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 .
Priority
This application is a Continuation (Bypass Application) of PCT/US2021/065703, filed 12/30/2021, which claims priority to U.S. Provisional Patent Application No. 63/132,408, filed on 12/30/2020. Applicant’s claim for the benefit of a prior-filed application under 35 U.S.C. 119(e) or under 35 U.S.C. 120, 121, 365(c), or 386(c) is acknowledged.
Information Disclosure Statement
The information disclosure statements (IDS) submitted on 3/3/2026 and 3/12/2024 are acknowledged. The submissions are in compliance with the provisions of 37 CFR 1.97. Accordingly, the information disclosure statements are being considered by the examiner.
Election/Restrictions
Applicant’s election without traverse of Invention I 1-36 and 59-66) in the reply filed on 3/3/2026 is acknowledged. For the election of species, Applicant elected SEQ ID NO:3 for the sequence of Sed1 in claim 10 and SEQ ID NO: 9 for claim 18; SEQ ID NO: 5 for Flo5-2 and SEQ ID NO: 12 for claim 20; SEQ ID NO: 7 for Flo11 and SEQ ID NO: 13 for claim 21.
The requirement is herein made FINAL.
It is noted that Applicant has elected multiple versions of the claimed fusion protein, although the election of species asked for a single fusion protein having one sequence. Regarding the election of species, a call was placed on 6/5/2026 to Applicant’s representative but no reply was received to the voicemail. Therefore, the requirement has been herein modified as these appear to be obvious substitutions, in view of the art and the Applicant’s disclosure. The search was performed for each of the variants of the protein indicated above (e.g. for Sed1::EndoH, Flo11::EndoH, and Flo5-2::EndoH).
Claims 37-58 and 67 are withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected invention, there being no allowable generic or linking claim. Claims 1-36 and 59-66 are pending and were examined on the merits herein.
Claim Interpretation
The claim term “endoglycosidase” is being interpreted under the Broadest Reasonable Interpretation (B.R.I.), in light of the specification. The specification states that “endoglycosidases cleave polysaccharide chains between residues that are not the terminal residue and break the glycosidic bonds between two sugar monomer in the polymer” and that “Numerous endoglycosidases have been characterized, cloned, and/or purified. These include Endoglycosidase D, Endoglycosidase F1, Endoglycosidase F2, Endoglycosidase F3, Endoglycosidase H, Endoglycosidase Hf, Endoglycosidase S, Endoglycosidase T, Endoglycoceramidase I, O-Glycosidase, Peptide-N-Glycosidase A (PNGaseA), and PNGaseF” ([0072]-[0073]). Thus, under the B.R.I. in view of the specification and the knowledge in the art, one would understand that the endoglycosidase includes at least any of these named enzymes.
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 2-4, 7-8, 11, 23-24, 26-27, 62-63, and 65-66 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 2 recites “The engineered eukaryotic cell of claim 2”. A claim cannot depend on itself. The claim is rejected as being indefinite. It is presumed that the claim was meant to depend on claim 1.
In claim 3, the phrase “a portion of the endoglycosidase in addition to its catalytic domain” renders the claim indefinite. The term “a portion of the endoglycosidase” is not defined by the claim, the specification does not provide a standard for ascertaining the requisite amount of the protein that comprises a “portion”, and one of ordinary skill in the art would not be reasonably apprised of the scope of the invention. The claim is indefinite because there is no way to determine what constitutes a portion of the endoglycosidase in addition to its catalytic domain. Is this just one extra amino acid? Is there a certain amount of structural identity required? The claim is therefore rejected for indefiniteness.
The term “substantially” in claim 4 is a relative term which renders the claim indefinite. The term “substantially” is not defined by the claim, 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 claim requires “the fusion protein comprises substantially the entire amino acid sequence of the endoglycosidase”, but there is no manner for one to determine the metes and bounds of this limitation.
Similar to the language in claim 3, claim 7 also recites” a portion of the cell surface protein in addition to its anchoring domain”. The claim is indefinite because there is no way to determine what constitutes the portion of the cell surface protein in addition to its anchoring domain. Is this just one extra amino acid? Is there a certain amount of structural identity required? The claim is therefore rejected for indefiniteness.
The term “substantially” in claim 8 is a relative term which renders the claim indefinite. The term “substantially” is not defined by the claim, 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. As described for claim 4, there is no way for one to determine the actual structural limitation required of this claim.
Claim 11 recites the limitation "the anchoring domain" in line 1. There is insufficient antecedent basis for this limitation in the claim. Claim 11 depends on claim 1, which does not recite any anchoring domain. Claim 2 recites “an anchoring domain of a cell surface protein”, so it is presumed that claim 11 was intended to refer to claim 2.
A broad range or limitation together with a narrow range or limitation that falls within the broad range or limitation (in the same claim) may be considered indefinite if the resulting claim does not clearly set forth the metes and bounds of the patent protection desired. See MPEP § 2173.05(c). In the present instance, claim 23 recites the broad recitation a yeast cell, and the claim also recites or “a Pichia species” which is the narrower statement of the limitation, because Pichia is a genus of yeast. The claim is considered indefinite because there is a question or doubt as to whether the feature introduced by such narrower language is (a) merely exemplary of the remainder of the claim, and therefore not required, or (b) a required feature of the claims. Although the claim appears to be reciting alternatives, claim 24 recites “wherein the yeast cell is a Pichia species”. Because of the confusion of the actual limitations of claim 23, as to what is required and what is the range of protection sought, claim 23 is deemed indefinite.
Regarding claims 26, 62, 65, the phrase “e.g.”, which is short for exempli gratia which translates to "for example", renders the claim indefinite because it is unclear whether the limitations following the phrase are part of the claimed invention or if these are merely exemplary. See MPEP § 2173.05(d).
Further, claims 27, 63, and 66 each recite the phrase “the egg protein”, which is part of the limitations following “e.g.” and thus is not clear if it actually required of the claim. There is thus insufficient antecedent basis for “the egg protein” in claims 27, 63, and 66, and instead the claims should recite “the animal protein”.
Claim Rejections - 35 USC § 102
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.
Claims 1-4, 11-14, 23-26, 28-30, 32-35, 59-62 and 64-65 are rejected under 35 U.S.C. 102(a)(1) as being anticipated by Qi et al. (CN1746302A, cited in the IDS filed 3/12/2024 - a translation from Espacenet is included herein and the paragraph numbers referenced here correspond to this translated document), with supporting evidence from “Catalog no. V175–20” (a product manual for pPIC9k from Invitrogen, published 3/6/2010).
Qi et al. (hereafter “Qi”) discloses a method for producing deglycosylated proteins using genetically modified yeast cells. ([0008]-[0009]: “recombinant yeast strains containing N-glycoamidase and exogenous protein genes can be constructed using molecular biology techniques. N glycoamidase is stably expressed on the surface of yeast cells, and the exogenous protein is secreted into the culture medium. During secretion and culture, the surface-expressed N glycoamidase removes the glycan chains of the secreted exogenous glycoprotein”).
Qi discloses a fusion protein having an active N-glycoamidase (Peptide-N-(N-acetyl-β-glucosaminyl)asparagine amidase, PNGase, EC 3.5.1.52) fused to a yeast agglutinin C-terminal anchoring site (e.g. a surface displayed anchoring domain) and the expression of this fusion protein in yeast cells (see [0011]-[0018] as well as Examples 1 and 2 at [0044]-[0062]). Qi discloses that the enzyme is preferably N-glycoamidase F, PNGase F ([0025]), which is a known endoglycosidase (see the claim interpretation above).
Qi discloses an engineered eukaryotic cell comprising a surface displayed catalytic domain of an endoglycosidase (e.g. the enzyme comprising PNGase F and having active N-glycoamidase activity) wherein the surface displayed catalytic domain is a fusion protein expressed in the cell (e.g. the yeast cell), and the fusion protein comprises an anchoring domain of a cell surface protein (e.g. the yeast agglutinin C-terminal anchoring region).
Qi is thus found to anticipate claims 1 and 2.
Regarding claims 3-4, as best understood due to the indefiniteness issues described above, the fusion protein of Qi contains what appears to be essentially the entire coding region of the PNGase F (see [0011] and [0026]) and thus meets these claim limitations (the entire coding regions comprises “a portion” of the enzyme in addition to the catalytic domain for claim 3).
Regarding claims 11 and 12, Qi discloses that the anchoring domain stably attaches the fusion protein to the extracellular surface (see [0008]: “N-glycoamidase is stably expressed on the surface of yeast cells”) and teaches that the fusion protein comprises a signal peptide ([0026]: “The first 40 amino acids are presumed to be a signal peptide”).
Regarding claims 13-14, Qi discloses that the anchoring domain (AGa1) is N-terminal to the catalytic domain (see Figure 3) of the enzymatic domain (PNGase) in the fusion protein, and thus has a linker that is C-terminal of the anchoring domain.
Regarding claims 23-24, Qi discloses that the yeast cells are the Pichia pastoris strain Gs115 ([0031], [0045]).
Regarding claims 25 and 26, Qi discloses that the yeast cells also express a secreted glycoprotein (see [0016]). Specific secreted glycoproteins in Qi include an interferon gene, an erythropoietin (EPO) gene, and a ribonuclease B gene ([0032]). Each of these are animal proteins, especially interferon and erythropoietin.
Regarding claims 28-30, Qi teaches the production of yeast strains that have a nucleic acid sequence on the vector pPIC9k-PNG-A that is electroporated into the genome of a yeast strain to construct a recombinant yeast strain ([0013]). This strain is not taught to have any modifications for overexpressing a secretory glycoprotein and thus fulfills the requirement of claim 28. Regarding claim 30, Qi states that the yeast recombinant engineered strain that secretes and expresses exogenous proteins according to the present invention receives exogenous genes by integration into the genome ([0037]).
Regarding claims 32-34, the vector demonstrated in Qi includes an AOX1 promoter (a known inducible promoter) and an AOX 1 terminator (see Figure 3 of Qi). There appears to be a typo in the figure drawing of Qi for Figure 3, as it says AOH1, which is not a recognized yeast gene. Further, the expression vector pPIC9k is designed for secreted protein expression in Pichia (see the product manual for Catalog no. V175–20, pPIC9k from Invitrogen, cited on the included PTO-892) and it is evident from general knowledge in the art and the cited catalog that this vector contains the inducible AOX1 promoter and an AOX1 terminators.
Regarding claim 35, the nucleic acid sequence of Qi, from the vector pPIC9k, includes the α-Factor mating signal peptide sequence (see pg,. 8 of Catalog no. V175–20, pPIC9k from Invitrogen) to ensure secretion of the expressed protein.
Regarding claims 59-60, Qi discloses culturing and growing engineered yeast cells in fermentation batches (e.g. a population of the engineered cells), using fermentation methods having control of shaking speed and temperature, wherein the culturing is for 36-60 hours (see [0018]). This constitutes a bioreactor, as the fermentation is controlled and the cell population is cultured under adjustable conditions to produce a biologically useful material.
Regarding claim 61, Qi discloses that in such culturing of the engineered yeast cells, N-glycosidase is expressed on the surface of yeast cell wall, while exogenous glycoprotein is secreted into the culture medium; in the culture medium, the surface-expressed N-glycosidase cleaves the glycan chains of the secreted glycoprotein, generating non-N-glycosylated protein ([0018]). Thus, this anticipates the composition comprising an engineered eukaryotic cell and a secreted glycoprotein, as recited in claim 61. Regarding claim 64, the composition of Qi will also naturally contain the cleaved oligosaccharides which are cleaved by the action of the enzyme-expressed on the cell surface (see e.g. the description at [0004]).
Regarding the dependent claims 62 and 65, the glycoproteins secreted and deglycosylated in the examples of Qi include multiple animal proteins, as described above for claim 26.
For these reasons, Qi is deemed to anticipate claims 1-4, 11-12, 13-14, 23-26, 28-30, 32-35, 59-62 and 64-65.
Claim Rejections - 35 USC § 103
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
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.
Claims 1-18, 23-26, 28-36, 59-62 and 64-65 are rejected under 35 U.S.C. 103 as being unpatentable over Qi et al. (CN1746302A), in view of Wang et al. (“High-level expression of endo-β-N-acetylglucosaminidase H from Streptomyces plicatus in Pichia pastoris and its application for the deglycosylation of glycoproteins”. PLoS One. 2015;10(3):e0120458, on IDS filed 3/12/2024, hereafter “Wang”) and Su et al. (“Surface display of active lipase in Pichia pastoris using Sed1 as an anchor protein” Biotechnol Lett (2010). 32:1131-1136, on IDS filed 3/12/2024, hereafter “Su”), with supporting evidence from UniProt Entry EBAG_STRPL (Endo-beta-N-acetylglucosaminidase H from Streptomyces plicatus), and UniProt Entry N1PAC8_YEASC (Sed1p from Saccharomyces cerevisiae).
The teachings of Qi et al. include all of those cited above. As described in the rejections under 35 U.S.C. § 102, Qi teaches engineered Pichia cells that overexpress fusion protein comprising a surface displayed catalytic domain of an endoglycosidase, wherein the surface displayed catalytic domain of an endoglycosidase is a portion of a fusion protein expressed by the cell (specifically one having PNGase F catalytic domain and an Aga1 agglutin targeting and anchoring sequence see [0011]-[0018]). Qi teaches that the engineered cells also contain a secreted glycoprotein that the glycosidase enzyme deglycosylates, removing undesired oligosaccharides from one or more animal protein. Thus all of claims 1-4, 11-14, 23-26, 28-30, 32-35, 59-62 and 64-65 are considered to be obvious, if not anticipated by Qi et al..
However, Qi et al. does not teach that the cell surface protein is derived from Sed1p, Flo5-2, or Flol1, nor that the endoglycosidase enzyme comprises endoglycosidase H. Further, Qi et al. does not teach the specific sequences disclosed herein comprising part of or all of the fusion proteins.
Wang et al. teaches that Endo-β-N-acetylglucosaminidase H (Endo H, EC3.2.1.96) is a glycohydrolase that is widely used in the study of glycoproteins, and demonstrates the effects of endo-β-N-acetylglucosaminidase H expression in Pichia pastoris (Title, Abstract). Wang teaches that the N-linked glycan side chains of several recombinant proteins expressed in Pichia pastoris can be efficiently removed through the co-fermentation of this recombinant strain with strains expressing substrates (Abstract, Fig. 8). Wang teaches that Endo H cleaves the β-1,4-glycosidic bond of the N-acetyl glucosamine core of oligosaccharides and leaves one N-acetylchitobiose attached to the asparagine residue of the glycoprotein, see e.g. Introduction, pg. 1).
Wang also teaches that when expressing secreted recombinant proteins in yeast systems, the post-translational modification system of eukaryotic cells leads to the aberrant glycosylation of target proteins, which in turn may cause them to malfunction, and thus to overcome this known problem in the art glycohydrolases such as glycopeptidase F (PNGase F) and Endo H may be used to remove N-glycan side chains from glycoproteins (see first paragraph on pg. 2).
Thus, Wang teaches that expression of Endo H from Streptomyces plicatus (e.g. the same as SEQ ID NO:1 herein) in Pichia pastoris is known in the art for the removal of unwanted N-linked glycan side-chains of recombinant proteins (see 2nd paragraph on pg. 2 and “Discussion”).
The sequence of Endo H from Streptomyces plicatus is demonstrated in Figure 1 of Wang et al., which appears to be identical to that of UniProt Entry EBAG_STRPL, which is 100% identical to the sequence of SEQ ID NO: 1 recited herein (see appendix below).
Regarding the selection of cell anchoring proteins, Su et al. teaches methods for the surface display of enzymes in P. pastoris using Sed1 as an anchor protein (Abstract). Su teaches that cell surface display is known to the art (pg. 1131, right col.). Su teaches that other known anchor proteins include Flo1, alpha-agglutinin, and Cwp2 (pg. 1131, right col). Su teaches an expression vector, derived from pPIC9K, in which the Sed1 anchor protein is fused to an lipase enzyme and is expressed with the inducible AOX1 promoter (pg. 1132, left col and Fig. 1). It is noted that the Sed1 sequence is provided C-terminal to the enzyme (CALB) sequence. Su teaches achieving high expression levels of the cell surface-anchored protein than a secreted free enzyme (Fig. 5), and Su teaches that the thermal stability of the cell surface anchored protein was improved (see Conclusions, pgs. 1135-1136). Therefore, Su suggests that anchoring an enzyme to the cell surface with Sed1 is a known powerful technique in yeast P. pastoris for improved protein expression and activity.
The sequence of Sed1 is disclosed in UniProt Entry N1PAC8, and this sequence is 100% identical to that of SEQ ID NO: 3, disclosed in the instant application (see appendix below).
To one of ordinary skill in the art, before the effective filing date of the instant invention, it would have been obvious to modify the engineered yeast cells taught in Qi by substituting Endo H as taught in Wang for the PGNaseF used in Qi, and it would have been obvious to select a Sed1 sequence as the cell anchor target, as suggested in the teachings of Su for the benefit of higher enzyme expression with cell surface display, a technique known to the art.
One would have been motivated to select Endo H as the endoglycosidase because it is taught in Wang for the removal of unwanted N-glycans from glycoproteins produced by and secreted from yeast cells. Because Wang teaches that glycohydrolases such as glycopeptidase F (PNGase F) and Endo H are both known and used for removing N-glycan sidechains. Therefore, substituting the Endo H of Wang for PNGase F would be a matter of judicial selection and simple substitution to one of ordinary skill in the art. Both enzymes are taught to perform the same function and thus it would be obvious to substitute the two. MPEP § 2143.I.b (B) describes the simple substitution of one known element for another to obtain predictable results is obvious when the equivalent components have the same function. See e.g. In re Fout, 675 F.2d 297, 213 USPQ 532 (CCPA 1982) : “Express suggestion to substitute one equivalent for another need not be present to render such substitution obvious." Id. at 301, 213 USPQ at 536”.
Therefore claims 5-6 are found obvious because Endo H is expressly taught in Wang and the sequence is 100% identical to that of the instant claim 1, as evidenced by UniProt Entry EBAG_STRPL, which is 100% identical to the sequence of SEQ ID NO: 1 recited herein.
Regarding claims 8-10, from the teachings of Qi and Su, it would have been obvious to provide Sed1 as the cell surface anchoring protein. Su teaches that Sed1 is one known option for anchoring enzymes, and also demonstrated improved thermal stability of a cell surface enzyme. Because Su teaches that alpha-agglutinin and Sed1 are both known cell-anchoring options, it would have been prima facie obvious to one of ordinary skill in the art to substitute Sed1 as used in Su for the alpha-agglutinin anchor taught in Qi. See MPEP § 2143.I.(B), as described previously. The selection from among known viable options in the art would have been a matter of routine optimization and judicious selection to an artisan of ordinary skill and knowledge.
For claims 7-8, Su teaches using the entire protein of Sed1 for the anchor (Fig. 1; Materials and Methods, pg. 1133). Because of the improved thermostability taught therein, one following the teachings of Su would be prompted to use the entire protein, as recited in claim 8.
Regarding claim 10, the sequence of Sed1 according to UniProt Entry N1PAC8 is 100% identical to that of the instant SEQ ID NO: 3 (see appendix below). Thus it would have been obvious to use the instantly claimed cell surface anchoring sequence.
Regarding claims 11-16, these would have been obvious to one having ordinary skill in the art over the teachings of Qi, Su, and Wang. Signal peptides and secretory signals are taught in each of the references. Su uses the α-factor signal peptide (see Fig. 1) for the secretion of the fusion protein. The orientation of the catalytic domain and the anchoring domain would be a matter of routine optimization and judicious selection. Regarding claims 13-14, this arrangement is disclosed in Qi. Regarding claims 15-16, Su teaches that the anchor protein is C-terminal to the enzyme. Thus, it would have been obvious to provide the two domains in either sequence.
Regarding claim 17, one motivated by the teachings of Qi, Wang, and Su would arrive at the instantly recited surface-domain fusion protein, having Endo H with Sed1p expressed on the cell surface. These are obvious selections to one having ordinary skill in the art, as the fusion protein would be reasonably predicted to have the desired function of removing N-glycans.
Regarding claim 18, a fusion protein having the sequences of UniProt Entry EBAG_STRPL- Endo-beta-N-acetylglucosaminidase H from Streptomyces plicatus, and UniProt Entry N1PAC8_YEASC - Sed1p from Saccharomyces cerevisiae, following from the suggestions of the prior art as discussed above, must have sequences that are at least 95% identical to sequences found in SEQ ID NO: 9. Indeed, both Endo H (EBAG_STRPL) and Sed1p (N1PAC8_YEASC) are found having 100% matches to regions in SEQ ID NO: 9. Thus, under the B.R.I. of the claim terms, such a fusion protein would have obvious.
Regarding claims 29-35, these would have all been obvious selections in view of the teachings of Qi, Su, and Wang, to one having ordinary skill in the art. The selection of nucleic acids, and the process for expressing the fusion protein (as in claims 30-31) amount to routine optimization in the art of protein expression. Such methods are known to Pichia systems, as evidenced in the art of record. Both Qi and Su disclose using an inducible AOX1 promoter and an AOX1 terminator, while the α-factor signal peptide is expressly taught in Su.
Regarding claim 36, Wang teaches that for the EndoH expression, the DNA coding sequence of Endo H from S. plicatus was optimized to match the codon usage preference of P. pastoris (see pg. 3, top paragraph). Such codon optimization techniques are known in the art and thus this would have been an obvious step to one having ordinary skill in the art, for the purpose of improved expression.
Claims 59-62 and 64-65 would have been obvious for all of the reasons explained previously, because Qi teaches that such systems are used for the expression of secreted glycoproteins, and the optimization of fermentation systems with bioreactors is known in the art.
From the teachings of the cited references, Qi, Wang, and Su, it is apparent that there would have been a reasonable expectation of success in combining the teachings therein to arrive at the claimed invention because cell surface expression of endoglycosidases fused to a cell anchoring domain is taught in Qi, and the expression of Endo H is successfully performed in Wang with P. pastoris. Further, Su demonstrates that Sed1p may be successfully used as a cell-surface anchor in the same organism. Providing of these particular sequences for the fusion protein would have been predictably successful in the engineered P. pastoris cells of Qi.
Therefore, the invention as a whole would have been prima facie obvious to one of ordinary skill in the art prior to the effective filing date, as evidenced by the cited references, especially in the absence of evidence to the contrary.
Claims 1-18, 23-26, 28-36, 59-62 and 64-65 are rejected as being obvious over the combined teachings of Qi, Wang, and Su, with supporting evidence from the UniProt database entries, for all of the reasons described above.
Claim 1-18, 22-26, 28-36, 59-62 and 64-65 are rejected under 35 U.S.C. 103 as being unpatentable over “Qi”, “Wang”, and “Su”, as applied to claims 1-18, 23-26, 28-36, 59-62 and 64-65 above, and further in view of Karbalaei et al. (“Pichia pastoris: A highly successful expression system for optimal synthesis of heterologous proteins.” Journal of cellular physiology vol. 235,9 (2020): 5867-5881. doi:10.1002/jcp.29583), to include the rejection of claim 22.
The teachings of Qi, Wang, and Su include all of those discussed above. The combined teachings make obvious at least the subject matter of claim 1.
However, none of Qi, Wang, or Su teaches using a eukaryotic cell having a AOX1 or AOX2 gene mutation, as required of claim 22.
Karbalaei is a review article that teaches the P. pastoris expression system is one of the most popular and standard tools for the production of recombinant protein in molecular biology (Title, Abstract). Karbalaei teaches that the commonly used P. pastoris GS115 strain has two encoding genes (AOX1 and AOX2) of alcohol oxidase (AOX) enzyme and the presence of methanol, the transcription of these genes is induced and produces a high amount of AOX enzyme (pg. 5869, right col). Karbalaei teaches that by knocking out the AOX1 gene, the growth on methanol is slowed down drastically, and by knocking out both genes, the strains are unable to grow on methanol (methanol utilizing minus [Mut−]) (pgs. 5869-5870). Karbalaei also teaches a KM71 strain, a derivative of GS115, wherein the aox1 gene has been deleted; therefore, the strain is known as MutS strain (pg. 5870). Karbalaei teaches that the selection of Mut strains has an known impact on the growth rate of different strains and the amount of methanol necessary for fully expressing a protein linked to the AOX1 promoter (pg. 5873, under section 4.1 “The impact of Mut forms and methanol on protein concentration”; and Table 5).
To one of ordinary skill in the art, before the effective filing date of the instant invention, it would have been obvious to modify the engineered yeast cells made obvious by the teachings of Qi, Wang, and Su, by providing an AOX1, AOX2, or AOX1/AOX2 mutant strain, as taught in Karbalaei, for the benefits of improving protein production with methanol induction.
One would have been motivated to use an AOX1 mutant strain such as the KM71 when using the AOX1 promoter for protein expression, because this gives improved control over protein expression, affecting the cell’s growth rate on methanol. This optimizes productivity and helps avoid methanol toxicity, according to the teachings of Karbalaei.
Because these are known mutations in strains of P. pastoris that allow for optimization of protein production, the inclusion of such mutations amounts to judicious selection and routine optimization to one of ordinary skill. There is no evidence of the criticality of these mutations to the claimed invention, nor of any unexpected success.
From the teachings of Karbalaei, it is apparent that there would have been a reasonable expectation of success in using yeast strains with AOX1 and/or AOX2 mutations because selecting such strains for methanol-induced protein production is routine.
Therefore, the invention of claim 22 as a whole would have been prima facie obvious to one of ordinary skill in the art prior to the effective filing date, as evidenced by the cited references, especially in the absence of evidence to the contrary.
Claims 1-4, 11-14, 23-27, 28-30, 32-35, 59-66 are rejected under 35 U.S.C. 103 as being unpatentable over Qi et al. (CN1746302A) in view of Choi et al. (US PGPub No. US20160183567A1), to include the rejections of claims 27, 63, and 66.
The teachings of Qi et al. include all of those cited above.
As described in the rejections under 35 U.S.C. § 102, Qi teaches engineered Pichia cells that overexpress fusion protein comprising a surface displayed catalytic domain of an endoglycosidase, wherein the surface displayed catalytic domain of an endoglycosidase is a portion of a fusion protein expressed by the cell (specifically one having PNGase F catalytic domain and an Aga1 agglutin targeting and anchoring sequence see [0011]-[0018]). Qi teaches that the engineered cells also contain a secreted glycoprotein that the glycosidase enzyme deglycosylates, removing undesired oligosaccharides from one or more animal protein.
However, Qi et al. does not teach nor suggest that the secreted glycoprotein is an egg protein selected from one of the group recited in claims 27, 63, and 66.
Choi (US PGPub No. US20160183567A1) pertains methods for preparing a low antigenic food, the method including removing a sugar linked to a glycoprotein of an allergenic food (Title, Abstract). Choi teaches that the method includes removing a glucose linked to a glycoprotein selected from the group consisting of ovalbumin, ovomucoid, ovotransferrin, β-conglycinin, Ara h1, and Ara h2 (Abstract). Choi teaches that glycoproteins present on food such as eggs, peanuts, and beans, are involved in allergic reactions and teaches that the removal of such glycoproteins is sought in order to produce low antigenic food materials ([0005]-[0008]).
Particularly, Choi teaches using exoglycosidases, including mannosidase, galactosidase, and N-acetylglucosaminidase, to remove oligosaccharides from the egg proteins ovalbumin, OVA, (FIG.s 5-7; [0101]-[0116]) and from ovomucoid, OM, (FIGs. 11-13; [0123]-[0138]) to produce low antigenic protein products (e.g. for food products). Choi teaches that “the removal of sugars linked to glycoproteins of allergenic foods can reduce antigenicity of the glycoproteins, and low antigenic foods can be prepared by a method comprising the step of removing a sugar linked to a glycoprotein of an allergenic food. Preferably, low antigenic ovalbumin or low antigenic ovomucoid can be prepared by the method for removing the sugar linked to ovalbumin or ovomucoid.” ([0069]).
Therefore, to one of ordinary skill in the art, before the effective filing date of the instant invention, it would have been obvious to modify the engineered yeast cells taught in Qi by providing as the secreted glycoprotein, one of the egg proteins ovalbumin or ovomucoid, as taught in Choi, because Choi teaches that removal of such allergenic oligosaccharides from egg proteins is desirable and Qi pertains to producing deglycosylated glycoproteins using a cell-surface anchored endoglycosidase enzyme.
One would have been motivated to provide at least the egg proteins ovalbumin or ovomucoid because Choi demonstrates that removing the oligosaccharides results in low antigenic food materials, as demonstrated in the mouse experiments therein. One having ordinary skill in the art would have been thus motivated to apply the deglycosylating yeast cell expression system of Qi for preparing these egg glycoproteins in which removal of oligosaccharides through deglycosylation is desirable. Because the PNGase F fusion protein taught in Qi removes the entire N-linked oligosaccharide, it would have been predictable to one of ordinary skill in the art that such treatment (e.g. expressing the egg proteins OVA or OM) would result in substantial removal of N-glycans on these proteins for the desired removal of antigenic oligosaccharides.
Further, one would reasonably predict that using the P. pastoris system of Qi would result in a cheaper fermentation cell-based production system that doesn’t require the recombinant enzymes used in Choi, thus amounting to a predictable improvement from the methods of Choi.
From the teachings of Qi and Choi, it is apparent that there would have been a reasonable expectation of success in expressing ovalbumin or ovomucoid in a P. pastoris system having a cell-surface anchored endoglycosidase, as Qi teaches that such a system effectively removes N-linked oligosaccharides from glycoproteins, and Choi teaches that removal of the oligosaccharides results in egg proteins with lowered antigenicity.
Therefore, the invention of claim 22 as a whole would have been prima facie obvious to one of ordinary skill in the art prior to the effective filing date, as evidenced by the cited references, especially in the absence of evidence to the contrary.
Nonstatutory Double Patenting
The nonstatutory double patenting rejection is based on a judicially created doctrine grounded in public policy (a policy reflected in the statute) so as to prevent the unjustified or improper timewise extension of the “right to exclude” granted by a patent and to prevent possible harassment by multiple assignees. A nonstatutory double patenting rejection is appropriate where the conflicting claims are not identical, but at least one examined application claim is not patentably distinct from the reference claim(s) because the examined application claim is either anticipated by, or would have been obvious over, the reference claim(s). See, e.g., In re Berg, 140 F.3d 1428, 46 USPQ2d 1226 (Fed. Cir. 1998); In re Goodman, 11 F.3d 1046, 29 USPQ2d 2010 (Fed. Cir. 1993); In re Longi, 759 F.2d 887, 225 USPQ 645 (Fed. Cir. 1985); In re Van Ornum, 686 F.2d 937, 214 USPQ 761 (CCPA 1982); In re Vogel, 422 F.2d 438, 164 USPQ 619 (CCPA 1970); In re Thorington, 418 F.2d 528, 163 USPQ 644 (CCPA 1969).
A timely filed terminal disclaimer in compliance with 37 CFR 1.321(c) or 1.321(d) may be used to overcome an actual or provisional rejection based on nonstatutory double patenting provided the reference application or patent either is shown to be commonly owned with the examined application, or claims an invention made as a result of activities undertaken within the scope of a joint research agreement. See MPEP § 717.02 for applications subject to examination under the first inventor to file provisions of the AIA as explained in MPEP § 2159. See MPEP § 2146 et seq. for applications not subject to examination under the first inventor to file provisions of the AIA . A terminal disclaimer must be signed in compliance with 37 CFR 1.321(b).
The filing of a terminal disclaimer by itself is not a complete reply to a nonstatutory double patenting (NSDP) rejection. A complete reply requires that the terminal disclaimer be accompanied by a reply requesting reconsideration of the prior Office action. Even where the NSDP rejection is provisional the reply must be complete. See MPEP § 804, subsection I.B.1. For a reply to a non-final Office action, see 37 CFR 1.111(a). For a reply to final Office action, see 37 CFR 1.113(c). A request for reconsideration while not provided for in 37 CFR 1.113(c) may be filed after final for consideration. See MPEP §§ 706.07(e) and 714.13.
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Claim 1-27, 29-31, and 59-66 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1, 2, 9-10, 13, 22-23, 26, 28-29, 34-40, 43-46, 48, 50-51, 58, 68 of copending Application No. 18/346,022 (reference application).
Although the claims at issue are not identical, they are not patentably distinct from each other because the scopes of application claims are substantially overlapping with the instantly claimed subject matter, such that the subject matter of the reference claims anticipates or makes obvious all of the instant claims.
Claim 1 of the reference ‘022 patent recites “An engineered eukaryotic cell comprising a surface displayed catalytic domain of an endoglycosidase, wherein the surface displayed catalytic domain of an endoglycosidase is a portion of a fusion protein expressed by the cell, wherein the endoglycosidase is endoglycosidase H.” and claim 2 recites “wherein the fusion protein further comprises an anchoring domain of a cell surface protein.”
Claim 22 of the reference patent recites “An engineered eukaryotic cell that expresses a fusion protein comprising a catalytic domain of an endoglycosidase and a portion of a cell surface protein, wherein the portion of the cell surface protein lacks its native anchoring domain, wherein the portion of the cell surface protein that lacks its native anchoring domain is capable of adhering to an extracellular component of the cell.”, and claim 23 recites “wherein the fusion protein comprises a portion of the endoglycosidase in addition to its catalytic domain”.
Thus claims 1-5, 7-8, and 11 are considered unpatentable over these claims, as the subject matter clearly anticipates each and every one of the limitations therein.
Regarding the instant claim 6, claim 26 of the reference patent recites “the fusion protein comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 1 or SEQ ID NO: 2.” These appear to be identical to the instantly recited sequence.
Regarding the instant claims 9-10 and 17-21, claim 9 of the reference patent recites “wherein the cell surface protein is selected from Sed1p, Flo5-2, or Flo11.” and reference claim 10 recites that the amino acid sequence of the fusion protein can be any of one of SEO ID NO: 1, SEO ID NO:2, SEQ ID NO: 3, SEO ID NO: 4, SEO ID NO: 5, SEO ID NO: 6, SEQ ID NO: 7, SEO ID NO: 9, SEO ID NO: 10, SEO ID NO: 11, SEO ID NO: 12, SEO ID NO: 13, SEO ID NO: 14, or SEQ ID NO: 20.
Thus, the alternatives for the sequences of the cell surface protein and the resulting fusion proteins of both the reference application and the instant claims appears to be identical.
Regarding the instant claim 12, the disclosed sequences of the fusion protein in the reference claim (i.e. those of claims 9-10 for example) appear to contain signal peptides and/or secretory signals. Because the fusion protein of the reference application is also expressed on the cell surface, it must inherently have a signal peptide that directs it to the membrane.
Regarding the instant claims 13-16, the reference claim 13 recites that the anchoring domain is N-terminal to the catalytic domain in the fusion protein or C-terminal to the catalytic domain in the fusion protein and reference claims 35 and 37 recite that the fusion protein may comprise a linker that is C-terminal of N-terminal to the portion of the cell surface protein.
Regarding claims 22-24, the reference claims 43 and 44 recite that the engineered eukaryotic cell comprises a mutation in its AOX1 gene and/or its AOX2 gene and the engineered eukaryotic cell is a yeast cell or a Pichia species.
Regarding claims 25-27, claim 46 of the reference application recite that the cells further comprise a genomic modification that overexpresses a secretory glycoprotein and claim 48 recites that the secretory glycoprotein is an egg protein selected from the group consisting of ovalbumin, ovomucoid, lysozyme ovoglobulin G2, ovoglobulin G3, a-ovomucin, 3-ovomucin, ovotransferrin, ovoinhibitor, ovoglycoprotein, flavoprotein, ovomacroglobulin, ovostatin, cystatin, avidin, ovalbumin related protein X, and ovalbumin related protein Y.
Regarding the instant claims 29-31, claims 50 and 51 of the reference application recite that the cells comprise a nucleic acid sequence that encodes the fusion protein, wherein the nucleic acid sequence that encodes the fusion protein is integrated into the cell's genome or is extrachromosomal.
Regarding claims 59-66, the limitations of these claims would have been prima facie obvious over the subject matter of the reference claims. The reference claims inherently include populations of engineered eukaryotic cells. Claim 58 of the reference application discloses a method for deglycosylating a secreted glycoprotein comprising contacting a secreted protein with a fusion protein anchored to an engineered eukaryotic cell of claim 1, thereby providing a deglycosylated secreted glycoprotein. Thus, compositions having the engineered cell, the deglycosylated secreted glycoproteins and the detached oligosaccharides are intrinsically taught in the subject matter of the reference claims. Further, claims 48 and 68 of the reference recite that the protein includes one of the recited egg proteins.
Thus, claims 1-27, 29-31, and 59-66 are found to be patentable indistinct from the subject matter of the reference application claims.
This is a provisional nonstatutory double patenting rejection because the patentably indistinct claims have not in fact been patented.
Claim 1-27, 29-31, 32-36, and 59-66 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1, 2, 9-10, 13, 22-23, 26, 28-29, 34-40, 43-46, 48, 50-51, 58, 68 of copending Application No. 18/346,022 (reference application) in view of Wang et al. (“High-level expression of endo-β-N-acetylglucosaminidase H from Streptomyces plicatus in Pichia pastoris and its application for the deglycosylation of glycoproteins”. PLoS One. 2015;10(3):e0120458, on IDS filed 3/12/2024, ) and Su et al. (“Surface display of active lipase in Pichia pastoris using Sed1 as an anchor protein” Biotechnol Lett (2010). 32:1131-1136, on IDS filed 3/12/2024).
The subject matter of claims 1-27, 29-31, and 59-66 are not patentable distinct from those of the reference claims for all of the reasons discussed above.
Regarding the instant claims 32-36, the reference claims do not explicitly disclose an inducible promoter, an encoded signal peptide and/or secretory signal, nor that the nucleic acid has been optimized for the chosen species.
The teachings of “Wang” and “Su” include all of those discussed above.
Wang teaches Endo-β-N-acetylglucosaminidase H (Endo H, EC3.2.1.96), a glycohydrolase that is widely used in the study of glycoproteins, and demonstrates the effects of endo-β-N-acetylglucosaminidase H expression in Pichia pastoris. Wang teaches that for the EndoH expression, the DNA coding sequence of Endo H from S. plicatus was optimized to match the codon usage preference of P. pastoris (pg. 3, top paragraph).
Su teaches an expression vector, derived from pPIC9K, in which the Sed1 anchor protein is fused to an lipase enzyme and is expressed with the inducible AOX1 promoter with an AOX1 terminator, and has an a-mating factor signal peptide (pg. 1132, left col and Fig. 1).
Therefore, these claim limitations these would have all been obvious modifications to the nucleic acid of the reference claim in view of the teachings of Su and Wang, to one having ordinary skill in the art. The selection of nucleic acids, and the process for expressing the fusion protein amount to routine optimization in the art of protein expression. Such methods are known to Pichia systems, as evidenced by Wang and Su. Su teaches using an inducible AOX1 promoter and an AOX1 terminator, while the α-factor signal peptide is expressly taught in Su.
Regarding claim 36, codon optimization techniques are known in the art, and explicitly taught in Wang, and thus this would have been an obvious step to one having ordinary skill in the art, for the purpose of improved expression.
Because the subject matter of the instant claims would have been obvious over the reference application claims in view of the ordinary level of knowledge in the art, the claims are herein rejected under the doctrine of obviousness-type nonstatutory double patenting.
This is a provisional nonstatutory double patenting rejection.
Citation of Pertinent Art
The prior art made of record and not relied upon is considered pertinent to applicant's disclosure.
US Pat. No. 7,132,273 (Choi et al.) discloses novel cell wall anchor proteins derived from yeast, genes thereof and the genetic method for tethering polypeptide to the yeast cell wall, including several GPI (glycosyl phosphatidyl inositol)-anchor protein genes, SFD1, GAS1, TIP1 and CWP1, and their proteins (Abstract).
US Pat. No. 9,645,146 (Rakestraw et al.) pertains to compositions and methods for displaying engineered polypeptides on a cell surface (Abstract) and teaches that the yeast fusion proteins used for display include a cell-surface display system based on fusing an engineered protein to be displayed to the flocculation domain of a GPI anchor protein without the C-terminal portion of the GPI anchor protein, and include flocculation proteins Flo1, Flo5, or Flo11 (Col 11, lines 31-67).
Conclusion
No claims are allowable.
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/A.T.M./Examiner, Art Unit 1655
/ANAND U DESAI/Supervisory Patent Examiner, Art Unit 1655
Appendix A: Alignment of SEQ ID NO:1 and Endo H (EBAG_STRPL)
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Appendix B: Alignment of SEQ ID NO:3 and Sed1p (N1PAC8_YEASC).
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Prosecution Insights