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 .
Election/Restrictions
Applicant's election with traverse of LNT (intermediate), LNFP-III (desired oligosaccharide), GDP-fucose, fucosyltransferase and Escherichia in the reply filed on 02/16/2026 is acknowledged. The traversal is on the ground(s) that the species are connected by design, operation or effect citing MPEP 806.04(b). This is not found persuasive because “The analysis used to determine whether the Office may require restriction differs in national stage applications submitted under 35 U.S.C. 371 (unity of invention analysis) as compared to national applications filed under 35 U.S.C. 111(a) (independent and distinct analysis). See MPEP Chapter 1800, in particular MPEP § 1850, § 1875, and § 1893.03(d), for a detailed discussion of unity of invention under the Patent Cooperation Treaty (PCT).” MPEP 823. As such, the provisions of MPEP 806.04(b) are inapplicable.
The requirement is still deemed proper and is therefore made FINAL.
Claims 10 and 16 is withdrawn from further consideration pursuant to 37 CFR 1.142(b), as being drawn to a nonelected species, there being no allowable generic or linking claim. Applicant timely traversed the restriction (election) requirement in the reply filed on 02/16/2026. Regarding claim 10, claim 10 requires that within the process of claim 1 that the recited transglycosidase acts on the intermediate oligosaccharide that is imported by the recited saccharide importer, and the elected species of fucosyltransferase does not read on claim 10.
It is noted that the elected species are not consistent with each other. LNFP-III, as described in the specification (page 6, lines 9-12, claim 12), is produced by transfer of a galactose moiety to LNT-II to produce LNnT and then transfer of a fucose moiety to LNnT to produce LNFP-III. That is, Table 1 of the specification states that LNFP-II is produced from LNT and LNnT is produced from LNT-III. As such, election of LNT and LNFP-III are contradicting since LNFP-III is not produced from LNT. Claim 1 explicitly requires that “an enzyme which is able to transfer a monosaccharide moiety to the intermediate oligosaccharide” operates on the same intermediate oligosaccharide that is in the culture media and transported by the saccharide importer, which is LNnT in embodiments of claim 12 wherein LNFP-III is produced. Claims 11 and 15 are not withdrawn in the interest of compact prosecution.
Claim Objections
Claims 11, 12 and 15 objected to because of the following informalities:
Abbreviations LNT-II, LNT, LNnT, LFNP-II, LFNP-II and any other abbreviations in the claims should be defined.
Appropriate correction is required.
Claim Rejections - 35 USC § 112
The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
Claims 1-9 and 13 are rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
Claim 1 recites a “desired oligosaccharide.” The specification indicates that a “desired oligosaccharide” has a different structure from a “non-desired oligosaccharide.” “A major encounter that also contributes significantly to the cost in the large-scale production of oligosaccharides is the need to remove non-desired compounds such as non-desired oligosaccharides.” Specification, page 3. As such, it is possible to practice a process that meets the features of claim 1 except that only non-desired oligosaccharides would be produced, wherein such method would not infringe claim 1 do to lack of production of a desired oligosaccharide.
“When a subjective term is used in the claim, the examiner should determine whether the specification supplies some objective standard for measuring the scope of the term. Some objective standard must be provided in order to allow the public to determine the scope of the claim. A claim term that requires the exercise of subjective judgment without restriction may render the claim indefinite.” MPEP 2173.05(IV). Here, what structure constitutes a “desired oligosaccharide” as opposed to an “oligosaccharide” or a “non-desired oligosaccharide” is not defined in the specification and the exercise of subjective judgement without restriction is required to determine what structures may or may not be a “desired oligosaccharide.” As such, an ordinarily skilled artisan cannot determine how to avoid infringement of the rejected claims.
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 1-9, 11-13, 15 and 17 (all non-withdrawn claims) 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.
The purpose of the written description requirement is to ensure that the inventor had possession, at the time the invention was made, of the specific subject matter claimed. For a broad generic claim, the specification must provide adequate written description to identify the genus of the claim.
“A written description of an invention involving a chemical genus, like a description of a chemical species, 'requires a precise definition, such as by structure, formula, [or] chemical name,' of the claimed subject matter sufficient to distinguish it from other materials." Fiers, 984 F.2d at 1171, 25 USPQ2d 1601; In re Smythe, 480 F.2d 1376, 1383, 178 USPQ 279, 284985 (CCPA 1973) (“In other cases, particularly but not necessarily, chemical cases, where there is unpredictability in performance of certain species or subcombinations other than those specifically enumerated, one skilled in the art may be found not to have been placed in possession of a genus.”). Regents of the University of California v. Eli Lilly & Co., 119, F.3d 1559, 1568, 43 USPQ2d 1398, 1405 (Fed. Cir. 1997).
“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.” MPEP 2163(II)(3)(a).
Furthermore, a “‘representative number of species’ means that the species which are adequately described are representative of the entire genus. Thus, when there is substantial variation within the genus, one must describe a sufficient variety of species to reflect the variation within the genus. The disclosure of only one species encompassed within a genus adequately describes a claim directed to that genus only if the disclosure ‘indicates that the patentee has invented species sufficient to constitute the gen[us].’ See Enzo Biochem, 323 F.3d at 966, 63 USPQ2d at 1615; Noelle v. Lederman, 355 F.3d 1343, 1350, 69 USPQ2d 1508, 1514 (Fed. Cir. 2004) (Fed. Cir. 2004) (‘[A] patentee of a biotechnological invention cannot necessarily claim a genus after only describing a limited number of species because there may be unpredictability in the results obtained from species other than those specifically enumerated.’). ‘A patentee will not be deemed to have invented species sufficient to constitute the genus by virtue of having disclosed a single species when … the evidence indicates ordinary artisans could not predict the operability in the invention of any species other than the one disclosed.’ In re Curtis, 354 F.3d 1347, 1358, 69 USPQ2d 1274, 1282 (Fed. Cir. 2004).” MPEP 2163(II)(3)(a).
Claim 1 recites a genus of “saccharide importer[s] for the uptake of an . . . oligosaccharide consisting of at least three monosaccharide moieties.” Claim 15 states a genus of such saccharide importers/transporters that uptake LNT-II, which has three monosaccharide moieties.
Claim 12 requires that the recited saccharide importer of claim 1 imports LNnT or LNT (tetrasaccharides) since claim 1 requires that the intermediate oligosaccharide (that is LNnT or LNT in embodiments of claim 12) by present in a culture medium and “the genetically engineered microbial cell to take up the intermediate oligosaccharide being LNnT or LNT.
Papadakis (WO 2023/099680 A1) is not prior art but evidences the state of the prior art at time of filing. “Although these approaches usually involve the import of lactose (substrate) by the native lactose permease LacY of E. coli and its further decoration in the cell to form more complex HMOs varying from tri- to hexa-saccharides, there are very few disclosures of the import of oligosaccharides with more than two units by a single cell.” Papadakis, page 1.
As such, there is not a general description of importer/transporter proteins for importation of oligosaccharides with three or more monosaccharide moieties in the art.
Example 6 of the specification describes analysis of metagenomic DNA from stool samples of breast-fed infants placed into a fosmid library of cells with expression in a medium containing 20 mM lacto-N-triose II (LNT-II). Further:
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The identity of any particular transporter gene/protein from an uncharacterized metagenomic DNA sample is necessarily unpredictable. The specification does not state how the same metagenomic DNA from breast-fed infants described in Example 6 can be re-obtained by an ordinarily skilled artisan at time of filing. The following is noted:
The specification does not disclose the sequence of any gene of interest identified for import of an oligosaccharide with three or more monosaccharide moieties;
The specification does not contain a Sequence Listing;
Example 6 of the specification nor any other portion of the specification discusses identification of importer proteins of oligosaccharides having four or more monosaccharide moieties as encompassed by the genus of importers as recited in claim 1.
There is no discussion anywhere in the specification regarding identity or possession of any saccharide importer capable of importing tetrasaccharides LNnT or LNT as recited in claim 12. Examples 4 and 5 of the specification describes production of LNFP II or LNFP III containing LNnT or LNT but without any expression of a specific saccharide importer having the function to import LNnT or LNT. It is noted that the specification does not provide any quantification for amount of LNFP II or LNFP III produced.
There is no discussion in the specification regarding specific saccharide importers or transporters for uptake of LNT-II, LNT, LNnT or other oligosaccharides that may be active in yeast cells.
That is, it is not possible for an ordinarily skilled artisan to either 1) reproduce Example 6 of the specification wherein the source of the specific metagenomic DNA used is not disclosed, nor 2) produce an embodiment of the claims utilizing a gene encoding a transporter asserted to be identified in Example 6 of the specification having no sequence nor other identifying characteristics of the same disclosed by the as-filed specification.
A “‘representative number of species’ means that the species which are adequately described are representative of the entire genus. Thus, when there is substantial variation within the genus, one must describe a sufficient variety of species to reflect the variation within the genus. The disclosure of only one species encompassed within a genus adequately describes a claim directed to that genus only if the disclosure ‘indicates that the patentee has invented species sufficient to constitute the gen[us].’ “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.” MPEP 2163(II)(3)(a). “For some biomolecules, examples of identifying characteristics include a sequence, structure, binding affinity, binding specificity, molecular weight, and length. Although structural formulas provide a convenient method of demonstrating possession of specific molecules, other identifying characteristics or combinations of characteristics may demonstrate the requisite possession.” MPEP 2163(II)(A)(3)(a).
The recited genus of saccharide importers in claim 1 and recited genus of transporters in claim 15 are biomolecules, and such recited genera are understood as including saccharide importers/transporters putatively identified by applicant in Example 6 of the specification. However, such saccharide importers/transporters putatively identified by applicant in Example 6 are not identified by any identifying characteristics including sequence, structure, possession of specific structural characteristics nor other identifying characteristics or combinations of characteristics that may demonstrate the requisite possession. The specification states that transporters were identified by sequence. But without disclosure of such sequences the same is not a description of any specific species nevertheless species that are representative of the entire genus of transporters/importers recited including such that transport oligosaccharides with more than 4 saccharide monomers. Further, without the disclosure of such sequences nor the specific metagenomic material the same were identified from is not 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. For this reason, the specification does not provide an adequate written description of the genus of transporter/importers for oligosaccharides with three or more monosaccharide moieties or for LNT-II as recited.
Regarding claim 12 in particular, it may be asserted that E. coli contains a native saccharide importer capable of importing LNT or LNnT. However, such an importer is not identified in the specification by any characteristic. It is noted that E. coli is known to have non-specific porin proteins that may allow a low rate of diffusion of oligosaccharides. See Nikaido et al. (Nonspecific and Specific Diffusion Channels in the Outer Membrane of Escherichia coli J. Supramolecular Structure 13, 1980, 135-43) (When we used higher oligosaccharides, we found that the rate of diffusion of tri-saccharides through the porin channel was extremely slow (less than 0.5% of the rate for glucose) (page 311).
Although claims 1 and 12 do not require a saccharide importer to be heterologous, recitation of a “genetically engineered microbial cell that possess” is understood as directly encompassing that such an importer is heterologously expressed by genetic engineering and particularly adapted for transport of oligosaccharides as recited, rather than a negligible, non-specific function to transport an oligosaccharide. A lack of discussion of the identity of any saccharide importer or transporter for LNnT or LNT is neither a description of representative number of species nor of relevant, identifying characteristics such that there is not a sufficient written description of a genus of saccharide importers operable on LNnT or LNT as recited in claim 12.
The following is a quotation of 35 U.S.C. 112(d):
(d) REFERENCE IN DEPENDENT FORMS.—Subject to subsection (e), a claim in dependent form shall contain a reference to a claim previously set forth and then specify a further limitation of the subject matter claimed. A claim in dependent form shall be construed to incorporate by reference all the limitations of the claim to which it refers.
The following is a quotation of pre-AIA 35 U.S.C. 112, fourth paragraph:
Subject to the following paragraph [i.e., the fifth paragraph of pre-AIA 35 U.S.C. 112], a claim in dependent form shall contain a reference to a claim previously set forth and then specify a further limitation of the subject matter claimed. A claim in dependent form shall be construed to incorporate by reference all the limitations of the claim to which it refers.
Claim 7 is rejected under 35 U.S.C. 112(d) or pre-AIA 35 U.S.C. 112, 4th paragraph, as being of improper dependent form for failing to further limit the subject matter of the claim upon which it depends, or for failing to include all the limitations of the claim upon which it depends.
"[E]ven though product-by-process claims are limited by and defined by the process, determination of patentability is based on the product itself. The patentability of a product does not depend on its method of production. If the product in the product-by-process claim is the same as or obvious from a product of the prior art, the claim is unpatentable even though the prior product was made by a different process." MPEP 2113(I).
Claim 7, depending from claim 1, is understood as reciting the intermediate oligosaccharide as a product-by-process of an undefined microbial fermentation. Claim 7 is not understood as requiring active performance of any method step of a microbial fermentation. The structure of an intermediate oligosaccharide is unchanged regardless of its method of production. As such, claim 7 reciting as its only feature that the intermediate oligosaccharide as recited in claim 1 is a product-by-process does not require that the intermediate oligosaccharide be made by any microbial fermentation and encompasses intermediate oligosaccharides made by any method including, for example, isolation from milk or an in vitro enzymatic process. For this reason, claim 7 fails to further limit claim 1 since there is no embodiment of claim 1 that would not also infringe claim 7 such that claims 1 and 7 have the same scope.
This rejection may potentially be obviated by amending claim 7 to recite a method further comprising performing an active step of microbial fermentation to produce an intermediate oligosaccharide.
Applicant may cancel the claim(s), amend the claim(s) to place the claim(s) in proper dependent form, rewrite the claim(s) in independent form, or present a sufficient showing that the dependent claim(s) complies with the statutory requirements.
Claim Rejections - 35 USC § 102
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 the appropriate paragraphs of 35 U.S.C. 102 that form the basis for the rejections under this section made in this Office action:
A person shall be entitled to a patent unless –
(a)(1) the claimed invention was patented, described in a printed publication, or in public use, on sale, or otherwise available to the public before the effective filing date of the claimed invention.
Claim(s) 1-9, 11, 13, 15 and 17 is/are rejected under 35 U.S.C. 102(a)(1) as being anticipated by Jennewein (U.S. 2018/0305724 A1).
Jennewein, abstract, states:
The present invention relates to methods for the production of oligosaccharides in genetically modified bacterial host cells, as well as to the genetically modified host cells used in the methods. The genetically modified host cell comprises at least one recombinant glycosyltransferase, and at least one nucleic acid sequence coding for a protein enabling the export of the oligosaccharide.
“Since decades, enormous knowledge about the import of carbohydrates into bacteria could be acquired. But regarding the export of carbohydrates, especially about molecules that are non-surface-associated, only little information is available. This is not unexpected since sugars actually depict a favourable carbon- and energy source, thus, once in the cell they shouldn't be released to a competitive environment.” Jennewein, para. [0009].
“Apart from this, from a mechanistic and energetic point of view, only the ion-gradient-driven transport systems have the potential to translocate solutes in both directions across the membrane. This is exemplarily true for the above mentioned LacY, a galactoside/H+ symporter, which is part of the bacterial lac operon that allows the metabolism of lactose in E. coli. This permease primarily imports lactose into the cell but it is also capable to transfer its substrate in the opposite direction.” Jennewein, para. [0012].
“According to the invention, a method for the production of a desired oligosaccharide by a genetically modified microbial host cell, comprising the steps of a) providing a genetically modified microbial host cell that comprises at least one recombinant glycosyltransferase, and that has the expression or activity of at least one endogenous sugar export protein modified such, that the expression or activity of the sugar export protein is either (i) increased or (ii) decreased or inactivated as compared to an genetically unmodified host cell, so that (i) the export of a oligosaccharide into the medium is either decreased or abolished, or (ii) the transport of a desired oligosaccharide is increased, respectively, as compared to an genetically unmodified host cell, b) cultivating the host cell in a medium under conditions permissive for the production of the desired oligosaccharide, whereby the desired oligosaccharide is transported into the medium. The method may further comprise the step of c) obtaining the desired oligosaccharide from the medium.” Jennewein, para. [0017].
“In the method according to the invention, it is preferred if the desired oligosaccharide is a human milk oligosaccharide comprising a lacto-N-triose II (LNT-II; GlcNAc(β1-3)Gal(β1-4)Gluc) as a core trisaccharide. In this connection, an oligosaccharide having a “core trisaccharide” is meant to comprise the specific trisaccharide representing the reducing end of a desired oligosaccharide, and comprising, as the case may be, additional saccharide moieties, with the specific trisaccharide representing the major moiety.” Jennewein, para. [0018].
“Accordingly, in an embodiment of the method and the host cell according to the invention, the desired oligosaccharide is selected from the group consisting of: lacto-N-triose II, lacto-N-tetraose, lacto-N-neotetraose, lacto-N-fucopentaose I, lacto-N-fucopentaose II, lacto-N-fucopentaose III, lacto-N-fucopentaose V, lacto-N-difucosylhexose I, lacto-N-difucosylhexaose II, lacto-N-sialylpentaose LSTa, LSTb, LSTc, disialyllacto-N-tetraose, disialyllacto-N-neotetraose.” Jennewein, para. [0019].
“In order to overcome the above mentioned drawbacks of limited oligosaccharide export the object is further solved by a method according to the invention, wherein the host cell comprises: at least one homologous or heterologous nucleic acid sequence coding for a protein enabling the export of a desired oligosaccharide into the culture medium, wherein said host cell has been modified such, that the expression of the homologous or heterologous nucleic acid sequence is overexpressed or under control of a promoter enabling the overexpression of the nucleic acid sequence; and/or the deletion, disruption, diminishment or inactivation of at least one endogenous nucleic acid sequence coding for an exporter protein that exports precursors of the desired oligosaccharide outside the host cell; and/or at least one homologous or heterologous nuclei acid sequence coding for a protein mediating the import of a precursor of a desired oligosaccharide into said host cell, wherein preferably the nucleic acid sequence is overexpressed, and wherein preferably the precursor is larger than a disaccharide.” Jennewein, para. [0020].
“The genetically modified microbial host cell comprising the characteristics as set forth herein are cultured in the presence of glucose, sucrose, glycerin or a combination thereof—using these substrates as carbon- and energy sources—as well as in the presence of lactose or oligosaccharides larger than disaccharides, e.g., LNT-II.” Jennewein, para. [0021].
“With the overexpression of at least one homologous or heterologous nucleic acid sequence coding for a protein enabling the import of a precursor of a desired oligosaccharide into said host cell, it is possible to feed precursors of a desired oligosaccharide to the culture medium, which get imported into the host cell, such as, e.g., LNT-II.” Jennewein, para. [0044].
Fig. 1 of Jennewein discloses that LNT-II is transformed to Lacto-N-tetraose (LNT) by operation of a GalT glycosyltransferase. “the cell may also and/or alternatively comprise or β-1,4-galactosyltransferase activity enabling the galactosylation of LNT-II to intracellularly generate lacto-N-neotetraose LNnT.” Jennewein, para. [0087]. “In total, 11 out of 30 tested genes were observed to produce LNnT from LNT-II and UDP-galactose.” Jennewein, para. [0087]. That is, it is understood that LNnT is produced by transfer of a galactose moiety from UDP-galactose to LNT-II as an acceptor to produce LNnT.
The working embodiments of Jennewein are in E. coli (a prokaryotic cell).
The above is understood as an anticipatory disclosure of:
A process for the production of a desired LNnT oligosaccharide comprising more than three monosaccharide moieties, wherein the process comprises
providing a genetically engineered microbial cell that possesses
a saccharide importer for the uptake of an intermediate oligosaccharide being LNT-II (lacto-N-triose II) consisting of at least three monosaccharide moieties but less monosaccharide moieties than the desired oligosaccharide by the microbial cell, and
an enzyme [i.e. a β-1,3-galactosyltransferases] which is able to transfer a monosaccharide moiety from a donor substrate to the intermediate oligosaccharide;
cultivating the genetically engineered microbial cell in a culture medium which contains the LNT-II intermediate oligosaccharide for the genetically engineered microbial cell to take up the intermediate oligosaccharide LNT-II and to transfer at least one galactose monosaccharide moiety from UDP-galactose to the intermediate oligosaccharide LNT-II, thereby synthesizing the desired oligosaccharide LNnT; and
retrieving the desired oligosaccharide LNnT from the culture medium and/or the genetically engineered microbial cell.
The above is anticipatory of claims 1-4, 9, 11, 13, 15 and 17.
Regarding claim 5, “In a preferred embodiment, the genetically modified host cell comprises an increased UDP-N-acetylglucosamine and UDP-galactose, GDP-fucose or CMP-N-acetylneuraminic acid production capability as compared to a genetically unmodified host cell.” Jennewein, para. [0035]. This is a description that the donor UDP-galactose substrate is synthesized by a genetically engineered microbial cell.
Regarding claim 6, The genetically modified microbial host cell comprising the characteristics as set forth herein are cultured in the presence of glucose, sucrose, glycerin or a combination thereof—using these substrates as carbon- and energy sources—as well as in the presence of lactose or oligosaccharides larger than disaccharides, e.g., LNT-II.” Jennewein, para. [0021]. Cultured in the presence of LNT-II is understood as description that such LNT-II is exogenously added to the culture.
Regarding claim 7, the rejection under 35 U.S.C. 112(b) above is incorporated herein by reference. As stated, claim 7 includes any generic intermediate oligosaccharide including LNT-II made by any means. Any LNT-II employed as instructed in Jennewein, para. [0021], must be made by some method wherein LNT-II is unchanged in structure regardless of its method of production.
Regarding claim 8, the specification, Example 3, provides:
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“The invention also concerns a genetically modified microbial host cell, preferably a bacterial host cell, as described above in which the endogenous β-galactosidase gene is inactivated or deleted and in which a functional lactose permease gene is present.” Jennewein, para. [0033]. Deletion of such beta-galactosidase is understood to be a lack of enzyme activity that degrades LNT-II, which contains a galactose unit.
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.
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.
Claim(s) 1-9, 11, 13, 15 and 17 is/are rejected under 35 U.S.C. 103 as being unpatentable over Jennewein (U.S. 2018/0305724 A1) further in view of Dekany et al. (U.S. 2016/0215315 A1) and Olsen et al. (Lactose Permease Mutants Which Transport (Malto-)Oligosaccharides, J. Bacteriol. 175, 1993, 6269-75).
While claims 1-9, 11, 13, 15 and 17 are understood to be anticipated by Jennewein, Jennewein does not disclose a specific at least one homologous or heterologous nucleic acid sequence coding for a protein enabling the import of a precursor of a desired oligosaccharide LNT-II (intermediate oligosaccharide) into said host cell.
Dekany, in the claims, states:
1. A method for producing an oligosaccharide having at least four monosaccharide units, said method comprising the step of culturing, in a culture medium containing an exogenously added fucosylated, sialylated or N-acetyl-glucosaminylated lactose trisaccharide as acceptor [i.e. an intermediate oligosaccharide having at least three monosaccharide moieties], a genetically modified cell having a recombinant gene that encodes an enzyme capable of modifying said acceptor or one of the intermediates in the biosynthetic pathway of the oligosaccharide having at least four monosaccharide units from said acceptor and that is necessary for the synthesis of said oligosaccharide from said acceptor.
10. The method according to claim 1, wherein said acceptor is internalized by a protein assisted regulation according to an active transport mechanism [i.e. a saccharide importer for the uptake of an intermediate oligosaccharide consisting of at least three monosaccharide moieties].
13. The method according to claim 1 for the production of an oligosaccharide derivative having at least four monosaccharide units selected from difucosyllactose, sialyl-fucosyl lactose, LNT and LNnT, wherein said cell is a E. coli of LacZ−Y+ genotype, said enzyme is selected from β-1,3-galactosyl transferase, β-1,4-galactosyl transferase, α-1,2-fucosyl transferase, α-1,3-fucosyl transferase and α-2,3-sialyl transferase, and said acceptor is selected from 2′-FL, 3-FL, 3′-SL and lacto-N-triose.
14. The method according to claim 13, wherein said oligosaccharide is difucosyllactose, said enzyme is α-1,2-fucosyl transferase, and said acceptor is 2′-FL, said method further comprising the preliminary steps of making 2′-FL comprising:
culturing, in a separate culture medium containing exogenously added lactose as acceptor, a genetically modified cell having a recombinant gene that encodes an α-1,2-fucosyl transferase capable of fucosylating lactose, and
separating 2′-FL from said cell, from said culture medium or from both.
19. The method according to claim 13, wherein said oligosaccharide is LNnT, said enzyme is β-1,4-galactosyl transferase, and said acceptor is lacto-N-triose, said method further comprising the preliminary steps of making lacto-N-triose comprising:
culturing, in a separate culture medium containing exogenously added lactose as acceptor, a genetically modified cell having a recombinant gene that encodes a β-1,3-N-acetyl-glucosaminyl transferase capable of N-acetyl-glucosaminylating lactose, and
separating lacto-N-triose from said cell, from said culture medium or from both.
The acceptor molecule of Dekany is an intermediate oligosaccharide as recited in the claims such that the above claims of Dekany anticipate at least claim 1 and there is an overlap between the teachings of Jennewein and Dekany.
“The method of the invention also involves initially transporting an exogenous fucosyl, sialyl or N-acetyl-glucosaminyl lactose trisaccharide, as an acceptor molecule, from the culture medium into the genetically modified cell for glycosylation where it can be glycosylated to produce higher oligosaccharides. The acceptor can be added exogenously in a conventional manner to the culture medium, from which it can then be transported into the cell. . . . In other embodiment the exogenous acceptor can be internalized in the cell with the aid of an active transport mechanism, during which the exogenous acceptor diffuses across the plasma membrane of the cell under the influence of a transporter protein or permease of the cell. Lactose permease (LacY) has specificity towards galactose and simple galactosyl disaccharides like lactose. The specificity of a transporter protein or permease towards the sugar moiety of the substrate to be internalized can be altered by mutation by means of known recombinant DNA techniques. In a preferred embodiment the internalization of the exogenous lactose derivative acceptor takes place via an active transport mechanism.” Dekany, para. [0024].
Claim 13 of Dekany directly states that the acceptor can be lacto-N-triose and is an lactose derivative as in LNT-II as taught by Jennewein. Dekany suggests that such lactose derivatives may be actively transported into a cell (i.e. via a transporter or importer protein) with a mutated lactose permease LacY.
Olsen, abstract, teaches:
Lactose permease mutants, which were previously isolated in sugar specificity studies, were screened for their abilities to transport the trisaccharide maltotriose. Six multiple mutants (e.g., five double mutants and one triple mutant) were identified as forming fermentation-positive colonies on maltotriose MacConkey plates and were also shown to grow on maltotriose minimal plates. All of these multiple mutants contained a combination of two or three amino acid substitutions at position 177, 236, 306, or 322 within the permease. In contrast, none of the corresponding single mutants at these locations were observed to exhibit an enhanced rate of maltotriose transport. In whole-cell assays, the multiple mutants were shown to transport relatively long alpha-nitrophenylglucoside (alpha NPG) molecules. In certain cases, alpha NPG molecules containing up to four glucose residues in addition to the nitrophenyl group were shown to be transported to a significant degree. Overall, the abilities of lactose permease mutants to transport maltotriose and long alpha NPGs are discussed with regard to the dimensions of the sugar and the mechanism of sugar transport.
Olsen does not demonstrate uptake of lactose-derived trisaccharides such as LNT-II. However, LacY is known to import the disaccharide lactose and Olsen (Table 3) shows mutation expands the functionality of LacY from disaccharide only to trisaccharides including maltotriose, and Dekany suggest that LacY mutants be used for import of lactose derivatives such as trisaccharide lacto-N-triose.
Jennewein does not disclose a specific at least one homologous or heterologous nucleic acid sequence coding for a protein enabling the import of a precursor of a desired oligosaccharide LNT-II into said host cell. As discussed, Dekany and Olsen suggests that specific mutants of LacY can serve as importers/transporters of trisaccharides including lactose-derived trisaccharides that would include lacto-N-triose and LNT-II. As such, in the absence of a disclosure of a specific at least one homologous or heterologous nucleic acid sequence coding for a protein enabling the import of a precursor of a desired oligosaccharide LNT-II into said host cell by Jennewein, at the time of filing an ordinarily skilled artisan would have been motivated to select a polynucleotide encoding a LacY mutant as discussed by Dekany and Olsen as a species of homologous or heterologous nucleic acid sequence coding for a protein enabling the import of a precursor of a desired oligosaccharide LNT-II with a reasonably expectation of success. Some species of transporter/importer must be selected to implement embodiments of Jennewein wherein “With the overexpression of at least one homologous or heterologous nucleic acid sequence coding for a protein enabling the import of a precursor of a desired oligosaccharide into said host cell, it is possible to feed precursors of a desired oligosaccharide to the culture medium, which get imported into the host cell, such as, e.g., LNT-II.” Jennewein, para. [0044].
Further regarding claim 7, as discussed above, claim 7 is not understood as requiring an intermediate oligosaccharide such as LNT-II be made by any specific process including microbial fermentation. Regardless, Jennewein, para. [0087], teaches: “FIG. 1 shows a schematic drawing of an exemplary host cell 10 according to the invention, importing lactose and synthesizing lacto-N-triose II (LNT II) and lacto-N-tetraose (LNT). Lactose is imported from the medium the host cell is cultivated in into the cell via transporter 1. The enzyme N-acetylglucosaminyltransferase NacGlcT ligates N-acetylglucosamine to the acceptor substrate lactose, thus generating LNT-II. LNT-II is exported from the cell via exporter protein 20. Since LNT-II is a precursor of LNT or LNnT, the exporter exporting LNT-II represents an exporter protein exporting precursors of the latter oligosaccharides.” The preceding is understood as teaching production of LNT-II (intermediate oligosaccharide) by microbial fermentation. Since LNT-II is structurally the same regardless of its means of production, at the time of filing, an ordinarily skilled artisan would have been motivated to add LNT-II to a cell culture of a microorganism for production LNnT produced by any convenient means including LNT-II produced by microbial fermentation as described in Jennewein, para. [0087], since again, LNT-II is the same regardless of how it is produced.
Double Patenting
Claims 1-9, 11, 13, 15 and 17 are rejected on the ground of nonstatutory double patenting as being unpatentable over claim 1-16 of U.S. Patent No. 12,338,473 in view of Jennewein (U.S. 2018/0305724 A1), Dekany et al. (U.S. 2016/0215315 A1), and Olsen et al. (Lactose Permease Mutants Which Transport (Malto-)Oligosaccharides, J. Bacteriol. 175, 1993, 6269-75).
Claims 1-9, 11, 13, 15 and 17 are rejected on the ground of nonstatutory double patenting as being unpatentable over claim 1-19 of U.S. Patent No. 11,713,475 in view of Jennewein (U.S. 2018/0305724 A1), Dekany et al. (U.S. 2016/0215315 A1), and Olsen et al. (Lactose Permease Mutants Which Transport (Malto-)Oligosaccharides, J. Bacteriol. 175, 1993, 6269-75).
Claims 1-9, 11, 13, 15 and 17 are rejected on the ground of nonstatutory double patenting as being unpatentable over claim 1-12 of U.S. Patent No. 11,046,985 in view of Jennewein (U.S. 2018/0305724 A1), Dekany et al. (U.S. 2016/0215315 A1), and Olsen et al. (Lactose Permease Mutants Which Transport (Malto-)Oligosaccharides, J. Bacteriol. 175, 1993, 6269-75).
Claims 1-9, 11, 13, 15 and 17 are rejected on the ground of nonstatutory double patenting as being unpatentable over claim 1-19 of U.S. Patent No. 12,410,454 in view of Jennewein (U.S. 2018/0305724 A1), Dekany et al. (U.S. 2016/0215315 A1), and Olsen et al. (Lactose Permease Mutants Which Transport (Malto-)Oligosaccharides, J. Bacteriol. 175, 1993, 6269-75).
Claims 1-9, 11, 13, 15 and 17 are rejected on the ground of nonstatutory double patenting as being unpatentable over claim 1-11 of U.S. Patent No. 11,981,947 in view of Jennewein (U.S. 2018/0305724 A1), Dekany et al. (U.S. 2016/0215315 A1), and Olsen et al. (Lactose Permease Mutants Which Transport (Malto-)Oligosaccharides, J. Bacteriol. 175, 1993, 6269-75).
Claims 1-9, 11, 13, 15 and 17 are rejected on the ground of nonstatutory double patenting as being unpatentable over claim 1-13 of U.S. Patent No. 12,123,040 in view of Jennewein (U.S. 2018/0305724 A1), Dekany et al. (U.S. 2016/0215315 A1), and Olsen et al. (Lactose Permease Mutants Which Transport (Malto-)Oligosaccharides, J. Bacteriol. 175, 1993, 6269-75).
The rejections stated above under 35 U.S.C. 102 and 103 stated above are incorporated herein by reference.
The claims of the reference applications all recite an engineered microorganism producing an oligosaccharide of LNT and/or LNnT or a method of using such microorganism including expression of a heterologous nucleotide encoding a galactosyltransferase utilizing UDP-galactose to form such oligosaccharides or open to expression of such a galactosyltransferase. The claims of 11,713,475 are representative:
1. A method for production of lactose or an oligosaccharide of interest which comprises a galactose-β1,4-glucose moiety at its reducing end, the method comprising:
a) providing a genetically engineered microbial cell, wherein said microbial cell comprises:
at least one glucose transporter for translocating glucose from the culture medium into the microbial cell's cytoplasm;
an UDP-galactose biosynthesis pathway;
at least one β-1,4-galactosyltransferase being able to galactosylate free glucose to intracellularly produce lactose; and
one or more of:
i) a fructose transporter, fructokinase-6 activity, and 6-phosphofructokinase-1 activity; and
ii) a fructose specific phosphotransferase system and 1-phosphofructokinase;
b) cultivating the microbial cell in a culture medium and under conditions that are permissive for production of said lactose or oligosaccharide of interest, wherein the culture medium comprises a mixture of glucose and fructose as main carbon source; and
c) recovering the lactose or oligosaccharide of interest from the culture medium and/or the microbial cell.
2. The method according to claim 1, wherein the oligosaccharide of interest is a human milk oligosaccharide selected from 2′-fucosyllactose, 3-fucosyllactose, 2′,3-difucosyllactose, 3′-sialyllactose, 6′-sialyllactose, 3-fucosyl-3′-sialyllactose, lacto-N-tetraose, lacto-N-neotetraose, lacto-N-fucopentaose I, lacto-N-fucopentaose II, lacto-N-fucopentaose III, lacto-N-fucopentaose V, lacto-N-difucosylhexose I, lacto-N-difucosylhexaose II, lacto-N-sialylpentaose LSTa, LSTb, LSTc.
10. The method of claim 1, wherein said microbial cell possesses at least one additional glycosyltransferase.
11. The method of claim 10, wherein the at least one additional glycosyltransferase is selected from fucosyltransferases, sialyltransferases, glucosaminyltransferases and galactosyltransferases.
As discussed above, Jennewein teaches that it is beneficial to modify a microorganism for producing an oligosaccharide to express a transporter/importer to transport an intermediate oligosaccharide thereto (e.g. triose LNT-II) and to culture the microorganism in the presence of such intermediate oligosaccharide including such intermediate oligosaccharide being LNT-II wherein a product of LNnT or LNFP-III is to be obtained.
As such, at the time of filing, on ordinarily skilled artisan would have been motivated to modify embodiment microbial cells of the reference claims to express a polynucleotide saccharide importer or transporter for LNT-II or other intermediate oligosaccharide (particularly trioses) and to culture such microbial cells in the presence of such intermediate oligosaccharide, since Jennewein teach that the same is beneficial for the production of oligosaccharides that are tetraose derivates of such an intermediate oligosaccharide, i.e. LNnT or LNT. As reviewed above, Dekany and Olsen et al. suggest certain mutants of LacY as appropriate transporter/importer for triose intermediate oligosaccharides having a lactose core, i.e. LNT-II. The features of the rejected claims are suggested by Jennewein (U.S. 2018/0305724 A1), Dekany et al. (U.S. 2016/0215315 A1), and Olsen et al. (Lactose Permease Mutants Which Transport (Malto-)Oligosaccharides, J. Bacteriol. 175, 1993, 6269-75), as discussed above, such that an ordinarily skilled artisan would have been motivated to modify embodiments of the reference claims being microorganisms and methods for producing oligosaccharides including LNnT or LNT to have the same features of the rejected claims as discussed above.
Conclusion
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/TODD M EPSTEIN/Primary Examiner, Art Unit 1652
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