DETAILED ACTION
Applicant’s amendment submitted 4/15/2025 is acknowledged. Claim 1 currently amended. Claims 2-4, 6-7, and 9-13 are canceled. Claims 14-15 remain withdrawn pursuant to 37 CFR 1.142(b) as being drawn to a non-elected invention. Claims 1, 5, 8, and 14-17 remain pending in the instant application.
Claims 1, 5, 8, and 16-17 are the subject of this office 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and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114.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 4/15/2026 has been entered.
Priority
The instant application is a U.S. National Phase of PCT/EP2019/062160 filed on 5/13/2019 and claims priority to EP18172658.9 filed on 5/16/2018. A certified copy of the foreign priority in English has been received. The effective priority date of the instant claims is 5/16/2018.
Claim Rejections - 35 USC § 103
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
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.
The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. Considering objective evidence present in the application indicating obviousness or nonobviousness.
This application currently names joint inventors. In considering patentability of the claims the examiner presumes that the subject matter of the various claims was commonly owned as of the effective filing date of the claimed invention(s) absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and effective filing dates of each claim that was not commonly owned as of the effective filing date of the later invention in order for the examiner to consider the applicability of 35 U.S.C. 102(b)(2)(C) for any potential 35 U.S.C. 102(a)(2) prior art against the later invention.
Claims 1, 16, and 17 are rejected under 35 U.S.C. 103 as being unpatentable over to Jennewein (WO2015/036138A1; Of Record) in view of Ashida et al. 2009 (Glycobiology, Vol. 19(9), p.1010-1017; Of Record).
Regarding claims 1, 16 and 17, Jennewein teaches a process for the production of oligosaccharides via microbial fermentation (see paragraph [0001]). The process of producing oligosaccharides includes using a host microorganism which can endogenously produce a glycosidase that is not naturally occurring in the host cell (i.e. heterologous), the glycosidase being capable of degrading saccharide intermediates, saccharide side products, and unused saccharide substrates (see paragraphs [0029] and [0042]-[0043] and [0063]). This is interpreted as providing a microbial host cell genetically engineered to express at least one heterologous glycosidase which is able to intracellularly degrade metabolic saccharide by-products that are generated during the intracellular biosynthesis of the desired oligosaccharide, and wherein the microbial host cell is able to recycle the degradation products resulting from the enzymatic activity of said glycosidase for the production of the desired oligosaccharide as claimed. Jennewein teaches a genetically-engineered E. coli strain for synthesizing 2’-fucosyllactose (see paragraph [0090]). The E. coli strain is BL21 (DE3) and contains a genomic integration of the wbgL gene, which the instant specification at p.34, lines 30-31, teaches is the gene for the enzyme α-1,2-fucosyltransferase. Jennewein further teaches that in some embodiments a microorganism can be employed which is genetically modified to express a fucose kinase and a guanyltransferase in which the bacterial enzyme Fkp is used and represents the first identified bifunctional enzyme with both fucose kinase and L-fucose-1P-guanyltransferase (see paragraph [0065]). Jennewein further teaches that the glycosidase is selected from the group comprising galactosidases (such as E. coli beta-galactosidase), glucosidases, fucosidases, and sialidases which reads on the limitation of wherein the heterologous glycosidase is a fucosidase (see paragraphs [0054]-[0055]). Jennewein teaches the host microorganism is cultivated under conditions and in a medium permissive for the production of said oligosaccharide and the desired oligosaccharide is recovered (see paragraphs [0014], and [0042]-[0043]). Jennewein further teaches that the desired oligosaccharide can be 2'-fucosyllactose (2'-FL), 3-fucosyllactose (3-FL), 3’-sialyllactose (3'-SL), 6'-sialyllactose (6'-SL), 3-fucosyl-3'-sialyllactose (F-SL), lacto-N-tetraose ((LNT), lacto-N-neotetraose (LNneoT), lacto-N-fucopentaose(s) (LNFP-I, II, III, V), lacto-N-difucohexaose(s) (LNDH-I and II) which are human milk oligosaccharides and read on limitations of claim 1(see paragraphs [0053] and [00102]).
Jennewein does not teach wherein the genetically-engineered microbial host cell has been genetically engineered to express: a) a heterologous α-1,3-fucosyltransferase and a heterologous α-1,2-fucosidase, or b) a heterologous α-1,2-fucosyltransferase and a heterologous α-1,3-fucosidase as claimed.
Ashida teaches two distinct α-L-fucosidases from Bifidobacterium bifidum that are essential for the utilization of fucosylated milk oligosaccharides (see Abstract). Ashida teaches that the core structures of human milk oligosaccharides have α1,2-, α1,3-, and α1,4-fucosyl residues at the nonreducing termini and the removal of these fucosyl residues may be essential for further degradation of HMOs (see p.1011, left column, 1st passage). Ashida teaches cloning the gene afcB in the E. coli strain BL21 (DE3) and found that the heterologous enzyme AfcB is able to hydrolyze α1,3- and α1,4-fucosidic linkages (see p.1012, left column, 2nd passage, and right column, 2nd passage). The full sequence (1493 bp) of the afcB gene disclosed in Ashida has 100% sequence identity to SEQ ID NO: 4 of the claimed invention (see Appendix A). Ashida teaches a fragment from base pairs 38-1468 that excludes an N-terminal signal peptide and C-terminal transmembrane region was cloned in the E. coli strain BL21 (DE3) (see p.1015, right column, 3rd passage). The instant specification at p.19, lines 3-4, teaches that the enzyme AfcB is an α-1,3-fucosidase. The instant specification also confirms that B. bifidum AfcB expressed in E. coli DE3 possesses intracellular by-product degradation activity (see Examples 2-4 and Table 1).
Therefore, it would have been obvious to one of ordinary skill in the art, before the effective filing date of the claimed invention, to have expressed the full afcB gene, as taught by Ashida, in the genetically-engineered E. coli host microorganism, as taught by Jennewein, to arrive at the claimed invention. One of ordinary skill in the art would have been motivated to because Ashida teaches the fucosidase AfcB is essential for the utilization of fucosylated milk oligosaccharides and Jennewein teaches the genetically-engineered host microorganism can endogenously produce a glycosidase, such as a fucosidase, capable of degrading saccharide intermediates, saccharide side products, and unused saccharide substrates. One of ordinary skill in the art would have had a reasonable expectation of success modifying the genetically-engineered microbial host as each of Jennewein and Ashida teach their methods in the DE3 strain of E. coli. The proposed modification above yields a genetically-modified E. coli strain that has the heterologous genes fkp, wbgL (codes an α-1,2-fucosyltransferase), and afcB (codes an α-1,3-fucosidase) integrated which reads on the limitations of claim 1. Thus, claims 1, 16, and 17 are prima facie obvious.
Claim 5 is rejected under 35 U.S.C. 103 as being unpatentable over Jennewein (WO2015/036138A1; Of Record) in view of Ashida et al. 2009 (Glycobiology, Vol. 19(9), p.1010-1017; Of Record), as applied to claims 1 and 16 above, and further in view of Endo et al. (Curr. Opin. Struc. Biol., 2000, Vol. 10, p.536-541; Of Record).
Jennewein in view of Ashida teach the method of claim 1 as outlined in the rejection above.
Regarding claim 5, Jennewein teaches that oligosaccharides can be synthesized de novo using glycosyltransferases (see paragraph [0003]).
Jennewein and Ashida do not teach the host microorganism has been genetically engineered to express a heterologous glycosyltransferase selected from the group consisting of galactosyltransferases, N-acetyl-glucosaminyltransferases, and glucosyltransferases.
Endo teaches large-scale production of oligosaccharides using glycosyltransferases in engineered bacteria (see Abstract). Specifically, Endo teaches cloning galactosyltransferase genes from several bacteria in E. coli for the synthesis of oligosaccharides (see p.537, right column, 1st-2nd passages).
Therefore, it would have been obvious to one of ordinary skill in the art, before the effective filing date, to have expressed a galactosyltransferase, as taught by Endo, in the host microorganism of Jennewein as modified by Ashida, to arrive at the claimed invention. One of ordinary skill would have been motivated to do so because Endo taught that heterologous galactosyltransferases expressed in E. coli were capable of producing oligosaccharides and would have yielded predictable results. One of ordinary skill would have had a reasonable expectation of success as Jennewein taught that oligosaccharides could be synthesized de novo by glycosyltransferases and the host microorganism of Jennewein is also E. coli. Thus, claim 5 is prima facie obvious.
Claim 8 is rejected under 35 U.S.C. 103 as being unpatentable over Jennewein (WO2015/036138A1; Of Record) in view of Ashida et al. 2009 (Glycobiology, Vol. 19(9), p.1010-1017; Of Record), as applied to claims 1 and 16 above, and further in view of Sprenger et al. (J. Biotechnol., 2017, Vol. 258, p.79-91; Of Record on the IDS submitted 11/12/2020).
Jennewein in view of Ashida teach the method of claim 1 as outlined in the rejection above. The host organism taught by Jennewein and Ashida is a genetically-engineered E. coli comprising the integration of the heterologous genes fkp, wbgL (codes an α-1,2-fucosyltransferase), and afcB (codes an α-1,3-fucosidase).
Jennewein and Ashida do not teach the microbial host cell has been engineered to express a heterologous β-1,3-N-acetylglucosaminyltransferase and a heterologous β-1,3-galactosyltransferase.
Sprenger teaches that β1,3-N-acetylglucosaminyltransferases, β1,3-galactosyltransferases, β1,4-galactosyltransferases, α2,3-sialyltransferases, α2,6-sialyltransferases, α1,2-fucosyltransferases, α1,3-fucosyltransferases, and α1,4-fucosyltransferases, mainly from bacterial sources, have been used for the enzymatic
synthesis of HMO (see p.82, right column, 1st passage). Sprenger teaches the formation of the core structures, LNT or LNnT in recombinant bacteria requires the glycosyl transfer of N-acetylglucosaminyl and galactosyl moieties to the acceptor substrate, lactose, and that E. coli K-12 cells can provide intracellular UDP-Gal and UDP-GlcNAc from their central metabolic pathway, however they lack β1,3-N-acetylglucosaminyltransferase and β1,3- or β1,4-galactosyltransferase activities (see p.86, left column, last paragraph).
Therefore, it would have been obvious to one of ordinary skill in the art, before the effective filing date of the claimed invention, to have expressed β1,3-N-acetylglucosaminyltransferase and β1,3-galactosyltransferase, as taught by Sprenger, in the genetically-engineered E. coli for the production of human milk oligosaccharides, as taught by Jennewein and Ashida, to arrive at the claimed invention. One of ordinary skill would have been motivated because Sprenger teaches these enzymes are not present in E. coli and are needed for the synthesis of LNT or LNnT HMOs and Jennewein teaches the method of their invention can synthesize these HMOs, yielding predictable results. Thus, claim 8 is prima facie obvious.
Response to Arguments
Applicant's arguments filed 4/15/2026 have been fully considered but they are not persuasive.
In Applicant’s Remarks, see p.2, last paragraph,-p.3, 2nd paragraph, Applicant argues Ashida does not select to express the characterized, full-length afcB gene. Applicant argues the deletion of the N-terminal signal sequence and a C-terminal transmembrane domain provides a soluble AfcB variant. Applicant argues Ashida does not show a native B. bifidum AfcB can be expressed in E. coli and retain its activity. Applicant further argues, arguendo, that the expression of B. bifidum AfcB in E. coli would have unknown functions of the various deleted domains. Applicant further argues that the intracellular/extracellular location of a fucosidase expressed from a full-length afcB gene, as well as the enzymatic activity of such, and how those functions would be capable of combining would be unknown in view of the cited combination of references. Applicant further argues that one of ordinary skill in the art would determine from Ashida that attempted expression of the full-length [afcB] gene in E. coli is unlikely to produce a soluble or enzymatically active AfcB. This is not found persuasive.
While Ashida does teach a fragment from base pairs 38-1468 that excludes an N-terminal signal peptide and C-terminal transmembrane region was cloned in the E. coli strain BL21 (DE3), one of ordinary skill in the art would have found it obvious to express the full length polynucleotide. As evidenced in Appendix A, Ashida discloses the full length sequence of AfcB, and thus it was available to one of ordinary skill in the art. Ashida also teaches the heterologous expression of AfcB in E. coli DE3 and its retained hydrolytic activity (see p.1012, left column, 2nd passage, and right column, 2nd passage). While it is true that Ashida does not explicitly state the hydrolytic activity of AfcB expressed in E. coli DE3 is intracellular, MPEP 2112(II) provides that an inherent feature of the prior art need not be recognized at the relevant time and there is no requirement that a person of ordinary skill in the art would have recognized the inherent disclosure at the relevant time. Thus, Ashida does not need to expressly teach the intracellular environment where the AfcB expressed in E. coli DE3 is active, which is evidenced by the instant specification describing that B. bifidum AfcB expressed in E. coli DE3 possesses intracellular by-product degradation activity (see Examples 2-4 and Table 1). Furthermore, since Ashida discloses the N-terminal domain is the signal peptide and C-terminal domain is the transmembrane region, one of ordinary skill in the art would have had a reasonable predictability of the function and targeting of the full-length polynucleotide encoding AfcB. Since Ashida does not teach the N-terminal and C-terminal domains must not be cloned into E. coli, one of ordinary skill in the art would not have had any reason not to use the full-length afcB in the invention of Jennewein as set forth in the rejection above. Furthermore, Applicant provides no evidence that the domains excluded from the truncated afcB gene would impute functions other than those described in Ashida’s characterization and explanation of the excluded domains. As set forth in MPEP 716.01(c)(II), arguments by Applicant cannot take the place of evidence. Applicant’s arguments do not contend with the characterization of the excluded domains as provided in Ashida. For these reasons, the 35 U.S.C. 103 rejection of claims 1, 16, and 17 over Jennewein in view of Ashida is maintained.
In Applicant’s Remarks, see p.3, last paragraph, Applicant’s argument is directed to the same points concerning Ashida discussed above and further argues Endo does not remedy those deficiencies. This argument is not found persuasive for the reasons stated above, and the rejection under 35 U.S.C. 103 of claim 5 over Jennewein in view of Ashida and further in view of Endo is also maintained.
In Applicant’s Remarks, see p.4, 3rd paragraph, Applicant’s argument is directed to the same points concerning Ashida discussed above and further argues Sprenger does not remedy those deficiencies. This argument is not found persuasive for the reasons stated above, and the rejection under 35 U.S.C. 103 of claim 8 over Jennewein in view of Ashida and further in view of Sprenger is also maintained.
Conclusion
All claims are identical to or patentably indistinct from, or have unity of invention with claims in the application prior to the entry of the submission under 37 CFR 1.114 (that is, restriction (including a lack of unity of invention) would not be proper) and all claims could have been finally rejected on the grounds and art of record in the next Office action if they had been entered in the application prior to entry under 37 CFR 1.114. Accordingly, THIS ACTION IS MADE FINAL even though it is a first action after the filing of a request for continued examination and the submission under 37 CFR 1.114. See MPEP § 706.07(b). Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action. In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any nonprovisional extension fee (37 CFR 1.17(a)) pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action. In no event, however, will the statutory period for reply expire later than SIX MONTHS from the mailing date of this final action.
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/J.P.S./ Examiner, Art Unit 1651
/MELENIE L GORDON/ Supervisory Patent Examiner, Art Unit 1651
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