC (acetonitrile/water) to obtain the compound 42 (0.004 g).
To a solution of the compound 42 (0.004 g) in a mixture of DMF/water (1.0 mL/1.0 mL), CDMBI (0.008 g) and triethylamine (0.001 mL) were added, and the resulting mixture was stirred at 0° C. for 30 minutes. This reaction solution was purified by gel filtration chromatography (LH-20, DMF) to obtain the compound 43 (0.004 g).
To an Eppendorf tube, the antibody-GlcNAc obtained in Example 2 (0.03 mg), one of the EndoF3 mutant enzymes prepared in Example 1 (0.003 mg), EndoS mutant enzyme prepared in Example 2 (0.003 mg), or WP1328 enzyme prepared in Example 12 (0.003 mg), and a solution of the compound 43 in DMSO (40 mg/mL, 0.002 mL) were added, and a 50 mM Tris-HCl buffer, pH 7.4 was further added to the mixture to adjust the antibody-GlcNAc concentration to 5.0 mg/mL. The reaction solution was incubated at 30° C. or 37° C. for 1.5 to 3.0 hours. The reaction solution was subjected to buffer replacement with a 50 mM phosphate buffer, pH 7.0 by ultrafiltration (Amicon Ultra 100 kDa), and the compound 44 was analyzed (
To a solution of the compound 8 (0.05 g) in DMF (6 mL), the compound 45 (azido-PEG4-amine, 0.30 g, Combi-Blocks) and DMT-MM (0.34 g) were added, and the resulting mixture was stirred overnight at 37° C. The reaction solution was purified by gel filtration chromatography (LH-20, methanol). The obtained purified fraction was further purified by silica gel column chromatography (water/acetonitrile) using Wakosil C-18 to obtain the compound 46 (0.02 g).
To a solution of the compound 17 (0.02 g) in DMF (3.0 mL), the compound 41 (0.006 g) was added, and the resulting mixture was stirred at room temperature for 17 hours. The reaction solution was concentrated under reduced pressure, and the resulting residue was purified by preparative HPLC (acetonitrile/water) to obtain the compound 47 (0.007 g).
To a solution of the compound 47 (0.004 g) in a mixture of DMF/water (0.5 mL/0.5 mL), CDMBI (0.008 g) and triethylamine (0.001 mL) were added, and the resulting mixture was stirred at 0° C. for 30 minutes. This reaction solution was purified by gel filtration chromatography (LH-20, DMF) to obtain the compound 48 (0.004 g).
To an Eppendorf tube, the antibody-GlcNAc obtained in Example 2 (0.03 mg), one of the EndoF3 mutant enzymes prepared in Example 1 (0.003 mg), EndoS mutant enzyme prepared in Example 2 (0.003 mg), or WP1328 enzyme prepared in Example 12 (0.003 mg), and a solution of the compound 48 in DMSO (40 mg/mL, 0.002 mL) were added, and a 50 mM Tris-HCl buffer, pH 7.4 was further added to the mixture to adjust the antibody-GlcNAc concentration to 5.0 mg/mL. The reaction solution was incubated at 30° C. or 37° C. for 1.5 to 3.0 hours. The reaction solution was subjected to buffer replacement with a 50 mM phosphate buffer, pH 7.0 by ultrafiltration (Amicon Ultra 100 kDa), and the compound 49 was analyzed (Figs. E and G).
To a solution of the compound 50 (Boc-GGFG-OH, 0.2 g, Broadpharm) in DMF (2.0 mL), the compound 51 (4-aminobenzyl alcohol, 0.1 g), diisopropylethylamine (0.3 mL), and HATU (0.2 g, Watanabe Chemical) were added, and the resulting mixture was stirred at room temperature for 4 hours. To the reaction solution, 0.1 M hydrochloric acid was added, and the resulting mixture was extracted with a mixed solvent of chloroform/methanol (9/1). The organic layer was concentrated under reduced pressure to obtain the compound 52 (0.3 g).
To a solution of the compound 52 (0.1 g) in DMF (5.0 mL), bis(4-nitrophenyl) carbonate (0.2 g) and diisopropylethylamine (0.5 mL) were added, and the resulting mixture was stirred at room temperature for 3 hours. The reaction solution was concentrated under reduced pressure to obtain the compound 53 (0.1 g).
To a solution of the compound 14 (0.1 g) in DMF (5.0 mL), the compound 53 (0.1 g), diisopropylethylamine (0.1 mL), and HOBt (0.2 g) were added, and the resulting mixture was stirred at room temperature for 48 hours. The reaction solution was concentrated under reduced pressure, and the resulting residue was purified by silica gel column chromatography (ODS-A, acetonitrile/water/formic acid) to obtain the compound 54 (0.02 g).
To a solution of the compound 54 (0.02 g) in dichloromethane (5.0 mL), TFA (0.3 mL) was added, and the resulting mixture was stirred at room temperature for 3 hours. Diisopropylethylamine (0.6 mL) and the compound 4 (0.02 g) were further added, and the resulting mixture was stirred at room temperature for 18 hours. The reaction solution was concentrated under reduced pressure, and the resulting residue was purified by preparative HPLC (acetonitrile/water/formic acid) to obtain the compound 55 (0.006 mg).
To a solution of the compound 55 (0.006 g) in DMF (1.0 mL), diethylamine (0.1 mL) was added, and the resulting mixture was stirred at room temperature for 1 hour. The reaction solution was concentrated under reduced pressure, to the resulting residue, DMF (1.0 mL) and the compound 6 (DBCO-NHS, 0.003 mg) were added, and the resulting mixture was stirred at room temperature for 17 hours. The reaction solution was purified by gel filtration chromatography (LH-20, DMF) to obtain the compound 56 (0.009 mg).
To a solution of the compound 56 (0.001 g) in DMF (0.5 mL), the compound 10 (0.007 g) was added, and the resulting mixture was stirred at room temperature for 24 hours. The reaction solution was concentrated under reduced pressure, and the resulting residue was purified by preparative HPLC (acetonitrile/water/formic acid) to obtain the compound 57 (0.003 g).
To a solution of the compound 57 (0.003 g) in a mixture of DMF/water (0.5 mL/0.5 mL), CDMBI (0.006 g) and triethylamine (0.002 mL) were added, and the resulting mixture was stirred at 0° C. for 30 minutes. This reaction solution was purified by gel filtration chromatography (LH-20, DMF) to obtain the compound 58 (0.001 g).
To an Eppendorf tube, the antibody-GlcNAc obtained in Example 2 (0.03 mg), one of the EndoF3 mutant enzymes prepared in Example 1 (0.003 mg), EndoS mutant enzyme prepared in Example 2 (0.003 mg), or WP1328 enzyme prepared in Example 12 (0.003 mg), and a solution of the compound 58 in DMSO (40 mg/mL, 0.002 mL) were added, and a 50 mM Tris-HCl buffer, pH 7.4 was further added to the mixture to adjust the antibody-GlcNAc concentration to 5.0 mg/mL. The reaction solution was incubated at 30° C. or 37° C. for 1.5 to 3.0 hours. The reaction solution was subjected to buffer replacement with a 50 mM phosphate buffer, pH 7.0 by ultrafiltration (Amicon Ultra 100 kDa), and the compound 59 was analyzed (
(1) Preparation of pRSFDuet-1-MBP-WP1328
A codon-optimized sequence (SEQ ID NO: 20) of the nucleotide sequence of WP1328 (amino acid sequence is WP 069215570.1) for E. coli expression was artificially synthesized (Genewiz), and this sequence was amplified by PCR using primers WP_1328_f and WP_1328-r (30 cycles of 98° C. for 10 seconds, 60° C. for 5 seconds, and 68° C. for 30 seconds).
Similarly, a vector obtained by cloning the maltose binding protein (amino acid sequence is that of SEQ ID NO: 5) and Factor Xa cleavage sequence ATCGAGGGAAGG (SEQ ID NO: 6; amino acid sequence is IEGR (SEQ ID NO: 7)) into the pRSF-1-MBP vector (pRSDuet-1,
The two fragments obtained were cloned by using the In-Fusion kit (TAKARA) so that the Factor Xa sequence was placed at the C-terminus of MBP and the WP1328 sequence of which N-terminal signal sequence was removed was placed downstream thereof. E. coli JM109 (TAKARA) cells were transformed with the resulting In-Fusion solution and cultured overnight at 37° C. Colony PCR was performed on the obtained colonies, and a plasmid pRSFDuet-1-MBP-WP1328 (SEQ ID NO: 23) was obtained from a positive clone.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2021-061693 | Mar 2021 | JP | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/JP2022/016786 | 3/31/2022 | WO |
