WO2017010559A1 - 新規EndoS変異酵素 - Google Patents
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- WO2017010559A1 WO2017010559A1 PCT/JP2016/070921 JP2016070921W WO2017010559A1 WO 2017010559 A1 WO2017010559 A1 WO 2017010559A1 JP 2016070921 W JP2016070921 W JP 2016070921W WO 2017010559 A1 WO2017010559 A1 WO 2017010559A1
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- C12Y302/01096—Mannosyl-glycoprotein endo-beta-N-acetylglucosaminidase (3.2.1.96)
Definitions
- the present invention relates to a novel mutant of endo- ⁇ -N-acetylglucosaminidase S (EndoS), a gene encoding the mutant, a recombinant plasmid, a transformant transformed with the plasmid, and an antibody using the mutant
- EndoS endo- ⁇ -N-acetylglucosaminidase S
- the present invention relates to a sugar chain modification method.
- glycoproteins are widely present in animal and plant tissues, cell membranes and walls of eukaryotic microorganisms.
- sugar chains of glycoproteins play an important role in the mechanisms such as cell differentiation, canceration, and intercellular recognition. Research on the correlation is ongoing.
- glycoprotein is translated as a protein consisting of a uniform amino acid sequence, and then a sugar chain is added by post-translational modification and produced in a culture solution. Since this post-translational modification is not uniquely determined according to the amino acid sequence, the structure of the added sugar chain varies even with the same protein expressed in the same culture system.
- the antibody is a glycoprotein molecule having an N-linked sugar chain (N297-linked sugar chain) bound to the 297th Asn side chain located in the Fc region of the heavy chain molecule.
- N297-linked sugar chain N-linked sugar chain
- Antibodies are important molecules in basic research and medical fields, and research and development as antibody drugs are being actively promoted, and various effects of sugar chains are being clarified (Non-Patent Document 1: JNArnold). et al., nu Annu. Rev. Immunol, 2007, 25, 21050).
- Currently used medical antibodies are IgG class molecules, and such antibodies are generally produced using cultured animal cells represented by CHO cells and NS0 cells.
- the N297-linked sugar chain of the antibody produced in 1) is a biantennary complex-type sugar chain, but heterogeneous ones are obtained in core fucose, terminal sialic groups and galactosyl groups, and bisecting GlcNAc (Non-patent Document 2: R. Jerfferis) Biotechnol. Prog., 2005, 21-11-6). It has been clarified that the N297-linked sugar chain of an antibody greatly affects the effector activity including ADCC (Antibody-Dependent Cell-Mediated Cytotoxicity) and CDC (Non-patent Document 3: Nimmerjahn, Nat. Rev. Immunol. 2008) , 8, 34- 47, Non-Patent Document 4: Jefferis Nat. Rev.
- Non-Patent Document 5 D. Bumbaca, AAPSJ, 2012, 14, 3
- an antibody in which the non-reducing end of the N297-linked sugar chain is 2,6-sialylated is the main medicinal component in IVIG
- Non-patent Document 6 Wang, ACS Chem. Biol. 2012, 7, 110- 122
- the heterogeneity of N297-linked sugar chains is thought to have a significant effect on the properties and quality as active ingredients. There is no denying the possibility that mixing will greatly change the properties of the final product.
- endo- ⁇ -N-acetylglucosaminidase A group of enzyme families called endo- ⁇ -N-acetylglucosaminidase is used particularly for the purpose of converting N-type sugar chains.
- the properties of this enzyme are required to have 1) the ability to perform a hydrolysis reaction on complex sugar chains as substrate specificity, and 2) the ability to perform a transglycosylation reaction on a specific structure.
- Endo- ⁇ -N-acetylglucosaminidase has been isolated from various species, and wild type and mutant can be used properly depending on the type of sugar chain used as a substrate.
- EndoA Arthrobacterhroprotophormiae-derived enzyme
- Non-patent document 9 Fan, J Biol Chem. 2012 Mar 30; 287 (14): 11272-81
- EndoM Mescoremhiemalis-derived enzyme
- Non-patent document 10 Umekawa, Biochem Biophys Res Commun. 1994 Aug 30; 203 (1): 244-52
- EndoH Non-patent document 11: Robbins, J Biol Chem. 1984 Jun 25; 259 (12): 7577-83
- EndoF2 Fevobacterium Meningosepticum-derived enzyme
- Non-patent document 13 Collin, JBC 2004, 279-21
- EndoS Streptococcus pygenes-derived enzyme
- Non-patent document 14 Collin, EMBO J. 2001 Jun 15; 20 (12): 3046-55
- EndoS is the only enzyme that has been confirmed to have both hydrolytic activity and transglycosylation activity using a complex N297-linked sugar chain as a substrate
- Non-patent Document 15 Allhorn, BMC Microbiol. 2008, 8, 3.
- Non-Patent Document 16 Goodfellow, J. Am. Chem. Soc. 2012,134, 8030- 8033).
- EndoS hydrolysis is performed in EndoS D233Q in which a mutation that substitutes Gln for the 233rd Asp, which is the nucleophilic catalytic residue, of the two catalytic residues, the 233rd Asp and the 235th Glu, is introduced. It is known that the activity is suppressed to some extent.
- This mutant is known to selectively undergo a transglycosylation reaction under conditions in which a large amount of an intermediate in which the reducing end of the sugar chain is oxazolineated exists in the reaction system (Non-patent Document 17: Huang) , J. Am. Chem. Soc.
- Non-Patent Document 18 B. Trastoy et al., PNAS (2014) vol 111, No. 18, pp6714 -6719)).
- EndoS2 derived from serotype M49 (Non-patent Document 19: Sjogren, Biochem J. 2013 Oct 1; 455 (1): 107-18, sometimes referred to as EndoS49) has been reported to have similar substrate properties.
- the present invention is an endo- ⁇ -N-acetylglucosaminidase having specificity for the sugar chain of the Fc region of IgG, compared with EndoS D233Q, which is known as a mutant in which the hydrolysis activity of EndoS is suppressed. It is an object of the present invention to provide a novel mutant enzyme that has a reduced hydrolysis activity and retains a certain transglycosylation activity.
- the present inventors designed and produced a large number of mutants with reference to the amino acid sequence information and three-dimensional structure of EndoS D233Q.
- the present inventors have completed the present invention by finding that a novel mutant enzyme having an additional mutation has the desired activity and further researching it.
- the present invention provides the following inventions. (1) In the amino acid sequence of amino acid numbers 37 to 995 of SEQ ID NO: 2, the 122nd (H122), 184th (P184), 279th (D279), 282nd (Y282), 303th (Q303), 348th ( Y348), having an additional mutation at a position containing at least one amino acid selected from the group consisting of 350th (E350), 402th (Y402), 405th (D405), and 406th (R406), and EndoS mutant enzyme characterized by exhibiting reduced hydrolytic activity as compared with the activity of EndoS D233Q as an activity for N-linked sugar chain (N297-linked sugar chain) that binds to 297th Asn of IgG .
- the additional mutation is a site consisting of 1 to 4 amino acids selected from the group consisting of H122, P184, D279, Y282, Q303, Y348, E350, Y402, D405, and R406, The EndoS mutant enzyme of (1).
- the amino acid after mutation of H122 is Gly, Cys, Thr, Ser, Val, Pro, Ala, Glu, Asp, Leu, Ile, Pro, Met or Phe;
- the amino acid after mutation of P184 is Gly, Cys, Thr, Ser, Val, Pro, Ala, Gln or Asn;
- the amino acid after mutation of D279 is Gly, Cys, Thr, Ser, Val, Pro, Ala or Gln;
- the amino acid after mutation of Y282 is Arg, Lys or His;
- the amino acid after mutation of Q303 is Met, Pro or Leu;
- the amino acid after mutation of Y348 is His or Trp;
- the mutated amino acid of E350 is Lys, Arg, His, Tyr, Gln, Asn, Gly, Cys, Thr, Ser, Val, Pro or Ala;
- H122 is Ala (H122A) or Phe (H122F); P184 is Gln (P184Q); D279 is Ser (D279S) or Gln (D279Q); Y282 is Arg (Y282R); Q303 is Leu (Q303L); Y348 is His (Y348H); E350 is Ala (E350A), Asn (E350N), Asp (E350D) or Gln (E350Q); Y402 is Phe (Y402F) or Trp (Y402W); D405 is Ala (D405A); and R406 is Gln (R406Q).
- the EndoS mutant enzyme according to (5) which contains at least one mutation selected from the group consisting of Q303L, E350A, E350N, E350D, E350Q, and D405A as an additional mutation.
- Additional mutations are Q303L, E350A, E350N, E350D, E350Q, D405A, H122A / Q303L, H122F / Q303L, P184Q / Q303L, D279Q / Q303L, D279S / Q303L, Y282R / Q303L, Q303L / Y348H, Q303L / E350A, Q303L / E350N, Q303L / E350D, Q303L / E350Q, Q303L / 402, Q303L / 402 Y402W, Q303L / D405A, Q303L / R406Q, H122A / E350A, H122F / E350A, P184Q / /
- Additional mutations are Q303L, E350A, E350N, E350D, E350Q, D405A, Q303L / E350A, Q303L / E350N, Q303L / E350D, Q303L / E350Q, Q303L / D405A, E350A / D405A, E350N / D405A, A350D / D405 Or EndoS mutant enzyme according to (7), which is E350Q / D405A.
- an N297-linked sugar chain is oxazolated with a molecule containing an Fc region of IgG having a core GlcNAc optionally having fucose added thereto.
- a method for producing an Fc region-containing molecule remodeled with a sugar chain characterized by comprising: (16) The production method of (15), wherein the Fc region-containing molecule is an IgG monoclonal antibody.
- the novel EndoS mutant enzyme of the present invention has a hydrolytic activity further reduced as compared with EndoS D233Q, which is known as a mutant having a reduced hydrolytic activity, and retains a certain level of transglycosylation activity.
- FIG. 1 is a diagram showing a chemical structural formula (left) of SG-Oxa that can be used as a sugar chain donor in sugar chain remodeling and a display (right) that symbolizes the sugar chain.
- FIG. 2 is a schematic diagram of a hydrolysis reaction of an antibody against an N297-linked sugar chain using EndoS or an EndoS mutant enzyme.
- FIG. 3 is a diagram schematically showing a glycosyltransferase reaction using EndoS or EndoS mutant enzyme.
- FIG. 1 is a diagram showing a chemical structural formula (left) of SG-Oxa that can be used as a sugar chain donor in sugar chain remodeling and a display (right) that symbolizes the sugar chain.
- FIG. 2 is a schematic diagram of a hydrolysis reaction of an antibody against an N297-linked sugar chain using EndoS or an EndoS mutant enzyme.
- FIG. 3 is a diagram schematically showing a glycosyltransferase reaction using EndoS or EndoS mutant enzyme.
- FIG. 4 shows various EndoS mutant enzymes (EndoS D233Q, EndoS D233Q / Q303L, EndoS D233Q / Q303L / E350Q, EndoS D233Q / Q303L / D405A, EndoS D233Q / E350Q, E350Q, E350Q, E350Q, E350Q2E33Q / E350Q, E350Q, E350Q, E350Q2 It is a figure which shows alignment. The amino acid number is assigned based on the entire coding region including the signal sequence (1 to 36).
- FIG. 4-1 shows an alignment of amino acid numbers 37 to 176 of each mutant enzyme.
- FIG. 4-2 is a continuation of FIG.
- FIG. 4-1 shows an alignment of amino acid numbers 177 to 316 of each mutant enzyme.
- FIG. 4-3 is a continuation of FIG. 4-2 and shows an alignment of amino acid numbers 317 to 456 of each mutant enzyme.
- FIG. 4-4 is a continuation of FIG. 4-3 and shows an alignment of amino acid numbers 467 to 596 of each mutant enzyme.
- FIG. 4-5 is a continuation of FIG. 4-4 and shows an alignment of amino acid numbers 597 to 736 of each mutant enzyme.
- FIG. 4-6 is a continuation of FIG. 4-5 and shows an alignment of amino acid numbers 737 to 876 of each mutant enzyme.
- FIG. 4-7 is a continuation of FIG. 4-6 and shows an alignment of amino acid numbers 877 to 995 of each mutant enzyme.
- the notation of an amino acid contained in a molecule is in accordance with the convention of this field, and in the case of showing a mutation site, a one-letter code of a wild-type amino acid (or nucleic acid) and its number (for example, the 233rd Asp) If so, it is represented by “D233”).
- the mutation the one-letter code of the wild-type amino acid (or nucleic acid), its number and the one-letter code of the amino acid (or nucleic acid) after the mutation (for example, the mutation in which the 233rd Asp is substituted with Gln is “D233Q”. ).
- a specific mutant having a mutation is represented by a molecular name and a mutation (for example, a mutant in which the 233rd Asp of EndoS is replaced with Gln is “EndoS D233Q”).
- EndoS D233Q the mutant having an additional mutation in which the 303rd Gln is replaced with Leu is represented by “EndoS D233Q / Q303L”).
- N297-linked sugar chain means a sugar chain that binds to the 297th Asn side chain of an IgG heavy chain.
- IgG is fragmented, in the peptide fragment containing Asn, even a sugar chain that binds to the corresponding Asn is included in the N297-linked sugar chain.
- the N297-linked sugar chain in IgG produced in animals or the like has a basic structure consisting of the structure of the following formula, and Gal or sialic acid may be further added to the non-reducing end.
- N297-linked sugar chains of IgG produced by cells include a sugar chain structure in which a sugar chain is further bound at the reducing terminal GlcNAc (core GlcNAc), non-reducing terminal, branched sugar, etc.
- core GlcNAc reducing terminal GlcNAc
- the 6-position of the core GlcNAc may be modified with a structure having a core fucose in which fucose is ⁇ 1,6-linked ((Fuc ⁇ 1,6) GlcNAc).
- a sugar chain containing Gal or sialic acid may be further bound.
- the “sugar chain donor” is a sugar chain-containing molecule having an oxazolineated GlcNAc at the reducing end of the sugar chain, and molecules having various sugar chain structures can be used.
- a human-type sugar chain that has few problems when applied to humans or a sugar-chain donor having a human-compatible sugar chain.
- Such a sugar chain is a sugar chain that is known not to show antigenicity in the human body.
- a high mannose type for an N-linked sugar chain, a high mannose type, a hybrid (hybrid) type, a complex (complex) type, etc. It has been known. These three have a common basic structure.
- the high mannose type is a sugar chain having a mannose-rich structure in which a plurality of mannoses are continuous with two branched chains (1-3 chain, 1-6 chain) branched from mannose ( ⁇ -mannose) located near the reducing end. It is.
- Hybrid molding is a structure in which one of two branched chains (1-3 chain, 1-6 chain) branched from mannose ( ⁇ -mannose) located near the reducing end has GlcNAc. .
- the complex type has a structure with GlcNAc in two branched chains (1-3 chain, 1-6 chain) branched from mannose ( ⁇ -mannose) located near the reducing end, with or without galactose, sial And a variety of structures including these bond isomerisms and positional isomerism. (See Table 1).
- a typical sugar chain donor is SG-Oxa produced in Example 2 (FIG. 1).
- an “acceptor molecule” is a molecule containing a sugar structure having GlcNAc at the non-reducing end.
- a sugar chain donor molecule By reacting with a sugar chain donor molecule in the presence of EndoS or its mutant enzyme, the non-reducing end
- the oxazoline ring of the sugar chain donor molecule reacts with the 4-position of GlcNAc to form a chitobiose structure.
- a typical acceptor molecule is IgG or Fc fragment thereof derived from a monoclonal antibody and having an N297-linked sugar chain consisting only of core GlcNAc to which core Fuc may be bound.
- the core GlcNAc may or may not be bound to the core Fuc depending on the antibody from which it is derived or its production method.
- the origin of the acceptor molecule is various monoclonal antibodies or Fc region-containing molecules (Fc, CLCH combined only with a constant region in which the variable region is deleted from the heavy chain and CL consisting only of the constant region of the light chain, etc.)
- Fc Fc region-containing molecules
- (Fuc ⁇ 1,6) -GlcNAc-IgG, (Fuc ⁇ 1,6) -GlcNAc-Fc, (Fuc ⁇ 1,6) -GlcNAc-CLCH, and the like can be mentioned.
- Mention may be made of (Fuc ⁇ 1,6) -GlcNAc-Trastuzumab prepared in Example 3.
- EndoS is a kind of endo- ⁇ -N-acetylglucosaminidase (ENGase) derived from Streptococcus pyogenes, and amino acid numbers 37 to 995 of SEQ ID NO: 1 (the 1st to 36th amino acids are signal sequences).
- EndoS specifically recognizes the N297-linked sugar chain at the Fc site derived from IgG, and has both hydrolytic activity and transglycosylation activity.
- the hydrolysis activity of EndoS is the activity of specifically hydrolyzing the ⁇ 1,4 glycosidic bond contained in the core chitobiose of the N297-linked sugar chain having the above basic structure (in this specification, unless otherwise specified, “hydrolysis activity”). "Means this activity.
- a schematic diagram of the reaction is shown in FIG.
- EndoS transglycosylation activity is due to the acceptor molecule containing an Fc moiety having only a core GlcNAc (with or without core fucose added) at N297, and a sugar having GlcNAc oxazolined at the reducing end.
- FIG. 3 shows an activity of glycosidically bonding the reducing end of a sugar chain derived from a chain donor (hereinafter referred to as “glycosyl transfer activity”, a schematic diagram of the reaction is shown in FIG. 3).
- EndoS consists of endoglycosidase enzymatic domain (catalyst domain: amino acid numbers 98 to 445 of SEQ ID NO: 1), Leucine-rich repeat domain (amino acid numbers 446 to 631 of SEQ ID NO: 1), hybrid Ig domain (amino acid number of SEQ ID NO: 1) 632-764), Carbohydrate-binding module (CBM: amino acid number 765-923 of SEQ ID NO: 1) and three helix-bundle domain (amino acid number 924-995 of SEQ ID NO: 1) It is reported (B. Trastoy et al., (PNAS (2014) vol111, No.18, pp6714-6719), and it is thought that the two important sites for interaction with the antibody are catalytic domain and CBM. .
- EndoS is an enzyme that is well conserved in Group A Streptococcus (GAS), but EndoS2 (also called EndoS49 found in NZ131 belonging to serotype M49 of GAS. Sjogren, Biochem J. 2013 Oct 1; 455 (1): 107-18, US 2014/0302519 A1)), although the sequence identity is 37%, important amino acids constituting the catalytic domain are well conserved and are similar to EndoS. Shows specificity. In the present specification, such an enzyme that retains the amino acid homology / identity of the active region of EndoS is referred to as “EndoS-related enzyme”.
- H122, D233, D279, Q303, E350, Y402, D405, and R406, which are important mutation sites of EndoS, are H75, D184, D226, Q250, E289, Y339, D342, and R343, respectively, in EndoS2. It corresponds to.
- a mutant enzyme in which a corresponding amino acid is mutated is also included in the mutant enzyme of the present invention.
- EndoS D233Q is an enzyme consisting of a sequence in which the 233rd Asp of wild-type EndoS is replaced with Gln (amino acid sequence of amino acid numbers 37 to 995 of SEQ ID NO: 1), and the hydrolysis of EndoS It is a mutant enzyme that has a certain degree of degradation activity and retains the same level of transglycosylation activity as wild-type.
- the present invention relates to a mutant enzyme of EndoS D233Q containing a region necessary for glycosyltransferase activity in the amino acid sequence of amino acid numbers 37 to 995 of SEQ ID NO: 2, further comprising a specific essential additional mutation site group in addition to D233Q.
- EndoS which has an additional mutation at a position containing at least one selected amino acid, and has reduced hydrolytic activity as an activity against N297-linked sugar chain of IgG as compared with EndoS D233Q
- the mutant enzyme is provided.
- “additional mutation” means an additional amino acid mutation occurring in an amino acid other than D233 in the amino acid sequence of EndoS D233Q.
- the “essential additional mutation site group” is a candidate group of sites that are necessarily additionally mutated in the mutant enzyme of the present invention, and the site indicated as Xaa in the amino acid sequence of SEQ ID NO: 2, ie, H122, A group consisting of P184, D279, Y282, Q303, Y348, E350, Y402, D405, and R406, and preferably a group consisting of Q303, E350, and D405.
- the mutant enzyme of the present invention is characterized by having both hydrolysis activity reduced to that of EndoS D233Q and a certain glycosyltransferase activity (hereinafter, having both activities is referred to as “the present enzyme activity”). And Whether or not the mutant enzyme has this enzyme activity can be evaluated for its hydrolysis activity and transglycosylation activity by the method of Example 4 described later.
- the hydrolysis activity is suppressed as compared with the hydrolysis activity of EndoS D233Q. That is, the hydrolysis rate under the condition of pH 7.4 described in Example 4 is low. In any time point from 1 to 48 hours after the start of the reaction, the hydrolysis rate is lower than the hydrolysis rate of EndoS D233Q.
- the hydrolysis rate after 24 hours from the start of the hydrolysis reaction is 50% or less, or the hydrolysis rate after 48 hours is 60% or less, more preferably the hydrolysis rate up to 24 hours after the start of the hydrolysis reaction is maintained at 40% or less, more preferably the same. Maintains 30% or less until time, even more preferably maintains 20% or less, and most preferably the hydrolysis rate is 0 at all time points. %, And the hydrolysis activity completely disappeared.
- the transglycosylation activity is comparable to that of EndoS D233Q, that is, pH 7.4 as described in Example 4, and the sugar chain donor is 5 equivalents of the acceptor molecule.
- the sugar transfer rate under the above conditions is equal to or higher than that of EndoS D233Q after any time point from 1 to 48 hours after the start of the reaction (for example, 70% or more of the value of EndoS D233Q).
- the rate of transglycosylation is indicated, but preferably the rate of transglycosylation exceeds 50% by 48 hours after the start of the reaction, more preferably the rate of transglycosylation exceeds 60% by 48 hours after the start of the reaction. More preferably, the transglycosylation rate exceeds 80% by 48 hours after the start of the reaction, and even more preferably, the transglycosylation rate is 9 by 48 hours after the start of the reaction. It exceeds 0%.
- the mutant enzyme of the present invention need not be the full-length sequence as long as it retains a region important for the glycosyltransferase activity of EndoS D233Q in the amino acid sequence of amino acid numbers 37 to 995 of SEQ ID NO: 2. From the domain analysis of EndoS, it is known that the catalytic domain (amino acids 98 to 445 of SEQ ID NO: 2) and CRM (amino acids 765 to 923 of SEQ ID NO: 2) are important, so long as these are included, It can be used as a mutant enzyme of the present invention.
- EndoS2 amino acids at the corresponding positions (EndoS H122, D233, D279, Q303, E350, Y402, D405, and R406 are Each of EndoS2 corresponds to H75, D184, D226, Q250, E289, Y339, D342, and R343) and is included in the present invention as long as it has this enzyme activity.
- Preferred is a polypeptide comprising amino acid numbers 98 to 923 of SEQ ID NO: 2, more preferred is a polypeptide comprising amino acid numbers 98 to 995 of SEQ ID NO: 2, and even more preferred is amino acid numbers 37 to 995 of SEQ ID NO: 2.
- the amino acid sequence of the mutant enzyme of the present invention 1 to several amino acids are substituted, deleted, inserted, and / or added at a location other than the essential mutation site described below, as long as the enzyme activity is not affected. May be.
- the site of such amino acid modification the entire site may be selected as long as it does not affect the enzyme activity, but preferably the catalytic domain (amino acids 98 to 445 of SEQ ID NO: 2) and CRM (sequence It is a site other than amino acid numbers 765 to 923) of No. 2, more preferably a site contained in the region of amino acid numbers 37 to 97 or amino acid numbers 924 to 995 of SEQ ID NO: 2.
- the term “several” means 20 or less, preferably 10 or less, more preferably 5 or less, and most preferably 4, 3, 2, or 1.
- the additional mutation site other than D233Q is a site containing at least one amino acid selected from the essential additional mutation site group.
- the number of additional mutation sites is not particularly limited as long as the final mutant has the enzyme activity of the mutant enzyme, but is preferably 10 sites or less, more preferably 5 sites or less, and still more preferably, 4 places, 3 places, 2 places, or 1 place.
- the other mutation sites are not particularly limited as long as the final mutant enzyme has this enzyme activity, but all additional It is preferable that the mutation is an essential additional mutation site group.
- the region containing the additional mutation other than the essential additional mutation site is an amino acid other than the catalytic domain related to the enzyme activity (amino acid numbers 98 to 445 of SEQ ID NO: 2). More preferably, it is a region of amino acid numbers 37 to 97, 446 to 764, and 924 to 995 of SEQ ID NO: 2, and more preferably, a region of amino acid numbers 37 to 97 and 924 to 995 of SEQ ID NO: 2. is there.
- the amino acid after substitution by additional mutation is not particularly limited as long as the finally obtained mutant enzyme has the present enzyme activity, naturally occurring amino acids, artificially synthesized amino acids, those Various amino acids, such as modified amino acids, can be adopted, preferably naturally occurring amino acids, more preferably naturally occurring L-amino acids, and still more preferably essential amino acids.
- modified amino acids such as modified amino acids, can be adopted, preferably naturally occurring amino acids, more preferably naturally occurring L-amino acids, and still more preferably essential amino acids.
- the preferable post-mutation amino acids at essential additional mutation sites are shown below.
- the amino acid after mutation of H122 is an amino acid having a small side chain structure (Gly, Cys, Thr, Ser, Val, Pro, Ala), side chain, and the interaction between the site and surrounding amino acids is eliminated.
- An amino acid having a negative charge (Glu or Asp) or an amino acid having no highly reactive functional group in the side chain (Leu, Ile, Pro, Met, Phe) is preferable, and Ala or Phe is more preferable.
- the amino acid after mutation of P184 is preferably an amino acid having a small side chain structure (Gly, Cys, Thr, Ser, Val, Pro, Ala) or an amino acid having an amide group in the side chain (Gln, Asn), more preferably. Is Gln.
- the amino acid after mutation of D279 is preferably an amino acid having a small side chain structure (Gly, Cys, Thr, Ser, Val, Pro, Ala) or Gln, more preferably Ser or Gln.
- the amino acid after mutation of Y282 is preferably an amino acid having a basic side chain (Arg, Lys, His), more preferably Arg.
- the amino acid after mutation of Q303 is preferably a hydrophobic amino acid (Met, Pro, Leu), more preferably Leu.
- the amino acid after mutation of Y348 is preferably an amino acid having a ring structure in the side chain (His, Trp), more preferably His.
- the amino acid after mutation of E350 is an amino acid having a large side chain capable of hydrogen bonding (Lys, Arg, His, Tyr, Gln, Asn), or the interaction between the site and surrounding amino acids is eliminated.
- An amino acid having a small side chain structure (Gly, Cys, Thr, Ser, Val, Pro, Ala) is preferred, and Ala, Asn, Asp or Gln is more preferred.
- the amino acid after mutation of Y402 is preferably a large amino acid having an aromatic ring in the side chain, Phe or Trp.
- the amino acid after mutation of D405 is preferably an amino acid having a small side chain structure (Gly, Cys, Thr, Ser, Val, Pro, Ala) that eliminates the interaction between the site and surrounding amino acids, and more Ala is preferred.
- the amino acid after mutation of R406 is an amino acid having a small side chain structure (Gly, Cys, Thr, Ser, Val, Pro, Ala) or a side chain that eliminates the interaction between the site and surrounding amino acids.
- An amino acid having negative charge (Glu, Asp) or an amino acid having an amide group in the side chain (Gln, Asn) is preferable, and Ala or Gln is more preferable.
- the mutations at the essential additional mutation sites as described above may be alone or may be combined with mutations at other essential additional mutation sites and / or mutations at other sites.
- the combination of mutations at the essential additional mutation site may be any combination, but is preferably a combination including mutations at least in Q303, E350 or D405, and more preferably at least Q303L, E350A, E350N, E350Q or D405A. Is a combination including
- Examples of the combination of additional mutations including Q303L include, for example, H122A / Q303L, H122F / Q303L, P184Q / Q303L, D279Q / Q303L, D279S / Q303L, Y282R / Q303L, Q303L / Y348H, Q303L / E350A, Q303L / E350N, Q303L / E350D, Q303L / E350Q, Q303L / Y402F, Q303L / Y402W, Q303L / D405A, Q303L / R406Q, and the like.
- E350A examples include, for example, H122A / E350A, H122F / E350A, P184Q / E350A, D279Q / E350A, D279S / E350A, Y282R / E350A, Y348H / E350A, E350A / Y402F, E350A / Y402W, E350A / D405A, E350A / R406Q, and the like.
- E350N examples include, for example, H122A / E350N, H122F / E350N, P184Q / E350N, D279Q / E350N, D279S / E350N, Y282R / E350N, Y348H / E350N, E350N / Y402F, E350N / Y402W, E350N / D405A, E350N / R406Q, and the like.
- E350D examples include, for example, H122A / E350D, H122F / E350D, P184Q / E350D, D279Q / E350D, D279S / E350D, Y282R / E350D, Y348H / E350D, E350D / Y402F, E350D / Y402W, E350D / D405A, E350D / R406Q, and the like.
- E350Q examples include, for example, H122A / E350Q, H122F / E350Q, P184Q / E350Q, D279Q / E350Q, D279S / E350Q, Y282R / E350Q, Y348H / E350Q, E350Q / Y402F, E350Q / Y402W, E350Q / D405A, E350Q / R406Q, and the like.
- D405A examples include H122A / D405A, H122F / D405A, P184Q / D405A, D279Q / D405A, D279S / D405A, Y282R / D405A, Y348H / D405A, Y402F / D405A, Y402W, D405A / R406Q, and the like.
- Preferred examples of the mutant enzyme of the present invention include EndoS D233Q / Q303L, EndoS D233Q / E350A, EndoS D233Q / E350N, EndoS D233Q / E350D, EndoS D233Q / E350Q, EndoSQDE33A / D33Q / E350Q / E350A / S303ED Q303L / E350N, EndoS D233Q / Q303L / E350D, EndoS D233Q / Q303L / E350Q, EndoS D233Q / Q303L / D405A, EndoSd233Q / E350A / D405E350D350E350D350E350D350D350E350D350D350D350D350 50Q / D405A, or a EndoS D233Q / Y402F, more preferably EndoS D233Q / Q303L, EndoS D233Q / Q303L, End
- the present invention further includes a recombinant gene encoding a mutant enzyme having an additional mutation in EndoS D233Q, a gene construct such as a plasmid or expression vector containing the recombinant gene, a host cell transformed with the gene construct, and the host cell
- a method for producing the mutant enzyme of the present invention which comprises the step of recovering the mutant enzyme of the present invention from the culture of
- These recombinant genes, gene constructs, and host cell sugars can be prepared according to known genetic engineering techniques based on the amino acid sequence of the mutant enzyme of the present invention.
- the recombinant gene encoding the mutant enzyme of the present invention has a desired additional mutation based on the nucleotide sequence of EndoS D233Q described in nucleotide numbers 109 to 2985 of SEQ ID NO: 3 (1 to 108 is a region encoding a signal peptide).
- a polynucleotide such as cDNA encoding the amino acid sequence containing it can be made.
- the nucleotide sequence encoding EndoS D233Q / Q303L is a nucleotide sequence obtained by replacing nucleotides 907 to 909 from CAG to CTG in the nucleotide sequence of nucleotide numbers 109 to 2985 of SEQ ID NO: 3.
- the nucleotide sequence encoding EndoS D233Q / E350A (N, Q) is a nucleotide sequence obtained by substituting nucleotides 1048 to 1050 from GAA to GCA in the nucleotide sequence of nucleotide numbers 109 to 2985 of SEQ ID NO: 3. EAT for E350N, CAG for E350Q).
- the nucleotide sequence encoding EndoS D233Q / D405A is a nucleotide sequence in which nucleotides 1213 to 1215 in the nucleotide sequence of nucleotide numbers 109 to 2985 of SEQ ID NO: 3 are substituted from GAT to GCA.
- Host cells transformed by introduction of the gene encoding the mutant enzyme of the present invention animal cells, plant cells, Escherichia coli, yeast, etc., cells normally used for protein production, etc. can be appropriately selected
- the mutant enzyme of the present invention can be recovered from the culture under the appropriate conditions. Recovery of the mutant enzyme is performed by appropriately combining ordinary purification techniques using the physical properties of the enzyme, but for easy recovery, expression is performed in a form in which a tag peptide such as GST is linked to the mutant enzyme in advance. As described above, by designing the gene construct, recovery using the affinity of the tag peptide can be performed.
- the tag peptide may be removed after purification, but if the enzyme activity is not affected, a mutant enzyme with the tag peptide linked may be used for reactions such as sugar chain remodeling.
- the mutant enzyme of the present invention includes an enzyme having an amino acid sequence in which such tag peptides are linked.
- the present invention relates to a method for remodeling a N297-linked sugar chain in an IgG or Fc region-containing molecule using the EndoS mutant enzyme of the present invention, and a substantially uniform structure produced by the sugar chain remodeling.
- An IgG or Fc region-containing molecule having an N297-linked sugar chain is provided.
- Glycan remodeling refers to a specific monoclonal antibody IgG or an Fc fragment of IgG or an N297-linked sugar chain of an Fc region-containing molecule such as CLCH consisting only of a constant region, to which a core GlcNAc (core fucose is added).
- the acceptor molecule is excised leaving behind, and the sugar chain derived from the sugar chain donor is then applied to the core GlcNAc of the acceptor molecule using the glycosyltransferase activity of the EndoS mutant enzyme of the present invention.
- By transferring it means a method for producing an IgG or Fc region-containing molecule having a uniform sugar chain structure in which the N297-linked sugar chain is derived from a sugar chain donor.
- the IgG or Fc region-containing molecule used for sugar chain remodeling may be any molecule as long as it is derived from an IgG heavy chain consisting of the same amino acid sequence and has an N297-linked sugar chain, and its production method is limited.
- IgG produced by a generally known method for producing a monoclonal antibody, CLCH of IgG, or an Fc fragment obtained by enzymatic treatment thereof can be used.
- Such IgG or Fc fragment may be a mixture of samples obtained in different production methods or different lots.
- the above-mentioned IgG or Fc region-containing molecule retained the activity of specifically hydrolyzing the 1.4-glycoside bond between GlcNAc in the core chitobiose structure of the N297-linked sugar chain. It can be adjusted by treating with ENGase.
- ENGase various types such as EndoA, EndoD, EndoE, EndoS can be adopted, but wild-type EndoS is preferable.
- sugar chain donors used for sugar chain remodeling those having various sugar chain structures can be adopted.
- a sugar chain possessed by humans it is preferable to employ a sugar chain donor having a human-type sugar chain or a human-compatible sugar chain structure that is similar or identical to the structure.
- a typical example of such a sugar chain donor is a molecule in which the core GlcNAc is removed from the basic structure of the N-linked sugar chain and the second GlcNAc from the reducing end is oxazolineated.
- SG-Oxa having the structure shown in FIG. 1 is preferred.
- reaction conditions for the hydrolysis reaction in sugar chain remodeling a method generally known for EndoS can be employed.
- the reaction is performed in a buffer solution, but citrate buffer (pH 3.5 to 5.5), acetate buffer (pH 4.5 to 6.0), phosphate buffer (pH 6.0 to 7.5), It is possible to appropriately select from buffers used in normal enzyme reactions such as MOPS-NaOH buffer (pH 6.5 to 8.0), Tris-HCl buffer (pH 7.0 to 9.0). Tris-HCl buffer (pH 7.0 to 9.0) is preferable.
- An additive that does not inhibit the enzyme reaction may be added to the reaction solution for the purpose of stabilizing the enzyme, but it may not be added.
- the reaction temperature can be appropriately selected between 10 ° C. and 50 ° C., preferably 25 ° C. to 38 ° C.
- the reaction time can be appropriately selected from 10 minutes to 96 hours, but a small amount of the reaction solution may be collected over time, and the completion of the reaction may be judged while confirming the degree of hydrolysis.
- the progress of sugar chain hydrolysis can be monitored by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), fully automated electrophoresis system, or liquid chromatography mass spectrometry (LC-MS).
- SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis
- LC-MS liquid chromatography mass spectrometry
- reaction conditions for the glycosyl transfer reaction in sugar chain remodeling can be appropriately selected according to conditions known for other enzymes.
- the reaction is preferably carried out in a buffer solution, but preferably does not promote the degradation of SG-Oxa.
- Phosphate buffer solution pH 6.0 to 7.5
- MOPS-NaOH buffer solution pH 6.5 to 8.0
- Tris-HCl buffer pH 7.0 to 9.0
- Tris-HCl buffer pH 7.0 to 9.0
- an additive that does not inhibit the enzyme reaction may be added to the reaction solution, but it may not be added.
- the reaction temperature can be appropriately selected between 10 ° C. and 50 ° C., preferably 25 ° C. to 38 ° C.
- the reaction time can be appropriately selected from 10 minutes to 96 hours, but a small amount of the reaction solution may be collected over time, and the completion of the reaction may be judged while confirming the progress of the transglycosylation reaction.
- the progress of the transglycosylation reaction can be monitored by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), a fully automated electrophoresis system, liquid chromatography mass spectrometry (LC-MS), or the like.
- Example 1 is a preparation example of the EndoS mutant enzyme used in the present invention.
- Example 2 is a production example of a sugar chain donor used in the present invention.
- Example 3 is an example of preparing an acceptor molecule used when measuring transglycosylation activity.
- Example 4 is a measurement example of the hydrolysis rate of the EndoS mutant enzyme of the present invention and a measurement example of the sugar chain transfer activity of the EndoS mutant enzyme of the present invention.
- the protein concentration described in this specification was quantified using an ultra-trace spectrophotometer NanoDrop1000 (manufactured by Thermo-Fisher-Scientific) or NanoDrop2000 (manufactured by Thermo-Fisher-Scientific).
- the mass of the sugar chain remodeling antibodies ((Fuc ⁇ 1,6) GlcNAc-Trastuzumab and SG-Trastuzumab) was confirmed by the following method. After the sugar chain remodeling antibody was fragmented into a heavy chain and a light chain, each peak was separated with an analytical column and subjected to mass spectrometry. ACUITY UPLC (manufactured by Waters), SYNAPT G2-S (Waters), and BEH Phenyl column (1.7 ⁇ m, 1.0x50 mm) were used for the apparatus, and 0.05% trifluoroacetic acid-containing acetonitrile was added to the mobile phase in 3 minutes. The sample was used in a gradient varying from 25% to 35%, and analyzed at a flow rate of 0.34 ⁇ L / min at 80 ° C.
- the 6 sites of H122, Y348, E350, Y402, D405, and R406 are set as sites that are predicted to be involved in sugar chain recognition, in order to improve the efficiency of sugar chain binding and dissociation.
- the enzyme was designed to cleave the interaction formed by the above amino acid residues, or to replace an amino acid residue having a large side chain with an amino acid residue having a small side chain.
- P184, D279, and Q303 were set near the active center, and amino acids having similar properties to wild-type amino acid residues, or mutations that widely substitute amino acids having different properties were designed.
- Y282 was set as a site predicted to be involved in antibody recognition, and a mutation for substitution with a basic amino acid was designed to strengthen the interaction with the negative charge near Asn297 to which the antibody sugar chain binds.
- Each mutant enzyme shown in Table 2 was designed in consideration of combination mutations within the above three sites or between the three sites.
- coli cells are cultured overnight in an LB agar medium containing 100 ⁇ g / mL ampicillin, and the colonies obtained the next day are collected and OD600 is 0 in 1 L TB medium containing 100 ⁇ g / mL ampicillin.
- the cells were cultured with shaking at 37 ° C. until reaching 8. After OD600 rises, the culture temperature is lowered to 16 ° C., and after 1 hour, Isopropyl ⁇ -D-1-thiogalactopyranoside (IPTG) is added so that the final concentration becomes 0.1 mM. Expression was induced overnight.
- IPTG Isopropyl ⁇ -D-1-thiogalactopyranoside
- the cells are collected by centrifuging the culture at 5,000 ⁇ G for 10 minutes, and 50 mL of PBS buffer containing 40 U / mL DNase I and 0.5 mg / mL Lysozyme is added, and the cells are re-applied. Suspended. The resulting bacterial solution was sonicated to disrupt the cells and centrifuged at 20,000 ⁇ g for 30 minutes to obtain the desired EndoS mutant enzyme in the soluble fraction.
- the glutathione sepharose 4B column equilibrated with PBS was supplied with the whole amount of the soluble fraction of the EndoS mutant enzyme through the PVDF membrane-passing membrane, and the column was washed with PBS buffer 3 times the column volume or more.
- the GST-fused EndoS mutant enzyme was eluted with a PBS buffer containing 10 ⁇ mM reduced glutathione and concentrated by ultrafiltration (Amicon Ultra-15 30K). After concentration, the solution was passed through a HiLoad 26/60 Superdex 200 pg column (GE Healthcare Bioscience) equilibrated with PBS to remove glutathione used for elution and obtain a final purified sample. Table 2 shows the yields of the various EndoS mutant enzymes thus prepared.
- the obtained EndoS mutant enzyme solution was prepared to 2 mg / mL, and these were used for measurement of hydrolytic activity and measurement of transglycosylation activity.
- SG-Oxa used as a sugar chain donor in Examples after preparation of a reaction solution of SG-Oxa (compound having the structure of Fig. 1) was produced by the following method.
- the passing liquid was purified by gel filtration chromatography.
- the device uses Purif-Rp2 (manufactured by Shoko Scientific), the column uses HiPrep 26/10 Desalting (manufactured by GE Healthcare), the mobile phase uses 0.03%-NH3 aqueous solution, and the flow rate is 10 ml / min and the fraction volume were 10 ml.
- Fractions containing the target compound that was UV-detected (220 nm) during elution were combined into one, added with 0.1N sodium hydroxide aqueous solution (100 ⁇ l) and lyophilized to obtain the target SG-Oxa as a colorless solid (87.0 mg, 43.4mol ⁇ mol, potato yield 88%).
- elution buffer ImmunoPure IgG Eution buffer, manufactured by PIERCE
- the eluate was immediately neutralized with 1 M Tris buffer (pH 9.0).
- the fraction detected by UV detection (280 nm) during elution was confirmed using an ultra-trace spectrophotometer NanoDrop1000 (Thermo Fisher Scientific) and an Experion electrophoresis station (BIO-RAD).
- a 20 mg / ml Trastuzumab solution used as a substrate solution for the hydrolysis reaction was prepared as follows. Otsuka distilled water (10 ml) was added to 100 ⁇ m Tris buffer (pH 7.4, manufactured by CALBIOCHEM) (10 ml) to prepare 50 ⁇ m Tris buffer (pH 7.4) (40 ml). A commercially available Trastuzumab (440 mg / vial, manufactured by Genentech) was added with an accompanying solution (20 ml) to obtain a Trastuzumab (ca. 21 mg / ml) solution. Trastuzumab (ca.
- the obtained measurement sample was transferred to Experion TM Pro260 Chips and measured according to the procedure attached to the Experion TM electrophoresis station (BIO-RAD). From the obtained chromatogram, it is confirmed as a peak in which the unreacted product and the hydrolyzate are separated.
- the hydrolysis rate was calculated from the peak area ratio of the unreacted product and the hydrolyzate by the following calculation formula.
- the obtained measurement sample was transferred to Experion TM Pro260 Chips and measured according to the procedure attached to the Experion TM electrophoresis station (BIO-RAD). From the obtained chromatogram, it is confirmed as a peak in which the unreacted product and the sugar chain transfer product are separated.
- the sugar chain transfer rate was calculated from the peak area ratio of the unreacted product and the sugar chain transfer product by the following formula.
- Glycan transfer rate (%) [peak area of H chain derived from SG-Trastuzumab] / ⁇ [peak area of H chain derived from (Fuc ⁇ 1,6) GlcNAc-Trastuzumab + peak area of H chain derived from SG-Trastuzumab] ⁇ ⁇ 100
- the sugar chain transfer rate at each reaction time of the other EndoS mutant enzymes prepared in Example 1 was calculated (Table 3).
- SG-Trastuzumab which is a glycosyl transfer product, was purified in the same manner as the purification of (Fuc ⁇ 1,6) GlcNAc-Trastuzumab in Example 3.
- the obtained LC-MS analysis data of SG-Trastuzumab is shown below.
Abstract
Description
(1) 配列番号2のアミノ酸番号37~995のアミノ酸配列において、122番目(H122)、184番目(P184)、279番目(D279)、282番目(Y282)、303番目(Q303)、348番目(Y348)、350番目(E350)、402番目(Y402)、405番目(D405)、及び、406番目(R406)からなる群から選択される少なくとも1つのアミノ酸を含む箇所に追加変異を有し、且つ、IgGの297番目のAsnに結合するN結合型糖鎖(N297結合糖鎖)に対する活性として、EndoS D233Qの活性と比較して低減された加水分解活性を示すことを特徴とする、EndoS変異酵素。
(2) 追加変異が、H122、P184、D279、Y282、Q303、Y348、E350、Y402、D405、及び、R406からなる群から選択される1~4アミノ酸からなる部位であることを特徴とする、(1)のEndoS変異酵素。
(3) 追加変異の箇所が、Q303、E350及びD405からなる群から選択される1アミノ酸における変異を含むことを特徴とする、(1)のEndoS変異酵素。
(4) 追加変異が以下の群から選択される少なくとも一つである、(1)~(3)のEndoS変異酵素、
H122の変異後のアミノ酸は、Gly、Cys、Thr、Ser、Val、Pro、Ala、Glu、Asp、Leu、Ile、Pro、Met又はPhe;
P184の変異後のアミノ酸がGly、Cys、Thr、Ser、Val、Pro、Ala、Gln又はAsn;
D279の変異後のアミノ酸は、Gly、Cys、Thr、Ser、Val、Pro、Ala又はGln;
Y282の変異後のアミノ酸は、Arg、Lys又はHis;
Q303の変異後アミノ酸は、Met、Pro又はLeu;
Y348の変異後アミノ酸は、His又はTrp;
E350の変異後アミノ酸は、Lys、Arg、His、Tyr、Gln、Asn、Gly、Cys、Thr、Ser、Val、Pro又はAla;
Y402の変異後のアミノ酸は、Phe又はTrp;
D405の変異後アミノ酸は、Gly、Cys、Thr、Ser、Val、Pro又はAla;、及び、
R406の変異後アミノ酸は、Gly、Cys、Thr、Ser、Val、Pro又はAla、Glu、Asp、Gln又はAsn。
(5) 追加変異が以下の群から選択される少なくとも一つである、(4)のEndoS変異酵素、
H122がAla(H122A)又はPhe(H122F);
P184がGln(P184Q);
D279がSer(D279S)又はGln(D279Q);
Y282がArg(Y282R);
Q303がLeu(Q303L);
Y348がHis(Y348H);
E350がAla(E350A)、Asn(E350N)、Asp(E350D)又はGln(E350Q);
Y402がPhe(Y402F)またはTrp(Y402W);
D405がAla(D405A);、及び、
R406がGln(R406Q)。
(6) 追加変異として、Q303L、E350A、E350N、E350D、E350Q及びD405Aからなる群から選択される少なくとも一つの変異を含むことを特徴とする、(5)のEndoS変異酵素。
(7) 追加変異が、Q303L、E350A、E350N、E350D、E350Q、D405A、
H122A/Q303L、H122F/Q303L、P184Q/Q303L、D279Q/Q303L、D279S/Q303L、Y282R/Q303L、Q303L/Y348H、Q303L/E350A、Q303L/E350N、Q303L/E350D、Q303L/E350Q、Q303L/Y402F、Q303L/Y402W、Q303L/D405A、Q303L/R406Q、
H122A/E350A、H122F/E350A、P184Q/E350A、D279Q/E350A、D279S/E350A、Y282R/E350A、Y348H/E350A、E350A/Y402F、E350A/Y402W、E350A/D405A、E350A/R406Q、
H122A/E350N、H122F/E350N、P184Q/E350N、D279Q/E350N、D279S/E350N、Y282R/E350N、Y348H/E350N、E350N/Y402F、E350N/Y402W、E350N/D405A、E350N/R406Q、
H122A/E350D、H122F/E350D、P184Q/E350D、D279Q/E350D、D279S/E350D、Y282R/E350D、Y348H/E350D、E350D/Y402F、E350D/Y402W、E350D/D405A、E350D/R406Q、
H122A/E350Q、H122F/E350Q、P184Q/E350Q、D279Q/E350Q、D279S/E350Q、Y282R/E350Q、E350Q/Y348H、E350Q/Y402F、E350Q/Y402W、E350Q/D405A、E350Q/R406Q、
H122A/D405A、H122F/D405A、P184Q/D405A、D279Q/D405A、D279S/D405A、Y282R/D405A、Y348H/D405A、又は、D405A/R406Q、であることを特徴とする、(6)のEndoS変異酵素。
(8) 追加変異が、Q303L、E350A、E350N、E350D、E350Q、D405A、Q303L/E350A、Q303L/E350N、Q303L/E350D、Q303L/E350Q、Q303L/D405A、E350A/D405A、E350N/D405A、E350D/D405A、又は、E350Q/D405Aである、(7)のEndoS変異酵素。
(9) 追加変異が、H122A、H122F、P184Q、D279Q、D279S、Y282R、Y348H、Y402F、Y402W、又はR406Qである、(4)のEndoS変異酵素。
(10) N297結合糖鎖に対する活性が、pH7.4の反応液中での加水分解反応において、24時間後まで、50%以下の加水分解率を維持することを特徴とする、(1)のEndoS変異酵素。
(11) N297結合糖鎖に対する活性として、さらに、pH7.4の反応液中での、5等量の糖鎖ドナーを用いた糖転移反応において48時間後の糖転移率が約60%以上である活性を示すことを特徴とする、(1)のEndoS変異酵素。
(12) (1)~(11)のいずれかのEndoS変異酵素をコードするポリヌクレオチド。
(13) (12)のポリヌクレオチドと相補的なヌクレオチドを含むベクター。
(14) (13)のベクターで形質転換された宿主細胞。
(15) (1)~(11)のいずれかのEndoS変異酵素の存在下で、N297結合糖鎖として、フコース付加していても良いコアGlcNAcを有するIgGのFc領域を含む分子とオキサゾリン化されたGlcNAcを含む構造を有する糖鎖ドナーを反応させ、当該Fc領域含有分子のN297結合糖鎖の当該コアGlcNAcに、当該糖鎖ドナーの有する糖鎖が転移した構造を有するFc領域含有分子を産生させることを特徴とする、糖鎖リモデリングされたFc領域含有分子の製造方法。
(16) Fc領域含有分子が、IgGモノクローナル抗体である、(15)の製造方法。
本発明は、配列番号2のアミノ酸番号37~995のアミノ酸配列において糖転移活性に必要な領域を含有するEndoS D233Qの変異酵素であって、D233Qに加えてさらに、特定の必須追加変異箇所群から選択される少なくとも1つのアミノ酸を含む箇所に追加変異を有し、且つ、IgGのN297結合糖鎖に対する活性として、EndoS D233Qと比較して加水分解活性が低減されていることを特徴とする、EndoSの変異酵素を提供する。
本発明は、さらに上記のEndoS D233Qに追加変異を有する変異酵素をコードする組み換え遺伝子、当該組み換え遺伝子を含むプラスミドや発現ベクター等の遺伝子構築物、当該遺伝子構築物により形質転換された宿主細胞、当該宿主細胞の培養物から本発明の変異酵素を回収する工程を含む、本発明の変異酵素の製造方法などを提供する。これらの組み換え遺伝子、遺伝子構築物、宿主細胞糖は、本発明の変異酵素のアミノ酸配列に基づき、公知の遺伝子工学的手法に従って作成することができる。
本発明は、本発明のEndoS変異酵素を使用した、IgG又はFc領域含有分子におけるN297結合糖鎖の糖鎖リモデリング方法、及び、当該糖鎖リモデリングにより製造された実質的に均一な構造からなるN297結合糖鎖を有するIgG又はFc領域含有分子、を提供する。
(1-1) EndoS変異酵素のデザイン
EndoS D233Qに変異を導入することで、EndoS D233Qの糖転移活性を維持しながら、加水分解活性を抑制することが達成されると考えられる変異型EndoSを設計した。EndoSの立体構造情報(PDB ID:4NUY)に基づいて、EndoSの触媒ドメインを、糖鎖の認識に関与することが予測される部位、活性中心付近、抗体の認識に関与することが予測される部位の3部位に区別し、それぞれ変異導入の対象とした。糖鎖の認識に関与することが予測される部位としては、H122、Y348、E350、Y402、D405、及びR406の6残基を設定し、糖鎖の結合と解離の回転を効率化するために、上記のアミノ酸残基が形成する相互作用を切断するべく、あるいは上記のアミノ酸のうち大きな側鎖を持つアミノ酸残基を小さい側鎖を持つアミノ酸残基に置換するべく酵素をデザインした。活性中心付近としては、P184、D279、及びQ303を設定し、野生型のアミノ酸残基と類似した性質を持つアミノ酸、あるいは異なる性質を持つアミノ酸へ幅広く置換する変異をデザインした。抗体の認識に関与することが予測される部位としてはY282を設定し、抗体糖鎖が結合するAsn297付近の負電荷との相互作用を強めるべく、塩基性のアミノ酸へ置換する変異をデザインした。上記の3部位内、あるいは3部位間での組合せ変異も考慮し、表2の各変異酵素をデザインした。
コンピテンシーが107cfu /μLの氷冷された大腸菌BL21(DE3)株、50 μLに対して、上記(1-1)に基づいてデザインした各EndoS変異酵素のアミノ酸配列をコードした遺伝子を含むタンパク質発現用プラスミド(pGEX4T3、50 μg/μL)を3 μL添加し、37℃で40秒間加熱することで、大腸菌を形質転換した。これら大腸菌を100 μg/mLのアンピシリンを含むLB寒天培地で一晩培養し、翌日得られたコロニーを採取して100 μg/mLのアンピシリンを含む1 LのTB培地でO.D.600が0.8となるまで37℃で振とう培養した。O.D.600が上昇した後、培養温度を16℃に下げ、1時間後に終濃度が0.1 mMとなるようにIsopropyl β-D-1-thiogalactopyranoside(IPTG)を添加することで、EndoS変異酵素の発現を一晩誘導した。翌日、培養液を5,000×Gで10分間遠心分離することで菌体を回収し、40 U/mLのDNaseI、および0.5 mg/mL Lysozymeを含むPBS バッファーを50 mL加え、菌体を再懸濁した。得られた菌液に対して超音波処理することで菌体を破砕し、20,000×gで30分間遠心分離することで可溶性画分に目的とするEndoS変異酵素を得た。
上記(1-2)で得られたEndoS変異酵素の可溶性画分について孔径が0.45 μmのPVDF膜を通し、通過後の溶液をグルタチオンアフィニティークロマトグラフィーとゲルろ過クロマトグラフィーの二段階工程で精製した。
以降の実施例で糖鎖ドナーとして使用するSG-Oxaは、以下の方法で製造した。
糖転移反応に使用するアクセプター分子として使用する(Fucα1,6)GlcNAc-Trastuzumabを、以下の方法で作製した。
精製装置:AKTA avant25(GE ヘルスケア製)
カラム:HiTrap Protein A HPカラム(5ml)(GE ヘルスケア製)
流速:5ml/min(チャージ時は1ml/min)
カラムへの結合時は、上記で得た反応液をカラム上部へ添加し、結合バッファー(20 mMりん酸緩衝液(pH7.0))を1 ml/minで1CV流し、更に5 ml/minで5CV流した。中間洗浄時は、洗浄溶液(20 mM りん酸緩衝液(pH7.0)、0.5 M 塩化ナトリウム溶液)を15CV流した。溶出時は、溶出バッファー(ImmunoPure IgG Eution buffer,PIERCE製)を6CV流した。溶出液を1 M トリス緩衝液(pH9.0)で直ちに中和した。溶出中にUV検出(280 nm)されたフラクションについて、超微量分光光度計NanoDrop1000(Thermo Fisher Scientific製)、及びExperion電気泳動ステーション(BIO-RAD製)を用いて確認した。
精製装置:AKTA avant25(GE ヘルスケア製)
カラム:Bio-Scale Mini CHT Type Iカートリッジ(5ml)(BIO-RAD製)
流速:5ml/min(チャージ時は1ml/min)
上記(1)で得られた溶液をカラムの上部へ添加し、A液(5 mM りん酸緩衝液、50 mM 2-モルホリノエタンスルホン酸(MES)、pH6.8)を4CV流した。その後、A液とB液(5 mM りん酸緩衝液、50 mM 2-モルホリノエタンスルホン酸(MES)、pH6.8、2 M塩化ナトリウム溶液)を用いて、溶出した。溶出条件は、A液:B液 = 100:0 ~ 0:100 (15CV)である。
LC-MS:
calculated for the heavy chain of (Fucα1, 6)GlcNAc-Trastuzumab, M = 49497.86 found(m/z), 49497 (deconvolution data).
calculated for the light chain of (Fucα1, 6)GlcNAc-Trastuzumab, M = 23439.1, found(m/z), 23439.1 (deconvolution data).
<実施例4> EndoS変異酵素の加水分解活性及び糖鎖転移活性の測定(pH7.4)
(4-1) 加水分解活性の測定
各種EndoS変異酵素の、市販のTrastuzumabのN297結合糖鎖に対する加水分解活性を、以下のように測定した。当該加水分解反応の模式図を図2に示す。
同様にして、実施例1で調製した他のEndoS変異酵素の、各反応時間における加水分解率を算出した(表3)。
実施例1で作製した各種EndoS変異酵素の糖転移活性を以下の方法で測定した。糖鎖ドナーとしては、実施例2で調整したSG-Oxaを、アクセプター分子としては実施例3で調製した、(Fucα1,6)GlcNAc-Trastuzumabを、それぞれ用いた。糖転移反応の模式図を図3に示す。
同様にして、実施例1で調製した他のEndoS変異酵素の、各反応時間における糖鎖転移率を算出した(表3)。
calculated for the light chain of SG-Trastuzumab, M = 23439.1 Da, found(m/z), 23439 (deconvolution data).
Claims (16)
- 配列番号2のアミノ酸番号37~995のアミノ酸配列において、122番目(H122)、184番目(P184)、279番目(D279)、282番目(Y282)、303番目(Q303)、348番目(Y348)、350番目(E350)、402番目(Y402)、405番目(D405)、及び、406番目(R406)からなる群から選択される少なくとも1つのアミノ酸を含む箇所に追加変異を有し、且つ、IgGの297番目のAsnに結合するN結合型糖鎖(N297結合糖鎖)に対する活性として、EndoS D233Qの活性と比較して低減された加水分解活性を示すことを特徴とする、EndoS変異酵素。
- 追加変異が、H122、P184、D279、Y282、Q303、Y348、E350、Y402、D405、及び、R406からなる群から選択される1~4アミノ酸からなる部位であることを特徴とする、請求項1のEndoS変異酵素。
- 追加変異の箇所が、Q303、E350及びD405からなる群から選択される1アミノ酸における変異を含むことを特徴とする、請求項1のEndoS変異酵素。
- 追加変異が以下の群から選択される少なくとも一つである、請求項1~3のEndoS変異酵素、
H122の変異後のアミノ酸は、Gly、Cys、Thr、Ser、Val、Pro、Ala、Glu、Asp、Leu、Ile、Pro、Met又はPhe;
P184の変異後のアミノ酸がGly、Cys、Thr、Ser、Val、Pro、Ala、Gln又はAsn;
D279の変異後のアミノ酸は、Gly、Cys、Thr、Ser、Val、Pro、Ala又はGln;
Y282の変異後のアミノ酸は、Arg、Lys又はHis;
Q303の変異後アミノ酸は、Met、Pro又はLeu;
Y348の変異後アミノ酸は、His又はTrp;
E350の変異後アミノ酸は、Lys、Arg、His、Tyr、Gln、Asn、Gly、Cys、Thr、Ser、Val、Pro又はAla;
Y402の変異後のアミノ酸は、Phe又はTrp;
D405の変異後アミノ酸は、Gly、Cys、Thr、Ser、Val、Pro又はAla;、及び、
R406の変異後アミノ酸は、Gly、Cys、Thr、Ser、Val、Pro、Ala、Glu、Asp、Gln又はAsn。 - 追加変異が以下の群から選択される少なくとも一つである、請求項4のEndoS変異酵素、
H122がAla(H122A)又はPhe(H122F);
P184がGln(P184Q);
D279がSer(D279S)又はGln(D279Q);
Y282がArg(Y282R);
Q303がLeu(Q303L);
Y348がHis(Y348H);
E350がAla(E350A)、Asn(E350N)、Asp(E350D)又はGln(E350Q);
Y402がPhe(Y402F)またはTrp(Y402W);
D405がAla(D405A);、及び、
R406がGln(R406Q)。 - 追加変異として、Q303L、E350A、E350N、E350D、E350Q及びD405Aからなる群から選択される少なくとも一つの変異を含むことを特徴とする、請求項5のEndoS変異酵素。
- 追加変異が、Q303L、E350A、E350N、E350D、E350Q、D405A、
H122A/Q303L、H122F/Q303L、P184Q/Q303L、D279Q/Q303L、D279S/Q303L、Y282R/Q303L、Q303L/Y348H、Q303L/E350A、Q303L/E350N、Q303L/E350D、Q303L/E350Q、Q303L/Y402F、Q303L/Y402W、Q303L/D405A、Q303L/R406Q、
H122A/E350A、H122F/E350A、P184Q/E350A、D279Q/E350A、D279S/E350A、Y282R/E350A、Y348H/E350A、E350A/Y402F、E350A/Y402W、E350A/D405A、E350A/R406Q、
H122A/E350N、H122F/E350N、P184Q/E350N、D279Q/E350N、D279S/E350N、Y282R/E350N、Y348H/E350N、E350N/Y402F、E350N/Y402W、E350N/D405A、E350N/R406Q、
H122A/E350D、H122F/E350D、P184Q/E350D、D279Q/E350D、D279S/E350D、Y282R/E350D、Y348H/E350D、E350D/Y402F、E350D/Y402W、E350D/D405A、E350D/R406Q、
H122A/E350Q、H122F/E350Q、P184Q/E350Q、D279Q/E350Q、D279S/E350Q、Y282R/E350Q、E350Q/Y348H、E350Q/Y402F、E350Q/Y402W、E350Q/D405A、E350Q/R406Q、
H122A/D405A、H122F/D405A、P184Q/D405A、D279Q/D405A、D279S/D405A、Y282R/D405A、Y348H/D405A、又は、D405A/R406Q、であることを特徴とする、請求項6のEndoS変異酵素。 - 追加変異が、Q303L、E350A、E350N、E350D、E350Q、D405A、Q303L/E350A、Q303L/E350N、Q303L/E350D、Q303L/E350Q、Q303L/D405A、E350A/D405A、E350N/D405A、E350D/D405A、又は、E350Q/D405Aである、請求項7のEndoS変異酵素。
- 追加変異が、H122A、H122F、P184Q、D279Q、D279S、Y282R、Y348H、Y402F、Y402W、又はR406Qである、請求項4のEndoS変異酵素。
- N297結合糖鎖に対する活性が、pH7.4の反応液中での加水分解反応において、24時間後まで、50%以下の加水分解率を維持することを特徴とする、請求項1のEndoS変異酵素。
- N297結合糖鎖に対する活性として、さらに、pH7.4の反応液中での、5等量の糖鎖ドナーを用いた糖転移反応において48時間後の糖転移率が約60%以上である活性を示すことを特徴とする、請求項1のEndoS変異酵素。
- 請求項1~11のいずれかのEndoS変異酵素をコードするポリヌクレオチド。
- 請求項12のポリヌクレオチドと相補的なヌクレオチドを含むベクター。
- 請求項13のベクターで形質転換された宿主細胞。
- 請求項1~11のいずれかのEndoS変異酵素の存在下で、N297結合糖鎖として、フコース付加していても良いコアGlcNAcを有するIgGのFc領域を含む分子とオキサゾリン化されたGlcNAcを含む構造を有する糖鎖ドナーを反応させ、当該Fc領域含有分子のN297結合糖鎖の当該コアGlcNAcに、当該糖鎖ドナーの有する糖鎖が転移した構造を有するFc領域含有分子を産生させることを特徴とする、糖鎖リモデリングされたFc領域含有分子の製造方法。
- Fc領域含有分子が、IgGモノクローナル抗体である、請求項15の製造方法。
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KR1020187001358A KR102546854B1 (ko) | 2015-07-16 | 2016-07-15 | 신규 EndoS 변이 효소 |
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CA3143262A1 (en) | 2019-08-05 | 2021-02-11 | Cho Pharma, Inc. | Fusion protein for remodeling antibody glycoform |
CN115443288A (zh) * | 2020-06-18 | 2022-12-06 | 上海宝济药业有限公司 | 一种免疫球蛋白降解酶IdeE的突变体 |
Citations (1)
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JP2015507925A (ja) * | 2012-02-10 | 2015-03-16 | ユニバーシティー オブ メリーランド,ボルティモア | 抗体及びそのFcフラグメントの酵素化学的糖鎖改変 |
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JP5795067B2 (ja) * | 2011-07-21 | 2015-10-14 | 京セラ株式会社 | 照明装置、イメージセンサヘッドおよびこれを備える読取装置 |
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Non-Patent Citations (2)
Title |
---|
HUANG, WEI ET AL.: "Chemoenzymatic Glycoengineering of Intact IgG Antibodies for Gain of Functions", JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, vol. 134, no. 29, 2012, pages 12308 - 12318, XP055035929 * |
See also references of EP3323886A4 * |
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EP4159749A2 (en) | 2016-07-01 | 2023-04-05 | Daiichi Sankyo Company, Limited | A method for producing fc-containing molecule with remodeled sugar chain |
KR102493562B1 (ko) | 2016-07-01 | 2023-01-30 | 다이이찌 산쿄 가부시키가이샤 | hANP-Fc 함유 분자 콘주게이트 |
US11208442B2 (en) | 2016-12-02 | 2021-12-28 | Daiichi Sankyo Company, Limited | Endo-beta-N-acetylglucosaminidase |
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WO2021140981A1 (ja) | 2020-01-10 | 2021-07-15 | 国立研究開発法人理化学研究所 | 左右非対称の糖鎖を有する抗体を均一に含む抗体集団、及びその製造方法 |
WO2022050300A1 (ja) | 2020-09-02 | 2022-03-10 | 第一三共株式会社 | 新規エンド-β-N-アセチルグルコサミニダーゼ |
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KR20180030536A (ko) | 2018-03-23 |
ES2848861T3 (es) | 2021-08-12 |
SG11201800394WA (en) | 2018-02-27 |
AU2016294206B2 (en) | 2021-12-02 |
CN108026518A (zh) | 2018-05-11 |
JP6752203B2 (ja) | 2020-09-09 |
EP3323886B1 (en) | 2020-10-21 |
EP3323886A4 (en) | 2019-02-27 |
JPWO2017010559A1 (ja) | 2018-04-26 |
US20180208915A1 (en) | 2018-07-26 |
US11001819B2 (en) | 2021-05-11 |
EP3323886A1 (en) | 2018-05-23 |
KR102546854B1 (ko) | 2023-06-22 |
TW201708540A (zh) | 2017-03-01 |
AU2016294206A1 (en) | 2018-02-08 |
HK1248271A1 (zh) | 2018-10-12 |
CN108026518B (zh) | 2022-09-06 |
AU2016294206B9 (en) | 2021-12-23 |
SG10201914047RA (en) | 2020-03-30 |
TWI730972B (zh) | 2021-06-21 |
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