DETAILED ACTION
Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Continued Examination Under 37 CFR 1.114
A request for continued examination under 37 CFR 1.114, including the fee set forth in 37 CFR 1.17(e), was filed in this application after final rejection. Since this application is eligible for continued examination under 37 CFR 1.114, and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114. Applicant's submission filed on 31 Oct. 2025 has been entered.
Claims 1-16 are currently pending.
Claims 6 and 9-16 are withdrawn as being drawn to a nonelected invention (claims 9-13 and 15) and species (claims 6, 14 and 16).
Claims 1-5, 7 and 8 are considered here with respect to the elected species of glucosamine-oxazoline as the precursor, lactose-N-triose II as the product, SEQ ID 1 as the GH20 enzyme, and B-galactosidase as the secondary enzyme. The amendments to independent claim 1 do not exclude the elected species of SEQ ID 1 as the enzyme (the enzyme can comprise “an amino acid sequence identity of at least 70% to any of SEQ ID NOs: 5 to 8”), and as such the examination proceeds herein with respect to the originally elected species (see MPEP 803.02, III.).
Any rejection not reiterated herein has been withdrawn.
Response to Arguments
Applicant's arguments filed 31 Oct. 2025 have been fully considered but they are not persuasive.
Applicant argues that the PCT Written Opinion indicated that the claims are free of the prior art. This is not persuasive because the PCT Written Opinion is non-binding and is based on an inventive step standard which is different than the US obviousness standard (see MPEP 1893.03(e)).
Applicant further argues that the cited combination fails to teach the conversion of glucosamine-oxazoline to LNT II. This is not persuasive because Chen teaches that the enzyme BbhI is useful for catalyzing the production of LNT II via a transglycosylation reaction in which a GlcNAc donor is transferred to lactose (acceptor) via a 1-3 linkage, and Champion teaches that in the same general reaction (using the same BbhI enzyme to transfer a glycosyl donor group to a milk oligosaccharide acceptor, which would include lactose) the donor compound can be either GlcNAc as taught by Chen or glucosamine-oxazoline. It would have thus been obvious to use either GlcNAc or glucosamine-oxazoline as the donor to form LNT II as taught by Chen.
Applicant further argues that Champion “is not related to a reaction for the purpose of making lacto-N-tetraose or lacto-N-triose II and does not disclose or suggest reacting with lactose”, and that it is unclear why one of ordinary skill looking to improve the process of Chen would look to Champion. This is not persuasive because Chen teaches that LNT II “is the backbone structure of one group of human milk oligosaccharides (HMOs) and thus can be elongated with sugar modifications to produce HMOs that can be used as prebiotics” (Chen, p. 5643, 3rd full ¶), and Champion teaches carrying out essentially the same transglycosylation reaction as Chen for the same purpose of making HMOs useful as prebiotics (Champion, [0002]-[0008]). Thus, one of ordinary skill practicing the methods of Chen and Champion would have reasonably consulted each other to investigate options for carrying out the reaction, including selection of donor compounds.
Applicant further argues that Champion discloses a large number of potential donor compounds (citing [0048] of Champion), and it is unclear why one of ordinary skill would have been led to the particular donor compounds recited in the claims. This is not persuasive because [0048] of Champion teaches four general classes of donor compounds (structures 1-4 in [0048]), and it would have been obvious that any of such compounds could be utilized as a glycosyl donor. A prima facie case of obviousness does not require an express motivation in Champion or elsewhere in the art to use a specific donor compound over another.
Applicant further argues that the claimed method produces unexpected results, as evidenced by [0051] and Table 1 of US publication US20220259580. This is not persuasive because any evidence of secondary considerations must be attributable to the claimed invention and the claims must be commensurate in scope with such evidence (see MPEP 2145). The evidence cited by Applicant at [0051] of US20220259580 shows that two specific variants of BbhI (D746E and Y827F) showed significantly enhanced transglycosylation and selectivity (RTH). However, the claims are currently being examined with respect to the elected species of the GH20 enzyme of SEQ ID 1, which does not include the D746E and/or Y827F substitutions (the amendments to independent claim 1 do not exclude the elected species of SEQ ID 1, as the enzyme can comprise “an amino acid sequence identity of at least 70% to any of SEQ ID NOs: 5 to 8”). Applicant further points to Figs. 6 and 7, but such results are derived using a different enzyme (LnbB) than in the instant claims. Thus, the asserted evidence of unexpected results is not attributable to the claimed invention, as required for establishing unexpected results.
Applicant also bears the burden of producing objective evidence establishing that the differences in results are in fact unexpected and unobvious (MPEP 716.02(b)). Such evidence must be factually supported by an appropriate affidavit or declaration to be of probative value (MPEP 716.01 (a)). The arguments of counsel cannot take the place of evidence in the record (MPEP 716.01 (a)). Applicant's response does not include any objective evidence indicating why one of ordinary skill would consider the asserted results unexpected and consists entirely of unsupported arguments of counsel.
Applicant further argues that the amendments to claim 1 render the claims commensurate in scope with the asserted unexpected results. This is not persuasive because the claims are not limited to the D746E and Y827F variants asserted to have enhanced transglycosylation and selectivity, but rather include any enzyme comprising “an amino acid sequence identity of at least 70% to any of SEQ ID NOs: 5 to 8”. SEQ IDs 5-8 are truncated versions of Bbhl that differ from SEQ ID 1 only in missing the N-terminal 32 amino acids of SEQ ID 1 and having mutations at D746 or Y827 (Spec., [0048]). Thus, the claims encompass a large genus of Bbhl enzymes which do not require a mutation at D746 or Y827, let alone the D746E and Y827F mutations shown to have enhanced activity/selectivity in the specification. Further, the claims encompass a D476Q variant, which is shown in the specification to abolish the desired transglycosylation activity (Spec., [0051] and Table 1). As such, the claims are not commensurate in scope with the asserted evidence of unexpected results at [0051] and Table 1 of the specification.
Claim Rejections - 35 USC § 112(a) (new matter)
The following is a quotation of the first paragraph of 35 U.S.C. 112(a):
(a) IN GENERAL.—The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor or joint inventor of carrying out the invention.
Claims 1-5, 7 and 8 are rejected under 35 U.S.C. 112(a) as failing to comply with the written description requirement. The claim(s) contains subject matter which was not described in the specification in such a way as to reasonably convey to one skilled in the relevant art that the inventor or a joint inventor, or for applications subject to pre-AIA 35 U.S.C. 112, the inventor(s), at the time the application was filed, had possession of the claimed invention.
Claim 1 has been amended to recite that the GH20 enzyme can be any enzyme having at least 70% identity to SEQ ID 1 and having a mutation at positions 746 and/or 827, or any enzyme having at least 70% identity to SEQ IDs 5-8. SEQ IDs 5-8 are truncated versions of Bbhl that differ from SEQ ID 1 only in missing the N-terminal 32 amino acids of SEQ ID 1 and having mutations at D746 or Y827 (Spec., [0048]). SEQ ID 1 is a 1627-residue protein. Thus, the at least 70% identity limitation in the claims defines a large genus of amino acid sequences.
The written description requirement for a claimed genus may be satisfied through sufficient description of a representative number of species sufficient to show the applicant was in possession of the claimed genus (MPEP 2163). A “representative number of species” means that the species which are adequately described are representative of the entire genus (Id.). The instant specification discloses the sequence of Bbhl and variants thereof with mutations at D746 or Y827, along with a sequence alignment of several homologous enzymes known in the art (Spec., Fig. 16 and SEQ IDs 12-24). However, the specification does not provide any additional species variants falling within the claimed genus of Bbhl enzymes capable of carrying out the claimed transglycosylation reaction using glucosamine-oxazoline as donor substrate, nor any other guidance such as a structure-function relationship or other
indication of what portions of the sequence are structurally important and/or subject to modification. As such, one of ordinary skill would not regard the specification as evidencing possession of the full scope of the claimed genus involving enzymes having at least 70% identity to SEQ IDs 1 and/or 5-8.
Claim Rejections - 35 USC § 103
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
Claims 1-5 and 7 are rejected under 35 U.S.C. 103 as being unpatentable over the combination of Chen et al., Applied and Environmental Microbiology 82.18 (2016): 5642-5652 in view of US20150182549 to Champion et al., optionally evidenced by Slámová et al., Biotechnology Advances 28.6 (2010): 682-693 (each previously cited).
Regarding claims 1 and 5, Chen teaches a method for producing lacto-N-triose II (LNT II) comprising, reacting N-acetyl-glucosamine (GlcNAc) as donor with lactose as acceptor, catalyzed by a glycoside hydrolase family 20 (GH20) enzyme to obtain lacto-N-triose II (p. 5644-5655, under Synthesis of HexNAc-Lac by BbhI; p. 5646, under HexNAc-Lac synthesis by BbhI; see also, instant Spec., [0006] referring to “Xiao and co-workers”). The GH20 enzyme is a β-N-acetylhexosaminidase from Bifidobacterium bifidum JCM1254, BbhI (Genbank accession BAI94822.1; see attached), which has 100% sequence identity to SEQ ID 1 (see attached alignment).
Regarding claim 2, the reaction was performed in an aqueous solution with lactose concentrations of 50-500 mM, which overlaps and renders obvious the claimed range of at least 50 g/L (approximately 150 mM) (Fig. 2C; p. 5644-5655, under Synthesis of HexNAc-Lac by BbhI; p. 5646, under HexNAc-Lac synthesis by BbhI).
Regarding claim 7, Chen teaches further teaches stopping the reaction by heating (p. 5645, 1st ¶).
Claims 1-5 and 7 differ from Chen in that: the donor substrate is glucosamine-oxazoline instead of GlcNAc (claim 1); the method is performed under conditions which are free or essentially free of organic solvent (claim 3); and the glucosamine-oxazoline is added to the enzyme/lactose over a period of at least 20 minutes (claim 4).
Champion teaches a method of producing modified milk oligosaccharides that is substantially similar to the reaction taught by Chen, comprising reacting a donor glycoside with an oligosaccharide acceptor in the presence of a GH20 enzyme which can be the same β-N-acetylhexosaminidase from Bifidobacterium bifidum JCM 1254 (BbhI; Accession no. 292673292 in Table 5 of Chen is the same Genbank accession BAI94822.1 as in Chen), such that a GlcNAc group is added via a 1-3 linkage to the acceptor (i.e. to form LNT II when lactose is the acceptor) ([0009]-[0019]; [0038]-[0059]; [0130]-[0152]). Champion teaches that the donor compound can be either GlcNAc as taught by Chen or glucosamine-oxazoline ([0048]-[0053], compounds 3-5).
It would have been obvious to one of ordinary skill in the art at the time the invention was made to use the method of Chen to produce LNTII by reacting a donor glycoside with lactose as acceptor in the presence of BbhI wherein the donor glycoside is glucosamine-oxazoline because it would have been obvious to combine prior art elements according to known methods to yield predictable results. One of ordinary skill would have been motivated to use glucosamine-oxazoline as donor in the reaction of Chen in order to achieve production of LNTII using a wider range of starting materials. Moreover, Chen teaches that LNT II “is the backbone structure of one group of human milk oligosaccharides (HMOs) and thus can be elongated with sugar modifications to produce HMOs that can be used as prebiotics” (Chen, p. 5643, 3rd full ¶), and Champion teaches carrying out essentially the same transglycosylation reaction as Chen for the same purpose of making HMOs useful as prebiotics (Champion, [0002]-[0008]). Using glucosamine-oxazoline as donor in the reaction of Chen would have led to predictable results with a reasonable expectation of success because Champion teaches the same general reaction with the same BbhI enzyme in which the donor compound can be either GlcNAc or glucosamine-oxazoline. Moreover, Slamova evidences that GH20 β-N-acetylhexosaminidase catalysis proceeds via an oxazoline intermediate (Fig. 1B), and thus one would expect BbhI to be permissive to both GlcNAc and glucosamine-oxazoline as the donor.
Regarding claim 3, while the reaction medium of Chen does include the organic solvent DMSO because “addition of organic solvents can increase the solubility of a substrate and reduce water activity, promoting transglycosylation over hydrolysis” (Chen, p. 5649, 1st full ¶), Champion teaches that the inclusion of organic solvent in the reaction medium is optional (Champion, [0120]), and it would have been obvious to exclude such solvent in order to reduce toxicity of the product, e.g. for food compositions of the type taught by Champion (e.g., [0159]-[0167]). Moreover, the instant specification states that “essentially free” means “that at any given point in time, in the reaction mixture of step (i) … there is 10 g/L or less … of an organic solvent present” (US20220259580, [0019]). Champion further teaches that an organic solvent can be used at a concentration range of 0.1 to 50 ml/l ([0120]), and it would have been obvious in view of Champion to use routine experimentation to minimize the concentration of organic solvent for the same purpose of minimizing toxicity of products intended for administration or consumption, as set forth above.
Regarding claim 4, Champion teaches that the reaction can be carried out in a continuous mode with “continuous flow of compounds and/or enzymes as necessary” ([0124]), and it would have been obvious to one of ordinary skill that the rate of feeding and time period could be varied using routine experimentation as needed to achieve a desired productivity or accommodate a desired reaction volume, etc.
Claim 8 is rejected under 35 U.S.C. 103 as being unpatentable over the combination of Chen in view of Champion, as applied to claims 1-5 and 7, further in view of EP2722394 to Hilla et al. (previously cited).
Claim 8 differs from the combination of Chen in view of Champion, as applied to claims 1-5 and 7, in that: the method further comprises adding a β-galactosidase or a galactosyl transferase and UDP-galactose to obtain lacto-N-tetraose or lacto-N-neotetraose.
Hilla teaches a method for making lacto-N-tetraose comprising i) adding lactose and N-acetylglucosamine in the presence of beta-(1,3)-N-acetylglucosaminyltransferase to form lacto-N-triose, as in the cited combination of Chen in view of Champion; and ii) adding galactose in the presence of beta-galactosyltransferase to form lacto-N-tetraose and/ or lacto-N-neotetraose ([0015]). Hilla teaches that the beta-galactosyltransferase enzyme can be a beta-galactosidase ([0021]-[0022]). Hilla further teaches that both lacto-N-triose and lacto-N-tetraose are of commercial interest since they are nutritionally important constituents of human breast milk which are absent from cow milk ([0005]-[0006]).
It would have been obvious to one of ordinary skill in the art at the time the invention was made to use the method Chen in view of Champion to make LNT II by reacting glucosamine-oxazoline and lactose in the presence of BbhI wherein the method further comprises converting the LNT to lacto-N-tetraose and/ or lacto-N-neotetraose in the presence of a galactosyltransferase as taught by Hilla because it would have been obvious to combine prior art elements according to known methods to yield predictable results. One of ordinary skill would have been motivated to further convert LNT obtained by the method of Chen in view of Champion to lacto-N-tetraose and/ or lacto-N-neotetraose as taught by Hilla because Hilla teaches that lacto-N-tetraose is a nutritionally important milk oligosaccharide that is lacking in commercial cow milk. Converting LNT obtained by the method of Chen in view of Champion to lacto-N-tetraose and/ or lacto-N-neotetraose as taught by Hilla would have led to predictable results with a reasonable expectation of success because Hilla teaches that the first step of the method comprises the same general reaction using the same enzyme as in Chen in view of Champion, and it would have been obvious that the LNT product of such reaction could be further converted to lacto-N-tetraose and/ or lacto-N-neotetraose as taught by Hilla (e.g., after optionally deactivating the BbhI).
Conclusion
No claim is allowed.
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/ROBERT J YAMASAKI/Primary Examiner, Art Unit 1657