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
Claims 1-8 and 11-21 are pending following the preliminary amendment filed 11/15/2023. Claims 9-10 and 22-24 have been cancelled by Applicant. Claims 1-8 and 11-21 are presently considered.
Priority
Receipt is acknowledged of certified copies of papers required by 37 CFR 1.55. The present application and all claims are being examined with the earliest effective filing date of 05/17/2021.
Information Disclosure Statement
The information disclosure statement (IDS) filed on 06/24/2024 is in compliance with the provisions of 37 CFR 1.97. Accordingly, the information disclosure statement is being considered by the examiner.
Claim Objections
Claim 1 is objected to because of the following informalities: the claim recites limitations “i-v” as “i… ii… and… iii… iv… and v”. Please remove the “and” recited between limitations “ii” and “iii” on line 12. Appropriate correction is required.
Claim 1 is objected to because of the following informalities: the claim recites “expression of and of i)-iv),” on line 19, which appears to be a typographical error. Please amend this phrase to read “expression of i)-iv);”. Appropriate correction is required.
Claim 1 is objected to because of the following informalities: the claim does not end in a period. Appropriate correction is required.
Claim 4 is objected to because of the following informalities: the claim does not end in a period. Appropriate correction is required.
Claim 16 is objected to because of the following informalities: the claim recites “wherein the HMO blend has molar % of...”. Please amend to “wherein the HMO blend has a molar % of…” Appropriate correction is required.
Claim 16 is objected to because of the following informalities: the claim does not end in period. Appropriate correction is required.
Claim 18 is objected to because of the following informalities: the claim recites “the level of lactose the fermentation medium” which is understood to mean “the level of lactose in the fermentation medium”. Appropriate correction is required.
Claim 19 is objected to because of the following informalities: the claim recites limitations “ii-vii” which appears to be in error, because the first limitation is numbered “ii” instead of “i". For clarity, please amend the numbering of the limitations to “i-vi”. Appropriate correction is required.
Claim 20 is objected to because of the following informalities: the claim recites “colanic acid gene cluster” in line 2, which is understood to be referring to the colanic acid gene cluster of claim 19. To provide unequivocal antecedent basis, please amend this phrase to read “the colanic acid gene cluster”. Appropriate correction is required.
Claim Rejections - 35 USC § 112(b)
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 1-8 and 11-21 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 the limitation “an amino acid sequence which is at least 80% identical to SEQ ID NO: 12 or 3,” in lines 8-9. This renders the claim indefinite because SEQ ID NO: 12 is a nucleic acid sequence. In the interest of compact prosecution, this limitation is reasonably interpreted to mean “an amino acid sequence which is at least 80% identical to SEQ ID NO: 1, 2 or 3”, as previously recited in the claim.
Claim 1 recites the limitation "the colanic acid gene cluster" in line 17. There is insufficient antecedent basis for this limitation in the claim.
Claim 1 recites the limitation “expresses functionally the colanic acid gene cluster” in line 17, wherein the phrase “expresses functionally” also renders the claim indefinite. Here, it is unclear if the limitation requires the cell to comprise a colanic acid gene cluster, or if the phrase “expresses functionally” broadens the scope to expressing functional equivalents of such genes. As such, one may consider a cell comprising and expressing a colanic gene cluster to meet the limitation, while another person may consider a cell expressing other genes with some similar function to also meet the limitation. One may also consider a cell having only some of the genes found in the colanic acid gene cluster, which raises further questions as to what minimal structure is required for a cell that “expresses functionally” said gene cluster. Hence, the metes and bounds have not been clearly set forth.
Claims 2 recites the limitation "the copy number" in line 2. There is insufficient antecedent basis for this limitation in the claim. See MPEP 2173.05(e) which states,
Inherent components of elements recited have antecedent basis in the recitation of the elements themselves. For example, the limitation "the outer surface of said sphere" would not require an antecedent recitation that the sphere has an outer surface. See Bose Corp. v. JBL, Inc., 274 F.3d 1354, 1359, 61 USPQ2d 1216, 1218-19 (Fed. Cir 2001) (holding that recitation of "an ellipse" provided antecedent basis for "an ellipse having a major diameter" because "[t]here can be no dispute that mathematically an inherent characteristic of an ellipse is a major diameter"). (Emphasis added)
In the instant case, it is understood that a given “gene cluster” inherently has a copy number (e.g., of at least one). However, the recitation of “the copy number” in the claim does not clearly refer back to the element of a gene cluster (e.g., “the copy number of said gene cluster”), rendering the limitation indefinite.
In the interest of compact prosecution, the limitation, “wherein the colanic acid gene cluster is overexpressed by increasing the copy number” is reasonably interpreted to mean “wherein the colanic acid gene cluster is overexpressed by increasing its copy number”. Applicant is advised the rejection may be obviated by amending the claim accordingly or as in the example above (“the copy number of said gene cluster”).
Claim 5 recites the limitation “the copy number” in line 2. There is insufficient antecedent basis for this limitation in the claim. Here, the limitation “wherein the expression of i) or ii) is overexpressed by increasing the copy number” may be interpreted as referring to the copy number of the proteins themselves or the copy number of the genes which encode them. However, no “genes” encoding i) or ii) are recited in the claim.
Claim 6 recites the limitation “wherein the expression of i) and/or ii) is obtained from a single copy” which renders the claim indefinite, because it has not been clearly set forth what element(s) the phrase “a single copy” is referring to. For example, this limitation may be interpreted as referring to a single copy of the protein(s) themselves or a single copy of the gene(s) which encode them. However, no “genes” encoding i) or ii) are recited in the claim.
Claim 6 recites the limitation "the regulatory element for expression of i) and/or ii)" in lines 2-3. There is insufficient antecedent basis for this limitation in the claim. Examiner notes that this element is recited in claim 5, while the present claim depends from claim 1, which recites “v. comprises a native or heterologous regulatory element for controlling the expression… of i)-iv)” in lines 18-19. Hence, it is unclear whether this limitation is referring to the same regulatory element (i.e., the regulatory element of limitation “v.”) or an additional regulatory element.
The terms “low or intermediate strength” and “high strength” in claims 6-7 are relative terms which render the claims indefinite. The terms are not defined by the claims, the specification does not provide a standard for ascertaining the requisite degree, and one of ordinary skill in the art would not be reasonably apprised of the scope of the invention. The examiner notes that while the specification discusses what Applicant considers to be low, intermediate, and strong “activity” of a regulatory element in Miller Units (MU), as well as examples of a strong and weak promoter (see pg. 33, lines 10-14), this discussion is not presented in a manner that clearly and specifically re-defines the terms (i.e., provides a special definition). Hence, while Applicant discloses that a “regulatory element with an activity above 10,000 MU is considered strong”, this disclosure does not clearly re-define the phrase “the regulatory element for expression… has high strength”. Applicant is reminded that while the claims are interpreted in light of the specification, limitations from the specification are not read into the claims. See In re Van Geuns, 988 F.2d 1181, 26 USPQ2d 1057 (Fed. Cir. 1993).
In the interest of compact prosecution, it is interpreted that any regulatory element of the prior art that is explicitly referred to as being “weak or strong” or having “low/intermediate/high strength” may meet the respective limitations, as well as any element explicitly described as such in Applicant’s disclosure. For example, the specification states that an “example of a strong regulatory element is the PglpF promoter” and “an example of a weak promoter is Plac” (see pg. 33, lines 10-14). Regulatory elements disclosed in the prior art as having the levels of activity in miller units (MU) as described in the instant specification may also meet the claim.
Claim 7 recites the limitation “wherein the expression of i) and/or ii) is obtained from two or more copies” which renders the claim indefinite, because it has not been clearly set forth what element the phrase “two or more copies” is referring to. For example, this limitation may be interpreted as referring to two or more copies of the protein(s) themselves or two or more copies of the gene(s) which encode them.
Claim 7 recites the limitation "the regulatory element for expression of i) and/or ii)" in lines 2-3. There is insufficient antecedent basis for this limitation in the claim. Examiner notes that this element is recited in claim 5, while the present claim depends from claim 1, which recites “a native or heterologous regulatory element for controlling the expression… of i)-iv)”. Hence, it is unclear whether this limitation is referring to the same regulatory element of limitation “v.” or a different regulatory element.
Claim 18 recites the limitation "the fermentation medium" in line 2. There is insufficient antecedent basis for this limitation in the claim. Examiner notes that the claim depends from claim 1, which recites a “cell culture medium”.
Claim 19 recites the limitation "the colanic acid gene cluster" in line 17. There is insufficient antecedent basis for this limitation in the claim. Applicant may amend this limitation to read, for example, “a colanic acid gene cluster” to obviate the rejection.
Claim 20 recites the limitation "the copy number" in line 2. There is insufficient antecedent basis for this limitation in the claim. Applicant may amend this limitation to read, for example, “its copy number” or “the copy number of said gene cluster” to obviate the rejection.
Claims 3-4, 8, 11-17 and 21 are rejected for depending from an indefinite claim and for failing to rectify the indefiniteness of the claim(s) from which they depend.
Claim Rejections - 35 USC § 112(a) – Written Description
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 1-8 and 11-21 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.
Brief Statement of the Issue(s)
The claims are directed to genetically engineered cells for the production of human milk oligosaccharides, wherein the cells comprise functional homologues having 80-100% sequence identity to the proteins represented by SEQ ID NOs 1-7, 28-33 and 49 and are capable of producing at least two HMOs. Further, the claims require that the HMO blend produced by the method has specific HMOs, 2’-FL and LNFP-I, as the predominant products produced by said method. However, the functional homologues of the claims encompass a vast genus, wherein there are insufficient structure-function teachings and not enough predictability in the art for one to have envisaged which species would be capable of performing the functions required by the claims. As such, the specification as filed would not lead a person of ordinary skill in the art to conclude that the inventor possessed the entire scope of the claimed invention.
Claim Scope
Independent claim 1 recites a method for the production of a human milk oligosaccharide (HMO) blend with 2’-FL and LNFP-I as the predominant HMOs and includes the step of providing a genetically engineered cell capable of producing at least two HMO’s. As recited in the claim, the cell comprises the functional homologues of i), ii), and iii), which are defined as being at least 80% identical to the proteins represented by SEQ ID NOs 1-7 and 49.
Claim 15 further requires the cell to comprise a gene product that upon expression acts as a sugar efflux transporter, which is selected from SEQ ID NOs 28-33 or to a functional homologue thereof having amino acid sequence which is at least 70% identical to the respective SEQ ID NO.
Claims 16-18 recite further functional limitations requiring the molar % of 2’-FL and LNFP-I produced in the HMO blend to be within a claimed range (e.g., 2’-FL between 25% to 70% and LNFP-I between 30% to 60%).
Independent claim 19 recites a genetically engineered cell comprising a recombinant nucleic acid sequence encoding the functional homologues of ii), iii), and iv), which are also defined as being at least 80% identical to the proteins represented by SEQ ID NOs 1-7 and 49.
The applicable claim interpretations have been set forth above under 35 U.S.C. 112(b) and in a separate Claim Interpretation section. Those discussions are incorporated herein.
The functional homologues of independent claims 1 and 19, having as little as 80% sequence similarity with SEQ ID NOs 1-7 and 49, may comprise as many as 87 modifications (e.g., amino acid substitutions) relative to the recited SEQ ID NOs. The functional homologues of claim 15, having as little as 70% sequence similarity with SEQ ID NOs 28-33, may comprise as many as 119 modifications relative to the recited SEQ ID NOs.
According to Reetz, M. (cited on Form 892), the random substitution of only three amino acids in a protein composed of 300 residues may result in 30 billion variants of said protein. Hence, it is unclear if the claim scope encompass trillions of species or perhaps only a few in view of the functional limitations set forth in the claim(s). Accordingly, the claim scope reasonably appears to be vast and highly varied.
Actual Reduction to Practice
Examples 1-4 of Applicant’s disclosure disclose the strains MP1 to MP18 (numbered 1 to 18) (see pgs. 76-81). The heterologous proteins comprised by each strain is provided in Tables 1-4 (see pgs. 58-60). These strains are all disclosed as comprising heterologous proteins represented by the same SEQ ID NOs of the claims. For example, strains MP12 to MP18 comprise HMO proteins represented SEQ ID NOs 1, 4 and 6, and any one of the sugar efflux transporters represented by SEQ ID NOs 28-33 (see pg. 60, Table 4). Therefore, zero examples of the claimed invention were reduced to practice wherein the genetically modified cell comprised a “functional homologue” of any of the recited SEQ ID NOs.
Assessment of whether disclosed species are representative of the claimed genus
MPEP § 2163 states that a “representative number of species” means that the species which are adequately described are representative of the entire genus (see, e.g., MPEP § 2163(II)(3)(a), MPEP §2163.03(V)). Thus, when there is substantial variation within the genus, one must describe a sufficient variety of species to reflect the variation within the genus.
In this case, the claims encompass an essentially infinite number of genetically engineered cells, but zero embodiments of the claimed invention specifically had a functional homologue of any of the recited SEQ ID NOs.
Although the MPEP does not define what constitutes a sufficient number of representative species, the Courts have indicated that the disclosure of two species within a subgenus did not describe that subgenus. In re Gostelli, 872 F.2d at 1012, 10 USPQ2d at 1618. Similarly, the disclosure of zero homologues of the claimed heterologous proteins does not provide sufficient disclosure to satisfy the written description requirement for the instantly claimed genus.
Identifying characteristics of the genus
In the absence of a reduction to practice of a representative number of species, the written description requirement for a claimed genus may be satisfied by disclosure of relevant, identifying characteristics, sufficient to show the applicant was in possession of the claimed genus.
According to the instant specification: a “functional homologue” of a protein sequence is a protein with alterations, which retains its original functionality; a functional homologue may be obtained by mutagenesis; the functional homologue should have a remaining functionality of at least 50% compared to the functionality of the protein sequence; a functional homologue of any one of the disclosed amino acid or nucleic acid sequences can also have a higher functionality; and a “functional homologue of any one of SEQ ID NOs: 1-47, should ideally be able to participate in the HMO production, in terms of HMO yield, purity, reduction in biomass formation, viability of the genetically engineered cell, robustness of the genetically engineered cell according to the disclosure, or reduction in consumables” (see page 30, lines 1-12).
However, zero discussion is provided in the originally filed disclosure identifying which structures (i.e., motifs, regions, secondary or tertiary structures, etc.) are required in order for the homologues to retain the functionality of their parent enzyme. There is also zero discussion regarding which mutations would result in an enzyme that retains at least 50% activity, or which mutations would be expected to result in higher functionality. Moreover, there is zero discussion of how the structure and activity of each functional homologue used in combination corresponds to a genetically engineered cell capable of producing an HMO blend with 2’FL and LNFP-I as the predominant HMOs, or any of the functional limitations required by the claims.
Accordingly, the functional limitations of a cell “capable of producing at least two HMO’s”, “2’-FL and LNFP-I as the predominant HMOs”, and “wherein the HMO blend has a molar % between 25% to 70% and LNFP-I between 30% to 60% of the total HMO” are only utilized as a vague attempt to capture unknown and undisclosed structures, sufficient to achieve some functional result that Applicant hopes and desires that the disclosed invention is able to achieve. However, the disclosure does not meaningfully disclose an unambiguous structure/function relationship permitting an artisan to identify, a priori, which exact structures do or do not satisfy the functional limitations at issue.
Predictability in the Art
It would have been prima facie unknown to a person of ordinary skill at the time of filing which homologues of the recited enzymes would be expected to have at least 50% functionality compared to the recited sequences, or have less than 50% functionality compared to the recited sequences. Moreover, it is prima facie unknown which functional homologues, and in which combinations, would be able to produce an HMO blend with 2’FL and LNFP-I as the predominant HMOs, and in the concentrations recited by the claims.
According to Applicant’s own disclosure, “knowledge as to how to make composition of Lacto-N-fucopentaose I (LNFP-I)-containing blends and how to fine-tune the levels of the different HMOs of the acquired blends, either by genetic engineering, or adjustment of fermentation parameters, is non-existent, because commercial fermentation process parameters for HMO manufacturing are normally kept secret and therefore the effects of e.g., fermentation parameters on LNFP-I blend compositions, or any other HMO blends, has not been described” (See page 2, lines 8-13; Emphasis added).
Although the level of skill in the art is high, the predictability in the art is low due to the complexity of biological systems, biochemistry, and the structural and functional properties of enzymes. Specifically, an artisan would not be able to predict or identify, a priori, and in the absence of any guidance or consensus structures exactly what compounds would be capable of obtaining the same results as those that were used in Applicant’s Examples.
Accordingly, in the absence of sufficient structure/function teachings, as required to practice the full scope of the claims, an artisan would not reasonably conclude that Applicant possessed the full scope of the broad and highly varied claim scope.
Conclusion
The description requirement of the patent statute requires a description of an invention, not an indication of a result that one might achieve if one made that invention. See In re Wilder, 736 F.2d 1516, 1521, 222 USPQ 369, 372-73 (Fed. Cir. 1984) (affirming rejection because the specification does "little more than outlin[e] goals appellants hope the claimed invention achieves and the problems the invention will hopefully ameliorate."). The courts have stated that “merely drawing a fence around a perceived genus is not a description of the genus. One needs to show that one has truly invented the genus, i.e., that one has conceived and described sufficient representative species encompassing the breadth of the genus. Otherwise, one has only a research plan, leaving it to others to explore the unknown contours of the claimed genus” (see, e.g., AbbVie v. Janssen, 111 USPQ2d 1780 (Fed. Cir. 2014) at 1789). In addition, the Courts have stated
“[r]egardless whether a compound is claimed per se or a method is claimed that entails the use of the compound, the inventor cannot lay claim to the subject matter unless he can provide a description of the compound sufficient to distinguish infringing compounds from non-infringing compounds, or infringing methods from non-infringing methods.” University of Rochester v. G.D. Searle Co., 69 USPQ2d 1886 1984 (CAFC 2004) (emphasis added).
This is pertinent because, in the instant case, Applicants have claimed a broad and highly varied genus comprising an unknown number of species defined by reference to one or more functional limitations; however, the originally filed disclosure has failed to identify any common structure-function relationship sufficient to permit an artisan to identify what structures are included or excluded by the claim scope. This also means that the skilled artisan cannot envisage what structures infringe or do not infringe upon the pending claim scope.
In conclusion, for the reasons discussed above, the skilled artisan would not reasonably conclude that the inventor(s), at the time the application was filed, had possession of the full scope of the claimed invention.
Claim Rejections - 35 USC § 103
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.
(a)(2) the claimed invention was described in a patent issued under section 151, or in an application for patent published or deemed published under section 122(b), in which the patent or application, as the case may be, names another inventor and was effectively filed before the effective filing date of the claimed invention.
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. Considering objective evidence present in the application indicating obviousness or nonobviousness.
Claim(s) 1-8, 11, 13, 16 and 18 is/are rejected under 35 U.S.C. 103 as being unpatentable over Pedersen et al. (WO 2019123324 A1; cited in the IDS filed 06/24/2024 at Cite No. 7), hereafter, “Pedersen”, and further in view of McCoy et al. (US Patent No. 11,643,675 B2; cited on Form 892), hereafter, “McCoy”, as further evidenced by GenBank WP_02248149.1 and GenBank AB050723 (both cited on Form 892).
Regarding claim 1, Pedersen teaches that the commercial importance of bacterial cells to produce recombinant molecules is increasing (see pg. 1, lines 11-12) and discloses a need for a simple and effective genome-based bacterial expression systems for the industrial production of recombinant polypeptides (see pg. 3, lines 1-2). Pedersen teaches nucleic acid constructs that can advantageously be used to produce a variety of human milk oligosaccharides (HMOs) recombinantly in industrial scales (see pg. 1, lines 5-8). Pedersen further teaches a bacterial recombinant cell comprising the nucleic construct, an expression system comprising the recombinant cell, and a method for the production of the oligosaccharides (see pg. 5, lines 14-22).
Regarding limitation (a), Pedersen discloses the engineering of Escherichia coli for the production of 2’FL and LNFP-I (see pg. 82, lines 8-9; pg. 83, lines 11-12). Pedersen teaches that proteins that are essential for the production of one or more HMOs by a host cell include (see pg. 29, lines 20-21):
(i) β-1,3-N-acetylglucosaminyltransferase (see pg. 29, Table 2, “IgtA”);
(ii) β-1,3-galactosyltransferase (see pg. 29, Table 2, “cpsIBJ”); and
(iii) α-1,2-fucosyl-transferase (see pg. 30, Table 2, “futC”).
Pedersen also teaches that the genetically engineered cell:
(iv) expresses the colanic acid gene cluster (see pg. 82, lines 10-19); and
(v) comprises a regulatory element, such as a PglpF promoter and/or a Plac promoter, to control expression of (i)-(iv) (see pg. 34, lines 6-8; pg. 82, lines 11-15).
Regarding limitations (b)-(c), Pedersen teaches the method for producing fucosylated HMOs comprises (b) culturing the bacterium in the presence of a carbon source and (c) retrieving a fucosylated HMO from the bacterium or from the culture supernatant of the bacterium (see pg. 54, lines 13-15).
(i) Regarding the limitation of “a heterologous β-1,3-N-acetyl-glucosaminyltransferase as shown in SEQ ID NO: 1, 2 or 3, or a functional homologue thereof”, Pedersen teaches the sequence ID for this enzyme is WP_02248149.1 in GenBank (see pg. 29, Table 2, “IgtA”). As shown in the following alignment, the GenBank sequence (bottom) is at least 80% identical to instant SEQ ID NO: 1 (top):
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494
641
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(ii) Regarding the limitation of “a heterologous β-1,3-galactosyltransferase as shown in SEQ ID NO: 4 or 5, or a functional homologue thereof”, Pedersen teaches the sequence ID for this enzyme is AB050723 in GenBank (see pg. 29, Table 2, “cpsIBJ”). The “cpsIBJ” gene is shown at the top of page 3 of this entry. As shown in the following alignment, the GenBank sequence (bottom) comprises an amino acid sequence that is at least 80% identical to instant SEQ ID NO: 4 (top):
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711
647
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Note that the prior art sequence comprises the full-length of the claimed sequence and the sequences share at least 82.6% similarity over this region.
Pedersen does not teach a functional homologue of the α-1,2-fucosyl-transferase having an amino acid sequence which is at least 80% identical to any one of SEQ ID NOs 6, 7 or 49.
McCoy teaches compositions and methods for engineering E. coli or other host production bacterial strains to produce fucosylated oligosaccharides (see Abstract). McCoy teaches that human milk contains a diverse and abundant set of oligosaccharides that serve critical roles in the establishment of a healthy gut microbiome, in the prevention of disease, and in immune function; however, the ability to produce human milk oligosaccharides (HMOs) has been problematic due to product impurities and high overall cost (see col. 1, lines 34-48).
McCoy teaches an efficient and economical method for producing fucosylated oligosaccharides using an isolated nucleic acid comprising a sequence encoding a lactose utilizing an α-1,2-fucosyltransferase gene product, which is operably linked to one or more heterologous control sequences that direct the production of the recombinant fucosyltransferase gene product in a host production bacterium such as Escherichia coli (see col. 1, lines 52-61). McCoy teaches that exemplary fucosylated oligosaccharides produced by the method include 2’-gucosyllactose (2’FL) and lacto-N-fucopentaose I (LNFP-I) (see col. 2, lines 4-7). McCoy teaches that the lactose-accepting α-1,2-fucosyltransferase is a “FutC” having the amino acid sequence represented by SEQ ID NO: 1 (see col. 2, lines 19-26).
As shown in the following alignment, McCoy’s SEQ ID NO: 1 (bottom) is at least 80% identical to instant SEQ ID NO: 6 (top):
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425
646
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It would have been obvious at the time of filing for a person of ordinary skill in the art to have arrived at the claimed invention by combining the teachings of Pedersen and McCoy, because both references teach methods for producing HMOs, including 2’-FL and LNFP-I, using genetically engineered cells, such as E. coli. One would have been particularly motivated to do so, because Pedersen and McCoy teach the importance of HMOs as an industrial product and the need for more efficient and inexpensive methods to produce said HMOs. As both references teach genetically engineered bacteria comprising an α-1,2-fucosyltransferase, one would have recognized that the enzyme taught by McCoy could be used in the genetically engineered bacterium taught by Pedersen with a reasonable expectation of success. One would have also recognized that the elements of each reference (i.e., HMO enzymes, colanic acid gene cluster, regulatory elements, etc.) could be combined by known methods and that the results of the combination would have been predictable. Hence, the combination would have been readily apparent and deemed to be a mere (A) combining of prior art elements according to known methods to yield predictable results (see MPEP 2143(I): Rationales to support rejections under 35 U.S.C. 103).
Regarding claim 2, Pedersen discloses that the integration of an extra genomic copy of the colanic acid genes expressed from the PglpF promoter resulted in a marked improvement of the LNFP-I titer (see pg. 82, lines 10-19). Hence, it would have been obvious to have overexpressed the colanic acid gene cluster by increasing the copy number and/or by choosing an appropriate element (i.e., PglpF promoter) in order to improve the production of LNFP-I.
Regarding claim 3, Pedersen teaches a β-1,3-N-acetylglucosaminyltransferase that is at least 80% identical to instant SEQ ID NO: 1, as discussed regarding claim 1.
Regarding claim 4, Pedersen teaches a β-1,3-galactosyltransferase that is at least 80% identical to instant SEQ ID NO: 4, as discussed regarding claim 1.
Regarding claim 5, Pedersen teaches that one approach to overcome the problem of insufficient level of production is the use of strong inducible promoters for controlling the transcription of integrated recombinant genes (see pg. 3, lines 4-6). Pedersen discloses a strain expressing the heterologous genes IgtA, galTK and futC from a single genomic copy under the control of the PglpF promoter (see pg. 82, lines 11-13). Note that these three genes encode heterologous proteins which read upon the proteins recited in claim 1(i)-(iii) (see Pedersen at pg. 67, Table 9). Pedersen also teaches embodiments wherein the host cell genome preferably comprises two or three copies of a genome-integrated expression cassette (see pg. 34, lines 22-24), wherein the expression cassette comprises genes “essential” for the production of HMOs (see pg. 34, lines 5-8). As previously discussed, Pedersen teaches the gene products which read upon the limitations of i) and ii) to be “essential” for the production of HMOs. Hence, it would have been obvious to have overexpressed these proteins by increasing their copy number and/or by choosing an appropriate regulatory element (i.e., a PglpF promoter).
Regarding claim 6, Pederson teaches that in some embodiments “it is preferred that the constructs are integrated in a single copy or a low copy number” (see pg. 34, lines 29-31). In one embodiment, Pedersen teaches the gene encoding a β-1,3-N-acetylglucosaminyltransferase or a galactosyltransferase (which read on the limitations of (i) and (ii), respectively) is under the control of a Plac promoter (see pg. 59, line 31 to pg. 60, line 6). In view of the instant specification, “an example of a weak promoter is Plac” (see pg. 33, lines 12-14). Hence, it would have been obvious to have obtained expression from a single copy of (i) and/or (ii) and/or from a regulatory element having low strength.
Regarding claim 7, Pedersen teaches embodiments wherein the host cell genome preferably comprises two or three copies of a genome-integrated expression cassette (see pg. 34, lines 22-24), wherein the expression cassette comprises genes “essential” for the production of HMOs (see pg. 34, lines 5-8). As previously discussed, Pedersen teaches the gene products which read upon the limitations of i) and ii) to be “essential” for the production of HMOs. Hence, it would have been obvious to have expressed these proteins from two or more copies.
Regarding claim 8, Pedersen teaches the genetically engineered cell comprising a regulatory element, such as a PglpF promoter and/or a Plac promoter, to control expression of (i)-(iv), as discussed regarding claim 1(v). Pedersen teaches the PglpF promoter is represented by SEQ ID NO: 12 (see, e.g., pg. 70, line 2). As shown in the following alignment, instant SEQ ID NO: 13 (top) is identical to Pedersen’s SEQ ID NO: 12 (bottom):
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Regarding claim 11, Pedersen discloses a working example wherein the removal of the transcriptional repressor GlpR in an E. coli strain increased expression from the PglpF promoter element (see pg. 78, line 30 to pg. 79, line 3). In view of the instant claims, GlpR meets the limitation of “a gene product that binds to v) or regions upstream of v) and represses the expression of any one of i), ii), iii) or iv)” (see claim 12). Hence, Pedersen teaches a gene product (GlpR) that represses the expression of (i)-(iv), which are under the control of a regulatory element (PglpF promoter), and teaches the deletion of this gene product within the cell to increase expression of said regulatory element. As Pedersen teaches the promoter DNA sequence to comprise binding sites for the GlpR protein (see, e.g., pg. 16, lines 20-24), it is understood that this gene product binds to said regulatory element, “v)”.
Regarding claim 13, Pedersen and McCoy teach the α-1,2-fucosyltransferase is a FutC and McCoy’s SEQ ID NO: 1 is at least 80% identical to instant SEQ ID NO: 6, as discussed regarding claim 1.
Regarding claim 16, Pedersen and McCoy do not explicitly teach the method, wherein the HMO blend has a molar % of 2’-FL between 25% to 70% and LNFP-I between 30% to 60% of the total HMOs produced. However, the further limitation of claim 16 is directed to an inherent property that does not affect the structure or steps of the claimed invention. As the claimed method is obvious over the prior art combination, the effects and properties of said method, as well as the composition used to perform said method, are presumed to be inherent. “[T]he claiming of a new use, new function or unknown property which is inherently present in the prior art does not necessarily make the claim patentable.” In re Best, 562 F.2d 1252, 1254, 195 USPQ 430, 433 (CCPA 1977). See also MPEP 2112(II) which states:
"Products of identical chemical composition can not have mutually exclusive properties." In re Spada, 911 F.2d 705, 709, 15 USPQ2d 1655, 1658 (Fed. Cir. 1990). A chemical composition and its properties are inseparable. Therefore, if the prior art teaches the identical chemical structure, the properties applicant discloses and/or claims are necessarily present.
In this case, Pedersen and McCoy teach genetically engineered bacteria comprising the components of claim 1 which are disclosed to produce the same HMO products when cultured. Because the limitations of the claimed method are necessarily present in the prior art combination, the functional limitations of the asserted claim are inherently met by the combination of references. See MPEP 2112. Hence, claim 16 is obvious for the same reasons discussed regarding claim 1.
Regarding claim 18, Pedersen teaches the cultured cells were inoculated into a medium containing 0.5% lactose prior to incubation and harvesting of the HMOs (see pg. 74, lines 1-2), which is equivalent to a level of lactose of 5 g/L. Hence, it would have been obvious for one to have selected this lactose concentration for the fermentation medium. Pedersen and McCoy do not explicitly teach the molar % of 2’-FL to be between 25% to 35% of the produced blend of HMOs. However, this limitation is directed to an inherent property that does not affect the structure or steps of the claimed invention. Pedersen and McCoy teach genetically engineered bacteria comprising the components of claim 1 which are disclosed to produce the same HMO products when cultured. Because the limitations of the claimed method are necessarily present in the prior art combination, the functional limitations of the asserted claim are inherently met by the combination of references. See MPEP 2112 and the discussion regarding claim 16 above.
Claim(s) 12 is/are rejected under 35 U.S.C. 103 as being unpatentable over Pedersen and McCoy, as evidenced by GenBank WP_02248149.1 and GenBank AB050723, as applied to claims 1-8, 11, 13, 16 and 18 above, and further in view of GenBank M96795.1 (cited on Form 892).
Regarding claim 12, Pedersen teaches the GlpR repressor is removed to increase expression of (i)-(iv), as discussed regarding claim 11.
Pedersen does not teach the nucleotide sequence encoding GlpR (SEQ ID NO: 48).
GenBank M96795.1 is identified in GenBank as a region of Escherichia coli comprising “repressor protein (glpR) genes” (see Title and Definition). As shown in the following alignment, instant SEQ ID NO: 1 (top) is at least 70% identical to GenBank M96795.1 (bottom):
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Hence, it would have been obvious for a person of ordinary skill to have deleted or made non-functional this region of the genome, because Pedersen discloses that doing so will improve the expression of the heterologous enzymes producing HMOs. One would have recognized that in order to remove the GlpR repressor gene from the genome of E. coli, the region encoding this gene product must be identified, and that the GlpR repressor gene in E. coli is readily identifiable in public databases. Hence, it would have been well within the ordinary skill in the art to have used any known genomic sequence corresponding to the species/strain used to produce HMOs in order to identify and remove this gene. Further, the claim is obvious for the same reasons discussed regarding claims 1 and 11.
Claim(s) 14, 17 and 19-21 is/are rejected under 35 U.S.C. 103 as being unpatentable over Pedersen and McCoy, as evidenced by GenBank WP_02248149.1 and GenBank AB050723, as applied to claims 1-8, 11, 13, 16 and 18 above, and further in view of Stefan et al. (WO 2018077892 A1; cited on Form 892), hereafter, “Stefan”.
Regarding claim 14, Pedersen teaches that the coding DNA of the construct may encode
a sugar transporter protein which is normally expressed by the host bacterial cell that naturally comprises in its genome a gene encoding said sugar transporter protein (see pg. 28, lines 24-27). However, there is no further discussion regarding this gene product in Pedersen.
Hence, Pedersen and McCoy do not explicitly teach the method wherein the cell further comprises a gene product that upon expression acts as a “sugar efflux transporter”.
Stefan teaches a method for producing fucosylated oligosaccharides by using a recombinant, genetically modified prokaryotic host cell, and the transport of the produced fucosylated oligosaccharide through the cell membrane is facilitated by an exogenous transport protein (see Abstract). Stefan teaches that numerous beneficial properties of HMOs have been reported, but the occurrence and concentration of these complex oligosaccharides are specific to humans and thus cannot be found in large quantities in the milk of other mammals (see pg. 1, para. [0002]-[0003]). Stefan teaches that the most prominent HMO is 2’-fucosyllactose (2’-FL), and other prominent HMOs in human milk include lacto-N-fucopentaoses (LNFP) (see pg. 2, para. [0004]). Stefan teaches that due to the challenges involved in the chemical synthesis of HMOs, fermentative approaches have been developed using mainly genetically engineered bacterial strains, such as recombinant Escherichia coli, to produce 2’-FL and LNFP-I (see pg. 2, para. [0007]). However, even the most efficient processes based on bacterial fermentation do not hardly achieve HMO titers greater than 20 g/L, while industrial-scale processes must typically exceed titers of 50 g/L, although 100 g/L is more desirable (see pg. 2, para. [0008]).
Stefan teaches the method for the production of HMOs comprises providing a genetically modified host cell comprising an exogenous gene encoding a fucosyltransferase, preferably an alpha-1,2-fucosyltransferase, which is preferably overexpressed in the host cell (see pg. 3, para. [0010]). Stefan teaches the host cell has been further modified to express a gene encoding a protein enabling or facilitating the transport of the desired fucosylated oligosaccharide into the medium the host cell is cultivated in (see pg. 4, para. [0014]). According to a preferred embodiment, the protein that enables or facilitates the export of the desired HMO into the culture medium is the sugar efflux transporter yberc0001_9420 (see pg. 18, para. [0066]). In Stefan’s Examples, the heterologous sugar exporter yberc0001_9420 is disclosed as enhancing 2’-FL production by faster transport of the oligosaccharide outside the cell (see pg. 28, para. [00122]).
It would have been obvious at the time of filing for a person of ordinary skill in the art to have arrived at the claimed invention by combining the teachings of Pedersen, McCoy and Stefan, because all references teach methods for producing HMOs, including 2’-FL and LNFP-I, using genetically engineered cells, such as E. coli. One would have been particularly motivated to have applied the teachings of Stefan, because Stefan teaches that a heterologous sugar efflux transporter can effectively increase the production of HMOs. One would have recognized there to be a reasonable expectation of success, because Stefan provides evidence that E. coli expressing the yberc0001_9420 gene led to a higher production of 2’FL. One would have also recognized that all three references teach the production of 2’-FL using genetically engineered E. coli comprising an alpha-1,2-fucosyltransferase. Therefore, one would have recognized that the elements of each reference (i.e., HMO enzymes, colanic acid gene cluster, regulatory elements, sugar efflux transporter, etc.) could be combined by known methods and that the results of the combination would have been predictable. Hence, the combination would have been readily apparent and deemed to be a mere (A) combining of prior art elements according to known methods to yield predictable results (see MPEP 2143(I): Rationales to support rejections under 35 U.S.C. 103).
Regarding claim 17, Stefan discloses that during the production of 2’FL by the strain comprising yberc0001_9420, fermentations were conducted at 30°C (see pg. 29, paras. [00125]-[00126]). Stefan does not explicitly teach the molar % of 2’-FL to be between 30% and 40% of the produced blend of HMOs. However, this limitation is directed to an inherent property that does not affect the structure or steps of the claimed invention. Pedersen, McCoy and Stefan teach genetically engineered bacteria comprising the components of claims 1 and 14, which are disclosed to produce the same HMO products when cultured. Because the limitations of the claimed method are necessarily present in the prior art combination, the functional limitations of the asserted claim are inherently met by the combination of references. See MPEP 2112 and the discussion regarding claim 16 above.
Regarding claim 19, limitations “ii.” through “vi.” are obvious for the same reasons discussed regarding claim 1. Regarding the limitation of “vii. a recombinant nucleic acid sequence encoding a sugar efflux transporter capable of exporting 2’FL and/or LNFP-I out of the cell”, Stefan teaches a sugar efflux transporter capable of exporting 2’-FL in E. coli, as discussed regarding claim 14. Hence, claim 19 is obvious for the same reasons discussed regarding claims 1 and 14.
Regarding claim 20, Pedersen discloses that the integration of an extra genomic copy of the colanic acid genes expressed from the PglpF promoter resulted in a marked improvement of the LNFP-I titer, as discussed regarding claim 2.
Regarding claim 21, Pedersen, McCoy and Stefan all teach the genetically engineered cell is E. coli, as discussed regarding claims 1 and 14.
Claim(s) 15 is/are rejected under 35 U.S.C. 103 as being unpatentable over Pedersen, McCoy, and Stefan, as evidenced by GenBank WP_02248149.1 and GenBank AB050723, and as further evidenced by GenBank EEQ08298 (cited in the IDS filed 06/24/2024 at Cite No. 10).
Regarding claim 15, Stefan teaches that the gene yberc0001_9420 encodes a sugar efflux transporter of the major facilitator superfamily from Yersinia bercovieri ATCC 43970 and is represented by accession number “EEQ08298” (see pg. 26, para. [00111]). As shown in the following alignment, instant SEQ ID NO: 30 (top) is identical to EEQ08298 found in GenBank (bottom):
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As Stefan explicitly refers to this accession number as representing the sugar efflux transporter of the disclosure, it would have been obvious for a person having ordinary skill in the art to have selected the sugar efflux transporter (yberc0001_9420) bearing the same sequence as the GenBank entry. Further, the claim is obvious for the reasons discussed regarding claim 14.
Conclusion
No claims are allowed.
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/DENNIS IGNATIUS ARMATO JR/Examiner, Art Unit 1651
/MELENIE L GORDON/Supervisory Patent Examiner, Art Unit 1651