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 .
Status of Claims
Receipt of Arguments/Remarks filed on 11/25/2025 is acknowledged. Claims 62-79, 81-83, 103, and 105 are pending. Claims 62, 63, 66, 67, 74, 77, 78, and 83 were amended. Claim 80 was canceled. New claim 105 was added.
Withdrawn Rejections
The rejections of claims 62-83 and 103 under 35 U.S.C. § 112(b) are withdrawn.
The rejection of claim 74 under 35 U.S.C. § 112(d) is withdrawn.
Maintained and modified rejections necessitated by amendment
Claim Rejections - 35 USC § 112
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.
The following is a quotation of the first paragraph of pre-AIA 35 U.S.C. 112:
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 of carrying out his invention.
Claims 67 and 105 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, 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.
Regarding written description, 35 U.S.C. 112(a) and the first paragraph of pre-AlA 35 U.S.C. 112 require that the "specification shall contain a written description of the invention ...." This requirement is separate and distinct from the enablement requirement. Ariad Pharm., Inc. v. Eli Lilly & Co., 598 F.3d 1336, 1340, 94 USPQ2d 1161, 1167 (Fed. Cir. 2010). To satisfy the written description requirement, a patent specification must describe the claimed invention in sufficient detail that one skilled in the art can reasonably conclude that the inventor had possession of the claimed invention (MPEP § 2163(I)).
MPEP 2163(II)(A)(3)(a)(i and ii) states that the written description requirement for a claimed genus may be satisfied through sufficient description of a representative number of species by actual reduction to practice, reduction to drawings, or by disclosure of relevant, identifying characteristics, i.e., structure or other physical and/or chemical properties, by functional characteristics coupled with a known or disclosed correlation between function and structure, or by a combination of such identifying characteristics, sufficient to show the applicant was in possession of the claimed genus.
A "representative number of species" means that the species which are adequately described are representative of the entire genus. See AbbVie Deutschland GmbH & Co., KG v. Janssen Biotech, Inc., .759 F.3d 1285, 1300, 111 USPQ2d 1780, 1790 (Fed. Cir. 2014). Thus, when there is substantial variation within the genus, one must describe a sufficient variety of species to reflect the variation within the genus.
Claims 67 and 105 recite an alpha-1,2-fucosyltransferase or alpha-1,3-fucosyltransferase (ii) comprising or consisting of a polypeptide comprising an amino acid sequence having at least 80% sequence identity to the full-length amino acid sequence of any one of SEQ ID NOs: 16 to 110 (α-1,2) or SEQ ID NOs: 111-132 (α-1,3) and having alpha-1,2- or alpha-1,3-fucosyltransferase activity on lactose and optionally on one or more further acceptor(s).
There is not sufficient written description support for a polypeptide variant with 80% identity to SEQ ID NOs: 16-110 or 111-132. For example, elected SEQ ID NO: 16 comprises 299 amino acids. This means that anywhere between 1-59 residues of SEQ ID NO: 16 could be modified or substituted relative to the wild type sequence within the scope of 80% identity. Given that there are 19 alternative amino acids that could be substituted at any of these positions, 80% identity encompasses millions of potential amino acid sequences.
The specification does not disclose a structure-function relationship for this large genus of fucosyltransferases. There is no disclosure of what positions or residues can or cannot be mutated, deleted, or truncated in any of these sequences while still maintaining function. 80% identity encompasses countless potential sequences, and it would not be clear to a skilled artisan which of these potential variants would maintain enzyme function. The disclosure has support for the specifically disclosed sequences: SEQ ID NOs: 16-110 or 111-132. However, there are a vast number of sequences encompassed within 80% identity, and these disclosed sequences are not representative of all of the possible sequences having 80% identity to the claimed sequences.
For these reasons, claims 67 and 105 fail to comply with the written description requirement.
Claim Rejections - 35 USC § 102
The text of those sections of Title 35, U.S. Code not included in this action can be found in a prior Office action.
Claims 62, 64-76, 78-79, and 82 are rejected under 35 U.S.C. 102(a)(1) as being anticipated by Jennewein et al., US 2019/0309336 A1.
Regarding claims 62 and 66, Jennewein teaches a method for producing fucosylated oligosaccharides, including difucosyllactose (Jennewein pg. 2 para. 24). See instant specification pg. 16 which states that the terms difucosyllactose and 2,3-difucosyllactose or DiFL are used interchangeably.
Jennewein teaches that the method involves providing an α-1,2-fucosyltransferase and an α-1,3-fucosyltransferase and a GDP-fucose donor by using a host cell that is genetically modified to express fucosyltransferases (Jennewein pg. 1 para. 11). For production of 2’3-difucosyllactose, both an α-1,2- and an α-1,3-glycosyltransferase are expressed (Jennewein pg. 3 para. 41). Jennewein teaches that fucosyltransferases are enzymes that transfer a L-fucose sugar from a GDP-fucose donor to an acceptor substrate (Jennewein pg. 3 para. 38).
Jennewein teaches contacting the cells expressing the fucosyltransferases and the GDP-fucose donor with lactose as an acceptor and cultivating the cells (Jennewein pg. 1 para. 13). Jennewein teaches that cultivating means growing or incubating a cell in a medium under conditions suitable for the production of a desired oligosaccharide, i.e. expressing fucosyltransferases and producing GDP-fucose donor (Jennewein pg. 3 para. 32). As set forth in the instant specification, “reaction mixture” is interchangeable with “cultivation” in the context of a cell-based method (see instant specification p. 25 lines 30-34). Thus, the cultivation taught by Jennewein is considered equivalent to a “reaction mixture”.
Jennewein teaches that the cells are cultivated for at least 60, 80, 100, or 120 hours, which is equivalent to 2.5 to 5 days (Jennewein p. 7 para. 89; claim 18). Jennewein teaches that DiFL production occurs when L-fucose is transferred to 2’-fucosyllactose (2’FL) by a fucosyltransferase catalyzed reaction (Jennewein pg. 9 para 124). Jennewein teaches that with the method according to the invention, as well as with the genetically modified host cell employed in the method, it is possible to produce fucosylated oligosaccharides at a titer exceeding 50 g/L, and even 100 g/L, and even more 150 g/L, thus providing a successful tool for the large-scale and, thus, industrial-scale fermentative production of fucosylated oligosaccharides (Jennewein p. 2 para. 23).
Jennewein teaches that the fucosylated oligosaccharides can be recovered from the cultivation medium, and that “recovering” includes separating (Jennewein pg. 1 para. 14; pg. 4 para. 34).
Regarding claim 64, Jennewein teaches that the fucosyltransferases and GDP-fucose are produced by a bacterial cell that is genetically modified to encode an enzyme for overexpression of GDP-fucose and exogenous fucosyltransferase genes (Jennewein pg. 1 para. 11).
Regarding claim 65, Jennewein teaches that the acceptors can include lactose, 2’FL, and 3-FL (Jennewein pg. 3 para. 39).
Regarding claim 67, Jennewein teaches that the α-1,2-fucosyltransferase is preferably the fucT2 gene Helicobacter pylori, which has the UniProt ID Q9X3N7 (Jennewein pg. 7 para. 96). SEQ ID NO: 16, which is an α-1,2-fucosyltransferase from H. pylori with UniProt ID Q9X435, and fucT2 from H. pylori, are 93% identical, and therefore fucT2 as taught by Jennewein is a polypeptide with at least 80% sequence identity to SEQ ID NO: 16. Additionally, fucT2 from H. pylori is 100% identical to instant SEQ ID NO: 108.
Jennewein teaches that the α-1,3-fucosyltransferase is derived from Akkermansia muciniphila, Bacteroides fragilis, Helicobacter pylori, or Helicobacter hepaticus (Jennewein pg. 7 para. 96). Instant SEQ ID NO: 111 is the α-1,3-fucosyltransferase fucT from H. pylori, UniProt ID O30511. Instant SEQ ID NO: 112 is another α-1,3-fucosyltransferase from H. pylori, UniProt ID O25142. As there are a finite, limited number of H. pylori α-1,3-fucosyltransferases, a skilled artisan could at once envisage the use of α-1,3-fucosyltransferases having SEQ ID NOs: 111 and 112 based on the disclosure of Jennewein. See MPEP 2131.02.
Therefore, the teachings of Jennewein anticipate a cell expressing an α-1,2-fucosyltransferase comprising SEQ ID NO: 108 or a functional homolog of SEQ ID NO: 16 with at least 80% identity, as well as an α-1,3-fucosyltransferase from H. pylori, comprising SEQ ID NOs: 111 or 112.
Regarding claim 68, Jennewein teaches that lactose is generated internally by the cell during cultivation, i.e. that the cell produces lactose, for the synthesis of 2’FL, 3-FL, and DiFL (Jennewein pg. 1 para. 15).
Regarding claim 69, Jennewein teaches that a precursor, i.e. GDP-fucose (see instant specification pg. 23) is used for synthesis of 2’FL, 3-FL, and DiFL (Jennewein pg. 3 para. 38).
Regarding claim 70, Jennewein teaches that the cell is modified in the expression of fucosyltransferases, as they are exogenously expressed in the cell (Jennewein pg. 1 para. 11).
Regarding claim 71, Jennewein teaches that the cell is metabolically engineered for oligosaccharide production (Jennewein pg. 1 para. 10).
Regarding claim 72, Jennewein teaches that the cell is modified to overexpress a gene encoding an enzyme for de novo synthesis of GDP-fucose, including a gene coding for GDP-L-fucose synthase (Jennewein pg. 1 para. 11, pg. 5 para. 66).
Regarding claim 73, resistance to lactose killing is a functional limitation of the cell used in the method for producing DiFL. The method involves the steps of providing a cell expressing at least 2 fucosyltransferases and contacting the fucosyltransferases and GDP-fucose with a lactose acceptor under conditions to catalyze the transfer of a fucose from GDP-fucose to the acceptor, as set forth in claim 64. Jennewein teaches such a cell cultivated according to these method steps, as discussed above. Therefore, it would be expected that a cell as recited in claim 64, cultivated according to the method of claim 62, would have the functional characteristic of resistance to lactose killing.
Further, the instant specification, page 27, provides a preferred embodiment of a cell resistant to lactose killing, comprising a cell with a genetic modification to express or overexpress a lactose transporter (for example, the lactose importer LacY from E. coli, instant specification page 90 line 20). The cell taught by Jennewein has E. coli LacY integrated and expressed (Jennewein pg. 8 para. 115). Therefore, the cell as taught by Jennewein would be expected to be resistant to lactose killing when grown in an environment where lactose is combined with other carbon sources.
Regarding claim 74, Jennewein teaches that the cell produces fucosylated oligosaccharides intracellularly, i.e. intracellular concentrations of 2’FL are detected (Jennewein pg. 9 para. 124). Jennewein teaches a fraction are transported out of the cell, i.e. DiFL and 2’FL are detected in the culture supernatant and therefore have been transported out of the cell (Jennewein pg. 9 para. 124; Fig. 2).
Regarding claims 75 and 76, Jennewein teaches that the cell is genetically modified to express a heterologous membrane protein, the sugar efflux transporter yberc0001_9420, which provides enhanced efflux of 2’FL to outside of the cell (Jennewein pg. 9 para. 124; “Abstract”).
Regarding claim 78, Jennewein teaches adding an acceptor, lactose, to the culture medium for producing the fucosylated oligosaccharides (Jennewein pg. 7 para. 87).
Regarding claim 79, Jennewein teaches that the cell is cultivated in a medium containing glycerol as a carbon source (Jennewein pg. 7 para. 106; pg. 9 para. 126).
Regarding claim 82, Jennewein teaches that the oligosaccharides can be recovered, or purified, from the microorganism culture (Jennewein pg. 3 para. 34).
Claim Rejections - 35 USC § 103
The text of those sections of Title 35, U.S. Code not included in this action can be found in a prior Office action.
Claims 63, 83, and 103 are rejected under 35 U.S.C. 103 as being unpatentable over Jennewein et al. US 2019/0309336 A1.
Jennewein teaches the method of claim 62 as set forth above. Jennewein does not expressly teach that the DiFL is present in the mixture of oligosaccharides at a purity or amount of 80% based on the total amount of 2’FL, 3-FL, and DiFL in the mixture as recited in claims 63 and 83, or that the acceptor is completely converted to an oligosaccharide as recited in claim 103.
Regarding claim 63, a “purity of at least 80%” is understood as the amount of DiFL in the mixture of 2'FL, 3-FL and DiFL of 80% or more (instant specification pg. 26), i.e. the concentration of DiFL compared to other oligosaccharides. While Jennewein does not expressly teach DiFL present at 80% in the oligosaccharide mixture, the concentration of various oligosaccharides is a result of the claimed method, the steps of which are taught by Jennewein as set forth above. Additionally, Jennewein teaches that DiFL is produced when L-fucose is transferred to 2’-fucosyllactose by a fucosyltransferase, and that higher concentrations of the 2’FL acceptor result in higher production of DiFL (Jennewein pg. 9 para. 124). Jennewein also teaches that the expression of an α-1,2- and α-1,3-fucosyltransferase in the cell is preferred for the production of DiFL (Jennewein pg. 3 para. 41).
It would have been obvious to a skilled artisan that the method as taught by Jennewein, expressing an α-1,2- and α-1,3-fucosyltransferase, could yield a mixture of DiFL, 2’FL, and 3-FL with 80% DiFL. Based on the teachings of Jennewein it is clear that cultivation conditions can be varied to obtain a desired amount of DiFL or other oligosaccharides. Thus, a skilled artisan would have a reasonable expectation that the method of Jennewein could produce a mixture of oligosaccharides with 80% DiFL. As Jennewein teaches the method steps as instantly claimed, it is expected that this method could similarly result in an oligosaccharide mixture comprising 80% DiFL.
Regarding claim 83, it would have been obvious to a skilled artisan to purify DiFL from the mixture comprising 80% DiFL, as purification or recovery of the oligosaccharides is taught by Jennewein (Jennewein pg. 1 para. 14, pg. 3 para. 34).
Regarding claim 103, Jennewein teaches that the fucosyltransferases transfer an L-fucose sugar from a GDP-fucose donor substrate to an acceptor substrate, thereby converting the acceptor into a fucosylated oligosaccharide (Jennewein pg. 3 para. 38). While Jennewein does not expressly teach that the acceptors are completely converted into 2’FL, 3-FL, or DiFL, it would have been obvious to a person having ordinary skill in the art that the method as taught by Jennewein could be carried out in order to completely convert the acceptors to the oligosaccharides, i.e. by reacting for sufficient time or providing sufficient amount of enzyme for all of the acceptor present in the reaction to be converted to the oligosaccharide of interest, as these are aspects of a method that would be routinely optimized by a person having ordinary skill in the art.
Claims 77, 81, and 105 are rejected under 35 U.S.C. 103 as being unpatentable over Jennewein et al. as applied to claims 62, 64-76, 78-79, and 82 above, and further in view of Petschacher et al., Journal of Biotechnology; 235:61-83.
Jennewein teaches the method of claim 62 as set forth above. Jennewein teaches that the production of DiFL requires both an α-1,2- and an α-1,3-glycosyltransferase with the claimed SEQ ID NOs, as set forth in the rejection of claim 67 (Jennewein pg. 3 para. 41). Jennewein teaches that the method involves a GDP-fucose donor and lactose (Jennewein pg. 1 para. 11, para. 13).
Jennewein does not teach that the fucosyltransferase and GDP-fucose are provided in a cell-free system as recited in claims 77 and 105. Jennewein does not teach that the separation comprises one of the techniques recited in claim 81.
Regarding claims 77 and 105, Petschacher teaches in vitro, or cell-free, production of fucosylated oligosaccharides including 2’FL and 3-FL using α-1,2- and α-1,3-fucosyltransferases and GDP-L-fucose (Petschacher pg. 71 Section 3.2. “In vitro production of fucosylated HMOs”; Table 4). Petschacher compares cell-free in vitro methods to whole cell systems for producing oligosaccharides, and teaches that in vitro methods have a unique advantage of flexibility, especially when characterizing new fucosyltransferases, or for smaller scale synthesis of complex human milk oligosaccharides (Petschacher pg. 72 first full para.).
Regarding claim 81, Petschacher teaches that conventional isolation of HMOs involves column chromatography using an activated charcoal step and further purification by size-exclusion chromatography (Petschacher pg. 71 first full para.).
It would have been obvious to a skilled artisan, before the effective filing date, to combine the teachings of Jennewein and Petschacher, using a cell-free system for producing of DiFL. Petschacher teaches both cell-free and cell-based systems of producing oligosaccharides. The use of isolated fucosyltransferase enzymes, rather than a cell expressing the fucosyltransferases, is established in the art as a method for producing human milk oligosaccharides. A skilled artisan would recognize that the method taught by Jennewein, wherein both α-1,2- and α-1,3-fucosyltransferases are contacted with GDP-fucose and lactose to produce DiFL, could be adapted as a cell-free system as taught by Petschacher for the production of oligosaccharides such as 2’FL and 3-FL.
Additionally, a skilled artisan would recognize that the techniques taught by Petschacher for separation of the oligosaccharides could be used in the cell-based method of Jennewein, as Petschacher teaches separation of oligosaccharides from mixtures obtained from both cell-free and cell-based methods.
A person of ordinary skill in the art would have been motivated to use a cell-free system for production of DiFL or other HMOs because Petschacher teaches that in vitro production of oligosaccharides offers unique advantages over cell-based methods, specifically flexibility when characterizing new fucosyltransferases, or for smaller scale synthesis of complex human milk oligosaccharides (Petschacher pg. 72 first full para.).
A skilled artisan would have a reasonable expectation of success in using a cell-free system as taught by Petschacher for producing DiFL, because cell-free enzymatic production of other fucosylated oligosaccharides is well-known and established in the art. A skilled artisan could reasonably expect success in using the α-1,2- and α-1,3-fucosyltransferases as taught by Jennewein in a cell-free system to yield the predictable result of production of DiFL by these fucosyltransferases. Additionally, a skilled artisan would have a reasonable expectation that the technique taught by Petschacher of isolating HMOs using column chromatography with an activated charcoal step and size-exclusion chromatography could be successfully used to separate the oligosaccharides produced by the method of Jennewein, as Petschacher teaches that this method has been used to purify fucosylated oligosaccharides.
Claim 67 is rejected under 35 U.S.C. 103 as being unpatentable over Jennewein et al. as applied to claims 62, 64-76, 78-79, and 82 above, and further in view of Whiteson et al., BMC genomics; 15(1):169.
Jennewein teaches the method of claim 64 as set forth above. Jennewein does not teach the elected α-1,3-fucosyltransferase corresponding with SEQ ID NO: 113.
Regarding claim 67, Whiteson teaches the genome of Basilea psittacipulmonis, which was annotated and deposited in NCBI under accession number AIL32582.1 (Whiteson “Abstract”). The polypeptide is identified as an α-1,3-fucosyltransferase FucT, and has a sequence that is 100% identical to instant SEQ ID NO: 113 (see Appendix with alignment below). As this polypeptide has the same sequence as instant SEQ ID NO: 113, it is expected to have the same α-1,3-fucosyltransferase activity.
It would have been obvious to a skilled artisan, before the effective filing date, to substitute the α-1,3-fucosyltransferases taught by Jennewein with the α-1,3-fucosyltransferase taught by Whiteson. This is considered a simple substitution of one known element for another having the same function, and there would be a reasonable expectation of obtaining predictable results when the α-1,3-fucosyltransferase according to SEQ ID NO: 113 is used according to the method taught by Jennewein, i.e. production of DiFL, 2’FL, or 3-FL. Jennewein discloses non-limiting examples of α-1,3-fucosyltransferases from various bacterial species that can be used in the method for producing DiFL, 2’FL, and 3-FL (Jennewein para. 42), so it would have been obvious that a different α-1,3-fucosyltransferase such as one from Basilea psittacipulmonis could be used in this method.
Double Patenting
The nonstatutory double patenting rejection is based on a judicially created doctrine grounded in public policy (a policy reflected in the statute) so as to prevent the unjustified or improper timewise extension of the “right to exclude” granted by a patent and to prevent possible harassment by multiple assignees. A nonstatutory double patenting rejection is appropriate where the conflicting claims are not identical, but at least one examined application claim is not patentably distinct from the reference claim(s) because the examined application claim is either anticipated by, or would have been obvious over, the reference claim(s). See, e.g., In re Berg, 140 F.3d 1428, 46 USPQ2d 1226 (Fed. Cir. 1998); In re Goodman, 11 F.3d 1046, 29 USPQ2d 2010 (Fed. Cir. 1993); In re Longi, 759 F.2d 887, 225 USPQ 645 (Fed. Cir. 1985); In re Van Ornum, 686 F.2d 937, 214 USPQ 761 (CCPA 1982); In re Vogel, 422 F.2d 438, 164 USPQ 619 (CCPA 1970); In re Thorington, 418 F.2d 528, 163 USPQ 644 (CCPA 1969).
A timely filed terminal disclaimer in compliance with 37 CFR 1.321(c) or 1.321(d) may be used to overcome an actual or provisional rejection based on nonstatutory double patenting provided the reference application or patent either is shown to be commonly owned with the examined application, or claims an invention made as a result of activities undertaken within the scope of a joint research agreement. See MPEP § 717.02 for applications subject to examination under the first inventor to file provisions of the AIA as explained in MPEP § 2159. See MPEP § 2146 et seq. for applications not subject to examination under the first inventor to file provisions of the AIA . A terminal disclaimer must be signed in compliance with 37 CFR 1.321(b).
The filing of a terminal disclaimer by itself is not a complete reply to a nonstatutory double patenting (NSDP) rejection. A complete reply requires that the terminal disclaimer be accompanied by a reply requesting reconsideration of the prior Office action. Even where the NSDP rejection is provisional the reply must be complete. See MPEP § 804, subsection I.B.1. For a reply to a non-final Office action, see 37 CFR 1.111(a). For a reply to final Office action, see 37 CFR 1.113(c). A request for reconsideration while not provided for in 37 CFR 1.113(c) may be filed after final for consideration. See MPEP §§ 706.07(e) and 714.13.
The USPTO Internet website contains terminal disclaimer forms which may be used. Please visit www.uspto.gov/patent/patents-forms. The actual filing date of the application in which the form is filed determines what form (e.g., PTO/SB/25, PTO/SB/26, PTO/AIA /25, or PTO/AIA /26) should be used. A web-based eTerminal Disclaimer may be filled out completely online using web-screens. An eTerminal Disclaimer that meets all requirements is auto-processed and approved immediately upon submission. For more information about eTerminal Disclaimers, refer to www.uspto.gov/patents/apply/applying-online/eterminal-disclaimer.
Claims 62, 64-72, 74-76, and 78 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claims 90-100 and 104-111 of copending Application No. 18/040,325 in view of Jennewein et al., US 2019/0309336 A1.
Regarding instant claim 62, claim 108 of ‘325 recites a method of producing a mixture of at least four different neutral fucosylated oligosaccharides by a cell, the method comprising: i) providing a cell that (a) is capable of expressing a glycosyltransferase being a fucosyltransferase and is capable of synthesizing a nucleotide-sugar GDP-fucose, (b) expresses at least one additional glycosyltransferase, and (c) is capable of synthesizing at least one or more nucleotide-sugar(s), wherein the nucleotide-sugar(s) is/are donor(s) for additional glycosyltransferases, and ii) cultivating the cell under conditions permissive to express the glycosyltransferases and to synthesize the nucleotide-sugars resulting in the cell producing the mixture of at least four different neutral fucosylated oligosaccharides (such as DiFL, 2’FL, 3-FL). Claims 105-107 of ‘325 recite that all of the neutral fucosylated oligosaccharides are lactose-based mammalian milk oligosaccharides and the precursor is lactose for producing a lactose-based oligosaccharides.
The claims of ‘325 do not recite that the method requires a 1-5 day cultivation and a yield of at least 50 g/L of DiFL (claim 62) or the fucosyltransferases of claim 67. However, these features are taught by Jennewein.
Regarding instant claim 62, Jennewein teaches a method for producing fucosylated oligosaccharides, including difucosyllactose (Jennewein pg. 2 para. 24), by providing an α-1,2-fucosyltransferase and an α-1,3-fucosyltransferase and a GDP-fucose donor using a host cell that is genetically modified to express fucosyltransferases (Jennewein pg. 1 para. 11). For production of 2’3-difucosyllactose, both an α-1,2- and an α-1,3-glycosyltransferase are expressed (Jennewein pg. 3 para. 41). Jennewein teaches contacting the cells expressing the fucosyltransferases and the GDP-fucose donor with lactose as an acceptor and cultivating the cells (Jennewein pg. 1 para. 13).
Jennewein teaches that the cells are cultivated for at least 60, 80, 100, or 120 hours, which is equivalent to 2.5 to 5 days (Jennewein p. 7 para. 89; claim 18). Jennewein teaches that with the method according to the invention, as well as with the genetically modified host cell employed in the method, it is possible to produce fucosylated oligosaccharides at a titer exceeding 50 g/L, and even 100 g/L, and even more 150 g/L (Jennewein p. 2 para. 23). Jennewein teaches that the fucosylated oligosaccharides can be recovered from the cultivation medium, and that “recovering” includes separating (Jennewein pg. 1 para. 14; pg. 4 para. 34).
Regarding instant claim 67, Jennewein teaches that the α-1,2-fucosyltransferase is preferably the fucT2 gene Helicobacter pylori, which has the UniProt ID Q9X3N7 (Jennewein pg. 7 para. 96). SEQ ID NO: 16, which is an α-1,2-fucosyltransferase from H. pylori with UniProt ID Q9X435, and fucT2 from H. pylori, are 93% identical, and therefore fucT2 as taught by Jennewein is a functional homolog having at least 80% sequence identity to SEQ ID NO: 16. Additionally, fucT2 from H. pylori is 100% identical to instant SEQ ID NO: 108.
Jennewein teaches that the α-1,3-fucosyltransferase is derived from Akkermansia muciniphila, Bacteroides fragilis, Helicobacter pylori, or Helicobacter hepaticus (Jennewein pg. 7 para. 96). Instant SEQ ID NO: 111 is the α-1,3-fucosyltransferase fucT from H. pylori, UniProt ID O30511. Instant SEQ ID NO: 112 is another α-1,3-fucosyltransferase from H. pylori, UniProt ID O25142.
It would have been obvious to a skilled artisan to combine the teachings of ‘325 and Jennewein, utilizing the cultivation techniques and specific fucosyltransferases taught by Jennewein in the method of ‘325. Both ‘325 and Jennewein teach fucosyltransferases for the production of fucosylated oligosaccharides, and it would be obvious to use fucosyltransferases taught by Jennewein as successfully used to produce DiFL, 2’FL, and 3-FL.
Regarding instant claims 64-66, 68-72, 74-76, and 78, all the limitations of these dependent claims are recited in claims 90-100 and 104-111 of copending ‘325.
This is a provisional nonstatutory double patenting rejection.
Claim 77 is provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claims 90-100 and 104-111 of copending Application No. 18/040,325 in view of Jennewein et al., US 2019/0309336 A1 and Petschacher et al., Journal of Biotechnology; 235:61-83.
Regarding instant claims 62 and 64, the teachings of ‘325 and Jennewein are set forth above. ‘325 and Jennewein do not teach the use of a cell-free system as recited in claims 77 and 80.
Regarding claim 77, Petschacher teaches in vitro, or cell-free, production of fucosylated oligosaccharides including 2’FL and 3-FL using α-1,2- and α-1,3-fucosyltransferases and GDP-L-fucose (Petschacher pg. 71 “3.2. In vitro production of fucosylated HMOs”; Table 4). Petschacher compares cell-free in vitro methods to whole cell systems for producing oligosaccharides, and teaches that in vitro methods have a unique advantage of flexibility, especially when characterizing new fucosyltransferases, or for smaller scale synthesis of complex human milk oligosaccharides (Petschacher pg. 72 first full para.).
It would have been obvious to a skilled artisan, before the effective filing date, to combine the teachings of ‘325, Jennewein, and Petschacher, using a cell-free system for producing of DiFL. Petschacher teaches both cell-free and cell-based systems of producing oligosaccharides. The use of isolated fucosyltransferase enzymes, rather than a cell expressing the fucosyltransferases, is established in the art as a method for producing human milk oligosaccharides. A skilled artisan would recognize that the methods taught by ‘325 and Jennewein, wherein both fucosyltransferases are contacted with GDP-fucose and lactose to produce DiFL and other oligosaccharides, could be adapted as a cell-free system as taught by Petschacher for the production of oligosaccharides such as 2’FL and 3-FL.
A person of ordinary skill in the art would have been motivated to use a cell-free system for production of DiFL or other HMOs, with a reasonable expectation of success, because cell-free methods are established for such a purpose and Petschacher teaches that in vitro production of oligosaccharides offers unique advantages over cell-based methods, specifically flexibility when characterizing new fucosyltransferases, or for smaller scale synthesis of complex human milk oligosaccharides (Petschacher pg. 72 first full para.).
This is a provisional nonstatutory double patenting rejection.
Claims 62, 64-66, 68-72, 74-76, 78, and 82 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claims 100-102, 104-107, 109, 113-116, and 118-121 of copending Application No. 18/040,332 in view of Jennewein et al., US 2019/0309336 A1.
Regarding instant claim 62, claim 114 of ‘332 recites a method of producing a mixture of at least three different mammalian milk oligosaccharides by a cell, the method comprising providing a cell capable of expressing at least two glycosyltransferases and capable of synthesizing one or more nucleotide-sugar(s), wherein the nucleotide-sugar(s) is/are donor(s) for the glycosyltransferases, and cultivating the cell under conditions permissive to express the glycosyltransferases and to synthesize the nucleotide-sugar(s), resulting in the cell producing at least three different mammalian milk oligosaccharides. Claim 115 of ‘332 recites that the mixture comprises neutral fucosylated oligosaccharides (such as DiFL, 2’FL, 3-FL). Claim 105 of ‘332 recites that the cell expresses a fucosyltransferase and the donor nucleotide sugar is GDP-fucose. Claim 106 of ‘332 recites uptake of a precursor or acceptor (including lactose, pg. 36 of ‘332 specification) for synthesis of the MMOs.
The claims of ‘332 do not recite that the method requires a 1-5 day cultivation and a yield of at least 50 g/L of DiFL (claim 62) or the fucosyltransferases of claim 67. However, these features are taught by Jennewein, as set forth above.
It would have been obvious to a skilled artisan to combine the teachings of ‘332 and Jennewein, utilizing the specific fucosyltransferases and cultivation conditions taught by Jennewein in the method of ‘332. Both ‘332 and Jennewein teach fucosyltransferases for the production of fucosylated oligosaccharides, and it would be obvious to use fucosyltransferases taught by Jennewein as successfully used to produce DiFL, 2’FL, and 3-FL.
Regarding instant claims 64-66, 68-72, 74-76, 78, and 82 all the limitations of these dependent claims are recited in claims 100-102, 104-107, 109, 113-116, and 118-121 of copending ‘332.
This is a provisional nonstatutory double patenting rejection.
Claim 77 is provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claims 100-102, 104-107, 109, 113-116, and 118-121 of copending Application No. 18/040,332 in view of Jennewein et al., US 2019/0309336 A1 and Petschacher et al., Journal of Biotechnology; 235:61-83.
Regarding instant claims 62 and 64, the teachings of ‘332 and Jennewein are set forth above. ‘332 and Jennewein do not teach the use of a cell-free system as recited in claim 77.
Regarding claim 77, the teachings of Petschacher are set forth above.
It would have been obvious to a skilled artisan, before the effective filing date, to combine the teachings of ‘332, Jennewein, and Petschacher, using a cell-free system for producing of DiFL. Petschacher teaches both cell-free and cell-based systems of producing oligosaccharides. The use of isolated fucosyltransferase enzymes, rather than a cell expressing the fucosyltransferases, is established in the art as a method for producing human milk oligosaccharides. A skilled artisan would recognize that the methods taught by ‘332 and Jennewein, wherein both fucosyltransferases are contacted with GDP-fucose and lactose to produce DiFL and other oligosaccharides, could be adapted as a cell-free system as taught by Petschacher for the production of oligosaccharides such as 2’FL and 3-FL.
A person of ordinary skill in the art would have been motivated to use a cell-free system for production of DiFL or other HMOs, with a reasonable expectation of success, because cell-free methods are established for such a purpose and Petschacher teaches that in vitro production of oligosaccharides offers unique advantages over cell-based methods, specifically flexibility when characterizing new fucosyltransferases, or for smaller scale synthesis of complex human milk oligosaccharides (Petschacher pg. 72 first full para.).
This is a provisional nonstatutory double patenting rejection.
Claims 62, 64-76, 78-79, 81-82, and 103 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1-3, 5, 7-9, 15, 18-19, 21-22, 26-27, 29-35, and 44-47 of copending Application No. 18/041,064 in view of Jennewein et al., US 2019/0309336 A1.
Regarding instant claim 62, claim 1 of ‘064 recites a method of producing a mixture of at least two different oligosaccharides by a cell, comprising providing a cell that is capable to express a glycosyltransferase and is capable to synthesize a nucleotide-sugar, wherein said nucleotide-sugar is donor for said glycosyltransferase, and cultivating said cell under conditions permissive to express said glycosyltransferase and to synthesize said nucleotide-sugar, and
addition of at least two acceptors (including disaccharides such as lactose) to said cultivation enabling said cell to produce at least two oligosaccharides, and separating at least one of said oligosaccharides from said cultivation. Claims 7 and 9 of ‘064 recite that the glycosyltransferase can be a fucosyltransferase, including α-1,2- and α-1,3-fucosyltransferases. Claims 9 and 15 of ‘064 recites that the nucleotide sugar donor is GDP-fucose. Claims 19 and 21-22 of ‘064 recite that the mixture comprises at least one fucosylated oligosaccharide, which includes DiFL, 2’FL, and 3-FL, see ‘064 specification pg. 16-17. Claims 26-27 of ‘064 recite that the mixture is all fucosylated oligosaccharides.
The claims of ‘064 do not teach that the cell expresses two fucosyltransferases, and do not expressly teach the production of DiFL, 2’FL, or 3-FL or that lactose, 2’FL, or 3-FL are the acceptors. The claims of ‘064 do not recite that the method requires a 1-5 day cultivation and a yield of at least 50 g/L of DiFL (claim 62) or the fucosyltransferases of claim 67. ‘064 does not teach that the cell produces lactose for production of DiFL, 2’FL, or 3-FL or the specific carbon sources for cultivation set forth in claim 79. However, these features are taught by Jennewein.
Regarding claim 62, the teachings of Jennewein are set forth above.
Regarding claim 65, Jennewein teaches that the acceptors can include lactose, 2’FL, and 3-FL (Jennewein pg. 3 para. 39).
Regarding claim 67, the teachings of Jennewein are set forth above.
Regarding claim 68, Jennewein teaches that lactose is generated internally by the cell during cultivation, i.e. that the cell produces lactose, for the synthesis of 2’FL, 3-FL, and DiFL (Jennewein pg. 1 para. 15).
Regarding claim 79, Jennewein teaches that the cell is cultivated in a medium containing glycerol as a carbon source (Jennewein pg. 7 para. 106; pg. 9 para. 126).
It would have been obvious to a skilled artisan to combine the teachings of ‘064 and Jennewein, resulting in a method of producing DiFL as instantly claimed. Both the method of ‘064 and Jennewein are directed to producing oligosaccharides, including fucosylated oligosaccharides, using a fucosyltransferase. It would have been obvious that the method of ‘064 could be modified as taught by Jennewein to incorporate two fucosyltransferases, including the specific examples taught by Jennewein, for the production of DiFL, using the acceptors for production of DiFL as taught by Jennewein. ‘064 is directed broadly to production of multiple oligosaccharides, but it would be obvious to a person having ordinary skill in the art that the specific oligosaccharides, fucosyltransferases, and acceptors taught by Jennewein could be chosen in the method of ‘064. The carbon sources used in the cultivation medium as taught by Jennewein are commonly used and established for cultivation of microorganisms, so it would have been obvious to cultivate the cells as taught by ‘064 using such a carbon source.
Regarding instant claims 64, 66, 69-76, 78, 81-82, and 103, all the limitations of these dependent claims are recited in claims 1-3, 5, 7-9, 15, 18-19, 21-22, 26-27, 29-35, and 44-47 of copending ‘064.
This is a provisional nonstatutory double patenting rejection.
Claim 77 is provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1-3, 5, 7-9, 15, 18-19, 21-22, 26-27, 29-35, and 44-47 of copending Application No. 18/041,064 in view of Jennewein et al., US 2019/0309336 A1 and Petschacher et al., Journal of Biotechnology; 235:61-83.
Regarding instant claims 62 and 64, the teachings of ‘064 and Jennewein are set forth above. ‘064 and Jennewein do not teach the use of a cell-free system as recited in claims 77 and 80.
Regarding claim 77, the teachings of Petschacher are set forth above.
It would have been obvious to a skilled artisan, before the effective filing date, to combine the teachings of ‘064, Jennewein, and Petschacher, using a cell-free system for producing of DiFL. Petschacher teaches both cell-free and cell-based systems of producing oligosaccharides. The use of isolated fucosyltransferase enzymes, rather than a cell expressing the fucosyltransferases, is established in the art as a method for producing human milk oligosaccharides. A skilled artisan would recognize that the methods taught by ‘064 and Jennewein, wherein both fucosyltransferases are contacted with GDP-fucose and lactose to produce DiFL and other oligosaccharides, could be adapted as a cell-free system as taught by Petschacher for the production of oligosaccharides such as 2’FL and 3-FL.
A person of ordinary skill in the art would have been motivated to use a cell-free system for production of DiFL or other HMOs, with a reasonable expectation of success, because cell-free methods are established for such a purpose and Petschacher teaches that in vitro production of oligosaccharides offers unique advantages over cell-based methods, specifically flexibility when characterizing new fucosyltransferases, or for smaller scale synthesis of complex human milk oligosaccharides (Petschacher pg. 72 first full para.).
This is a provisional nonstatutory double patenting rejection.
Claims 62, 64-76, 78-79, 82-83, and 103 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claims 80-83, 88-98, 100-101, 110-111, 114-124, and 127-136 of copending Application No. 18/041,154 in view of Jennewein et al., US 2019/0309336 A1.
Regarding instant claim 62, claim 120 of ‘154 recites a method of producing a di- and/or oligosaccharide by a cell comprising cultivating the cell of claim 80 under conditions permissive to produce the di- and/or oligosaccharide, and, optionally, separating the di- and/or oligosaccharide from the cultivation. Claims 80 and 88-89 of ‘154 recites a genetically modified cell to produce the di- and/or oligosaccharide by introducing a pathway for producing the di- and/or oligosaccharide. Claims 91 and 94 of ‘154 recite that the cell can express fucosyltransferases. Claim 92 of ‘154 recites that the nucleotide activated sugar can be GDP-fucose. Claims 121, 123, and 124 of ‘154 recite that the method involves an acceptor/precursor, which can be lactose, to produce the oligosaccharides.
The claims of ‘154 do not recite that the method requires a 1-5 day cultivation and a yield of at least 50 g/L of DiFL (claim 62). The claims of ‘154 do not explicitly recite a method for producing DiFL, 2’FL, or 3-FL, or that lactose, 2’FL, or 3-FL are the acceptors as recited in claims 62 and 65. The claims of ‘154 do not teach the specific fucosyltransferases recited in claim 67. ‘154 does not teach that the cell produces lactose for production of DiFL, 2’FL, or 3-FL as recited in claim 68. However, these features are taught by Jennewein.
Regarding claims 62, 65, 67, and 68, the teachings of Jennewein are set forth above.
It would have been obvious to a skilled artisan to combine the teachings of ‘154 and Jennewein, resulting in a method of producing DiFL as instantly claimed. Both the method of ‘154 and Jennewein are directed to producing oligosaccharides using fucosyltransferases. It would be obvious that the method taught by ‘154, directed broadly to producing oligosaccharides including through fucosyltransferases, could be used to produce DiFL with the acceptors, precursors, and specific fucosyltransferases taught by Jennewein.
Regarding instant claims 64, 66, 69-76, 78-79, 82-83, and 103, all the limitations of these dependent claims are recited in claims 80-83, 88-98, 100-101, 110-111, 114-124, and 127-136 of copending ‘154.
This is a provisional nonstatutory double patenting rejection.
Claim 77 is provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claims 80-83, 88-98, 100-101, 110-111, 114-124, and 127-136 of copending Application No. 18/041,154 in view of Jennewein et al., US 2019/0309336 A1 and Petschacher et al., Journal of Biotechnology; 235:61-83.
Regarding instant claims 62 and 64, the teachings of ‘154 and Jennewein are set forth above. ‘064 and Jennewein do not teach the use of a cell-free system as recited in claims 77 and 80.
Regarding claim 77, the teachings of Petschacher are set forth above.
It would have been obvious to a skilled artisan, before the effective filing date, to combine the teachings of ‘154, Jennewein, and Petschacher, using a cell-free system for producing of DiFL. Petschacher teaches both cell-free and cell-based systems of producing oligosaccharides. The use of isolated fucosyltransferase enzymes, rather than a cell expressing the fucosyltransferases, is established in the art as a method for producing human milk oligosaccharides. A skilled artisan would recognize that the methods taught by ‘154 and Jennewein, wherein both fucosyltransferases are contacted with GDP-fucose and lactose to produce DiFL and other oligosaccharides, could be adapted as a cell-free system as taught by Petschacher for the production of oligosaccharides such as 2’FL and 3-FL.
A person of ordinary skill in the art would have been motivated to use a cell-free system for production of DiFL or other HMOs, with a reasonable expectation of success, because cell-free methods are established for such a purpose and Petschacher teaches that in vitro production of oligosaccharides offers unique advantages over cell-based methods, specifically flexibility when characterizing new fucosyltransferases, or for smaller scale synthesis of complex human milk oligosaccharides (Petschacher pg. 72 first full para.).
This is a provisional nonstatutory double patenting rejection.
Claims 62, 64-66, 68-72, 75-76, and 78 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claims 100-102, 104-107, 109, 113-116, and 118-121 of copending Application No. 17/627,088 in view of Jennewein et al., US 2019/0309336 A1.
Regarding claim 62, claim 51 of copending ‘088 recites a host cell genetically modified for production of a fucosyllactose, wherein the host cell comprises at least one nucleic acid sequence coding for a fucosyltransferase that transfers a fucose residue from a GDP-fucose donor to a lactose acceptor, thereby synthesizing fucosyllactose. Claim 68 of copending ‘088 teaches that the fucosyllactose is 2'-fucosyllactose, 3-fucosyllactose or difucosyllactose. Claim 60 of copending ‘088 teaches that the cell is cultured in a medium.
The claims of ‘064 do not recite that the method requires a 1-5 day cultivation and a yield of at least 50 g/L of DiFL (claim 62). The claims of ‘088 do not teach that the cell expresses two fucosyltransferases as recited in claim 62, or the specific fucosyltransferases recited in claim 67. ‘088 does not recite a method of producing DiFL, 2’FL, and 3-FL as set forth in claim 62 or a medium with the carbon sources set forth in claim 79. However, these features are taught by Jennewein.
Regarding claims 62, 67, and 79, the teachings of Jennewein are set forth above.
It would have been obvious to a skilled artisan to combine the teachings of ‘088 and Jennewein, resulting in a method of producing DiFL as instantly claimed. While the claims of ‘088 are not directed to a method of producing DiFL, the cell is genetically engineered to produce DiFL, 2’FL, and 3-FL and is cultured. It would have been obvious to a skilled artisan to incorporate the methods of Jennewein and cultivate the cell of ‘088 to produce DiFL. Additionally, it would have been obvious to incorporate two fucosyltransferases, including the specific examples taught by Jennewein, for the production of DiFL, as ‘088 is generally directed to fucosyltransferases and Jennewein teaches fucosyltransferases that are successfully used to produce DiFL.
Regarding claims 64-66, 68-72, 75-76, and 78, all the limitations of these dependent claims are recited in claims 51-53 and 59-64 of copending ‘088.
This is a provisional nonstatutory double patenting rejection.
Response to Arguments
Applicant's arguments filed 11/25/2025 have been fully considered but they are not persuasive.
35 U.S.C. § 112(a)
Applicant argues that the amended claims are strictly limited to explicit sequences and closely related variants expressly tied to demonstrated enzymatic activity. Applicant argues that the specification enumerates 95 α-1,2-fucosyltransferase sequences (SEQ ID NOs: 16-110), and 22 α-1,3-fucosyltransferase sequences (SEQ ID NOs: 111-132), and that the extensive listing of these sequences demonstrates that the inventors were in possession of a broad yet structurally coherent genus of fucosyltransferases capable of producing DiFL and optionally 2'FL and 3-FL and that the 80% identity threshold corresponds to conservation of the catalytic architecture characteristic of these fucosyltransferase families. Applicant argues that the disclosed sequences fall into predictable structural families where the conserved catalytic machinery and sequence architecture indicate predictable retention of activity across each cluster. Such tight clustering reinforces the predictable nature of these enzymes and confirms that the inventors possessed not only the individually enumerated sequences but also the full set of closely related variants sharing ~80% identity and conserved functional motifs. Applicant argues that the amended claims cover only variants within structural families already thoroughly described in the application.
In response to this argument, it is noted that, as stated in the above rejection under 35 U.S.C. § 112(a), the full scope of sequences having 80% identity to the claimed sequence is extremely vast and encompasses millions of possible sequences. As such, the claims are not considered to be “strictly limited to explicit sequences and closely related variants expressly tied to demonstrated enzymatic activity” as asserted by applicant. Taking as an example the elected SEQ ID NO: 16, with 299 amino acids in SEQ ID NO: 16, anywhere from 1-59 of these positions may differ from the corresponding position in SEQ ID NO: 16, with 19 possible amino acids at any given position. There are countless variations that are encompassed within 80% identity, not all of which will have the claimed enzyme activity.
The specification has support for the specifically disclosed sequences, SEQ ID NOs: 16-132. These sequences have been shown to possess enzyme activity, as illustrated by applicant in Tables 1 and 2 of the arguments and in the Examples of the specification. However, the disclosure of these 117 sequences is in no way representative of “the full set of closely related variants sharing ~80% identity”, which encompasses countless possible amino acid sequences. Applicant’s arguments are directed to the disclosed sequences, which do have written description support, but this does not indicate that applicant was in possession of the entire genus encompassing sequences with 80% identity to the elected SEQ ID NOs.
35 U.S.C. § 102
Applicant argues that as amended, the claims now include elements that are not disclosed in Jennewein under any reading of the claims-whether the method of claim 62 is practiced in a cell-free format or in a cell-based format. Applicant argues that Jennewein teaches only engineered cell fermentation and does not disclose:
• any 1-5 day contacting step,
• any reaction conditions meeting the contacting step of claim 62,
• any disclosure of >50 g/L DiFL in a reaction mixture or cultivation.
Regarding the 1-5 day contacting step, applicant is pointed to Jennewein p. 7 para. 89, and Jennewein claim 18, which discloses that the cells are cultivated for at least 60, 80, 100, or 120 hours, which is equivalent to 2.5 to 5 days, thus meeting the requirement of a 1-5 day contacting step.
Regarding the reaction conditions meeting the contacting step of claim 62, this step requires “contacting the fucosyltransferases and GDP-fucose donor with a reaction mixture
comprising lactose as an acceptor and optionally one or more further acceptor(s), over the
course of 1 to 5 days under conditions wherein the fucosyltransferases catalyze the transfer of a fucose residue from the GDP-fucose donor to the acceptor”. Jennewein teaches all of these steps. Jennewein provides a cell expressing fucosyltransferases and expressing a GDP-fucose donor, and teaches providing lactose to the cultivation medium (Jennewein pg. 1 para. 13). As set forth in the instant specification, “reaction mixture” is interchangeable with “cultivation” in the context of a cell-based method (see instant specification p. 25 lines 30-34). Thus, the cultivation taught by Jennewein is considered equivalent to a “reaction mixture”, and this method taught by Jennewein involves contacting of the fucosyltransferases and GDP-fucose donor, which are produced by the cell, with a reaction mixture comprising lactose.
Regarding the >50 g/L DiFL, Jennewein teaches that with the method according to the invention, as well as with the genetically modified host cell employed in the method, it is possible to produce fucosylated oligosaccharides at a titer exceeding 50 g/L, and even 100 g/L, and even more 150 g/L (Jennewein p. 2 para. 23). Jennewein teaches that preferably, the
fucosylated oligosaccharide is one that is selected from 2'-fucosyllactose, 3-fucosyllactose or difucosyllactose. Therefore, Jennewein teaches that the method produces at least 50 g/L of DiFL.
Applicant argues that the claim covers both cell-free and cell-based embodiments, but Jennewein fails both. Applicant argues that Jennewein does not teach a cell-free method. Applicant argues that in the cell-based method, Jennewein does not teach the time period, contacting requirement, and 50 g/L DiFL requirement of claim 62, and specifies that Jennewein does not teach the required contacting with a reaction mixture comprising lactose as a step distinct from general fermentation conditions.
In response to this argument, regarding the cell-free method, the examiner recognizes that Jennewein does not teach a cell-free system, as stated on p. 15 of the Non-Final Office Action (“Jennewein does not teach that the fucosyltransferase and GDP-fucose are provided in a cell-free system as recited in claims 77 and 80”).
Regarding the deficiencies of the cell-based method: the time period, contacting requirement, and 50 g/L requirement are addressed above.
Regarding applicant’s assertion that Jennewein does not teach the required contacting with a reaction mixture comprising lactose as a step distinct from general fermentation conditions, it is noted that claim 62 does not require contacting with lactose as a step distinct from fermentation conditions. Claim 62 requires “contacting the fucosyltransferases and GDP-fucose donor with a reaction mixture comprising lactose as an acceptor and optionally one or more further acceptor(s), over the course of 1 to 5 days under conditions wherein the fucosyltransferases catalyze the transfer of a fucose residue from the GDP-fucose donor to the acceptor”, all of which is met by Jennewein. There is nothing to state that the lactose is added in a distinct step from general fermentation conditions. However, it is also noted that Jennewein specifically teaches cultivating said genetically modified host cell in a cultivation medium, and then providing lactose to the cultivation medium (p. 1 para. 13), indicating that the lactose is added as a separate step after cultivation is started.
35 U.S.C. § 103
Applicant argues that claim 67 requires defined α-1,2- and α-1,3-fucosyltransferases and requires their combination to yield DiFL at >50 g/L. Jennewein provides no teaching of specific enzyme pairings, of selection criteria, or of any combination that actually produces DiFL. Jennewein merely states that DiFL "could" be formed, without disclosure of the required α-1,2- and α-1,3-pair, any experimental demonstration, any yield, concentration, or purity.
In response to this argument, Jennewein discloses that “for synthesis of 2',3-difucosyllactose, both, a suitable alpha-1,2-fucosyltransferase and an alpha-1,3-fucosyltransferase or at least one gene encoding for a protein exhibiting an alpha-1,2- as well as an alpha-1,3-fucosyltransferase activity is expressed” (p. 3 para. 41). Thus, Jennewein expressly teaches that both of these enzymes are required for DiFL production. Jennewein additionally teaches specific fucosyltransferases that can be used, as set forth in the above rejection, corresponding with a sequence that is 93% identical to SEQ ID NO: 16, a sequence that is 100% identical to SEQ ID NO: 108, or an H. pylori α-1,3-fucosyltransferase (SEQ ID NOs: 111 or 112). Thus, Jennewein does teach the claimed combination of fucosyltransferases for producing DiFL.
Applicant argues that Petschacher does not teach or suggest DiFL production, required reaction conditions, or an enzyme pair that produces DiFL.
In response to this argument, all of these features are taught by the primary reference, Jennewein, as discussed above. Petschacher is relied upon to teach a cell-free method. A skilled artisan would have been motivated to combine the teachings of Jennewein and Petschacher and use a cell-free method instead of cell-based because Petschacher teaches that in vitro production of oligosaccharides offers unique advantages over cell-based methods, specifically flexibility when characterizing new fucosyltransferases, or for smaller scale synthesis of complex human milk oligosaccharides (Petschacher pg. 72 first full para.).
Applicant argues that Whiteson does not teach enzyme activity, specific pairing of fucosyltransferases, or reaction conditions, and there is no motivation to combine or reasonable expectation of success in combining the teachings of Jennewein and Whiteson.
In response to this argument, it is noted that the amino acid sequence disclosed by Whiteson is annotated as an α-1-3-fucosyltransferase, FucT, as shown in the Appendix of the Non-Final Office Action. The pairing of fucosyltransferases and reaction conditions are taught by Jennewein as set forth above. A skilled artisan would have found it obvious to make a simple substitution of one known fucosyltransferase for another with a reasonable expectation of success, as discussed in the rejection above.
Non-statutory double patenting
The rejections on the grounds of non-statutory double patenting are modified in view of claim amendments and maintained, for the reasons set forth above. It is considered that the invention as claimed is an obvious variation of the inventions recited in the copending applications in view of Jennewein or Jennewein and Petschacher.
Conclusion
Claims 62-79, 81-83, 103, and 105 are rejected. No claims are allowed.
Applicant's amendment necessitated the new ground(s) of rejection presented in this Office action. Accordingly, THIS ACTION IS MADE FINAL. See MPEP § 706.07(a). 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.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to EMILY F EIX whose telephone number is (571)270-0808. The examiner can normally be reached M-F 8am-5pm ET.
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/EMILY F EIX/Examiner, Art Unit 1653
/JENNIFER M.H. TICHY/Primary Examiner, Art Unit 1653