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 .
Application Status
This action is written in response to applicant’s correspondence received 24 March 2026. Claims 1 and 3-8 are currently pending. Claim 8 is withdrawn from prosecution as being drawn to non-elected subject matter. Accordingly, claims 1 and 4-7 are examined herein. In the response to restriction requirement filed 23 September 2025, Applicant's elected Group I, claims 1-7, alongside SEQ ID NOs: 2, 4, 6, 8, and 10 without traverse. For the purposes of examination, the species of SEQ ID NOs: 1, 3, and 5 have been rejoined into examination.
Any rejection or objection not reiterated herein has been overcome by amendment. Applicant' s amendments have been thoroughly reviewed, but are not persuasive to place the claims in condition for allowance for the reasons that follow.
Priority
Should applicant desire to obtain the benefit of foreign priority under 35 U.S.C. 119(a)-(d) prior to declaration of an interference, a certified English translation of the foreign application, CN202411087607.8, must be submitted in reply to this action. 37 CFR 41.154(b) and 41.202(e).
Claim Interpretation
Regarding the claimed recombinant engineering microorganism, Komagataella phaffii (i.e., K. phaffii), it is noted that the instant specification teaches that an alternative name for the organism is Pichia pastoris (i.e., P. pastoris) ([0032]).
Further, it is noted that the instant specification teaches that the claimed genes comprising SEQ ID NOs: 2, 4, 6, 8, and 10 were “obtained by retrieving currently known protein databases (such as Uniprot, The Human Protein Atlas, PhosphoSitePlus) and publicly available documents” ([0080]). The instant specification provides the Uniprot entries for each of the claimed SEQ ID NOs in Table 1, as seen below on page 16 of the specification.
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Accordingly the instant specification teaches that, and as described below in the 35 USC 103 rejections of record, that the claimed CCW14, EM1, and PUN1 genes were known in the prior art to be proteins related to the cell wall of P. pastoris. Further, both EXG1 and SPR1 were known genes in the prior art that encoded glucan-1,3-β-gucosidases in P. pastoris.
Claim Rejections - 35 USC § 103
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. Considering objective evidence present in the application indicating obviousness or nonobviousness.
This application currently names joint inventors. In considering patentability of the claims the examiner presumes that the subject matter of the various claims was commonly owned as of the effective filing date of the claimed invention(s) absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and effective filing dates of each claim that was not commonly owned as of the effective filing date of the later invention in order for the examiner to consider the applicability of 35 U.S.C. 102(b)(2)(C) for any potential 35 U.S.C. 102(a)(2) prior art against the later invention.
Claim(s) 1 and 3-4 is/are rejected under 35 U.S.C. 103 as being unpatentable over Weinhandl ("Pichia pastoris mutants as host strains for efficient secretion of recombinant branched chain aminotransferase (BCAT)." Journal of biotechnology 235 (2016): 84-91) in view of Zhu ("High level expression and purification of recombinant human serum albumin in Pichia pastoris." Protein Expression and Purification 147 (2018): 61-68), Liao ("A versatile toolbox for CRISPR-based genome engineering in Pichia pastoris." Applied microbiology and biotechnology 105.24 (2021): 9211-9218), “A0A1G4KPI2” (Uniprot Accession No. A0A1G4KPI2; published 18 January 2017), “A0A1G4KPG0” (Uniprot Accession No. A0A1G4KPG0; published 18 January 2017), “F2QLI3” (Uniprot Accession No. F2QLI3; published 31 May 2011), “XM_002490597.1” (NCBI Reference sequence XM_002490597.1; published 17 February 2023), “XM_002490426.1” (NCBI Reference sequence XM_002490426.1; published 17 February 2023), “XM_002490236.1” (NCBI Reference sequence XM_002490236.1; published 17 February 2023), Jiang ("Regulation of cell wall beta-glucan assembly: PTC1 negatively affects PBS2." (1995)), “A0A1B2JCC4” (Uniprot Accession No. A0A1B2JCC4; published 2 November 2016), and “A0A1G4KPG0” (Uniprot Accession No. A0A1G4KPG0; published 2 November 2016). This rejection is necessitated by amendment.
Regarding claims 1 and 3, Weinhandl is drawn towards a study concerned with mutating P. pastoris strains in order to improve recombinant protein production (Abstract). Weinhandl teaches the engineering of P. pastoris by the disruption (i.e., knockout (pg. 89) of genes encoding proteins related to cell wall assembly (SCW10, CWP1) and cell wall glycosylation (OCH1) in order to increase extracellular recombinant BCAT production (i.e., Weinhandl teaches the use of a recombinant P. pastoris for improving a yield of recombinant BCAT via the targeted knockout of genes related to cell wall assembly and glycosylation) (Abstract). Weinhandl teaches that SCW10 participates in building the glucan scaffold of the inner part of the cell wall (i.e., Weinhandl teaches that reducing the glucan content of the cell’s inner cell wall resulted in an increased secretion capacity) (pg. 85). Weinhandl teaches that the influencing cell wall plasticity by deleting cell wall proteins could lead to a more permeable structure that facilitates better recombinant protein secretion (pg. 85). Weinhandl teaches that combining OCH1 deletion with the deletion in a cell wall protein (CWP1) deletion resulted in a significantly increased growth rate and a diminished tendency to form cell clusters that would be advantageous for simplified cultivation on larger scale in bioreactors (pg. 90-91).
Weinhandl does not teach or suggest that the recombinant protein is human albumin (Claim 1). Weinhandl does not teach or suggest that the knocked out genes are the claimed CCW14, EMW1, and PUN1 genes that comprise SEQ ID NO: 2, 4, and 6 respectively using a CRISPR-Cas9 gene editing technology (Claim 1). Weinhandl does not teach or suggest that the claimed EXG1 and SPR1 genes, comprising SEQ ID NOs: 8 and 10 respectively, are integrated and over-expressed in the strain of the recombinant P. pastoris (Claim 1). Weinhandl does not teach or suggest that the nucleotide sequences encoding the claimed proteins are as shown in SEQ ID NOs: 1, 3, or 5 (Claim 3).
It was known in the art that P. pastoris could be utilized to express recombinant human albumin as taught by Zhu. Zhu is drawn towards a study concerned with the purification and expression of recombinant human albumin (i.e., HAS) in P. pastoris (Abstract). Zhu teaches that P. pastoris is an excellent system for the expression of HAS due to its outstanding expression capacity (Abstract). Zhu teaches that HAS is a major component of human plasma that is utilized in pharmaceutical applications including nano delivery of drugs, vaccine formulation and manufacturing, and cryopreservation (pg. 61). Zhu teaches that P. pastoris shows attractive potential for HSA expression by virtue of several advantages including the simplicity of its molecular genetic manipulation, its genetic stability, its strong inducible alcohol oxidase 1 (AOX1) promoter, the high cell density it displays in an inexpensive basal salts medium, its potential for posttranslational modification, its high secretory capacity and its low endogenous protein secretion (pg. 61).
It was known in the art that targeted knockout of genes of interest in P. pastoris genomes could be accomplished through the use of a CRISPR/Cas9 system as taught by Liao. Liao teaches that the CRISPR/Cas9 system allows for the metabolic engineering of P. pastoris in synthetic biology (Abstract). Liao teaches the use of a CRISPR/Cas9 gene editing system that utilizes two designed sgRNAs that and could achieve up to 100% knockout efficiency of an endogenous OCH1 gene in P. pastoris (pg. 9213-9214).
It was known in the art that the claimed CCW14, EMW1, and PUN1 proteins and the nucleotide sequences encoding the proteins, alongside their respective SEQ ID NOs, were known cell walls proteins in P. pastoris, as described by both the instant specification (i.e., see “Claim Interpretation” above) and by the Uniprot entries that the instant specification refers to as prior art: A0A1G4KPI2, A0A1G4KPG0, and F2QLI3. Further, these Uniprot entries teach that P. pastoris is a yeast cell and that the claimed SEQ ID NOs were known proteins known to be involved in the cell wall of the cells. Additionally, it was known in the art that the claimed SEQ ID NOs: 1, 3, and 5 were known genomic nucleotide sequences within P. pastoris that encoded the claimed proteins as taught by XM_002490597.1, XM_002490426.1, and XM_002490236.1.
A0A1G4KPI2 teaches that the claimed SEQ ID NO: 2 was a known cell wall glycoprotein linked protein termed “CCW14” that was known to be expressed by P. pastoris yeast cells (pg. 1).
XM_002490597.1 teaches that a nucleotide sequence that has 100% identity to the claimed SEQ ID NO: 1 is a nucleotide sequence found in K. phaffii that encodes a protein that has 100% identity to the claimed SEQ ID NO: 2 (i.e., the nucleotide sequence of XM_002490597.1 is a known nucleotide sequence that encodes the claimed CCW14 protein comprising the claimed SEQ ID NO: 2) (pg. 1-2).
A0A1G4KPG0 teaches that the claimed SEQ ID NO: 4 was a known cell wall integrity protein termed “EMW1” that was known to be expressed by P. pastoris yeast cells (pg. 1).
XM_002490426.1 teaches that a nucleotide sequence that has 100% identity to the claimed SEQ ID NO: 3 is a nucleotide sequence found in K. phaffii that encodes a protein that has 100% identity to the claimed SEQ ID NO: 4 (i.e., the nucleotide sequence of XM_002490426.1 is a known nucleotide sequence that encodes the claimed EMW1 protein comprising the claimed SEQ ID NO: 4) (pg. 1-2).
F2QLI3 teaches that the claimed SEQ ID NO: 6 was a known cell wall integrity protein termed “PUN1” that was known to be expressed by P. pastoris yeast cells (pg. 1).
XM_002490236.1 teaches that a nucleotide sequence that has 100% identity to the claimed SEQ ID NO: 5 is a nucleotide sequence found in K. phaffii that encodes a protein that has 100% identity to the claimed SEQ ID NO: 6 (i.e., the nucleotide sequence of XM_002490426.1 is a known nucleotide sequence that encodes the claimed PUN1 protein comprising the claimed SEQ ID NO: 6) (pg. 1-2).
It was known in the prior art that the overexpression of EXG1 in yeast cells was advantageous because it leads to a reduction of specific glucans on the cell wall that causes the yeast cell to be resistant to killer toxin, as taught by Jiang. Jiang teaches that EXG1 is a gene that is known to be involved in yeast cell wall β-glucan assembly and that overexpression of the protein in the cell resulted in a reduction of glucans in the cell’s cell wall (pg. 260). Jiang teaches that overexpression of an EXG1 protein via the use of a recombinant plasmid within yeast cells led to resistance to dominant killer toxin via the alteration of the yeast cell’s cell wall (pg. 260, 267). Jiang further teaches that using multicopy plasmids encoding multiple genes that express proteins associated with the yeast cell wall could be overexpressed in the yeast cell (pg. 260).
It was known in the art that the claimed EXG1 and SPR1 genes and their respective claimed SEQ ID NOs were known glucan-1,3-β-glucosidases in P. pastoris that had the exact same enzymatic activity, as described by both the instant specification (i.e., see “Claim Interpretation” above) and by the Uniprot entries that the instant specification refers to as prior art: A0A1B2JCC4 and A1B2JHM0.
A0A1B2JCC4 teaches that the claimed SEQ ID NO: 10 was a known glucan-1,3-β-glucosidase termed “EXG1” that was known to be expressed in P. pastoris (pg. 2-3). A0A1B2JCC4 teaches that the EC classification of the protein is 3.2.1.58 (pg. 2).
A1B2JHM0 teaches that the claimed SEQ ID NO: 8 was a known glucan-1,3-β-glucosidase termed “SPR1” that was known to be expressed in P. pastoris (pg. 2-3). A1B2JHM0 teaches that the EC classification of the protein is also 3.2.1.58 (pg. 2).
Therefore, with regard to the production of the claimed human albumin, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have arrived at the claimed recombinant engineering bacterium because it would have merely amounted to a simple combination of prior art elements according to known methods to yield predictable results. Because Weinhandl teaches using a strain of P. pastoris for a similar reason as Zhu, namely the production of a recombinant protein, then one would have had a reasonable expectation of success in using the P. pastoris strain of Weinhandl for the production of human albumin. And because Zhu teaches that the bacterium possess many features that allow for the efficient production of the albumin, one would have been motivated to do so.
With regard to the claimed method of knocking out the claimed cell wall proteins with CRISPR-Cas9, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have arrived at the requirements of the claimed CRISPR-Cas9 gene editing technology because it would have merely amounted to simple substitution of one method of knocking out an endogenous protein for another. Since Liao teaches using CRISPR-Cas9 to knockout the target nucleotide sequences encoding the exact same gene knocked out in Weinhandl, namely OCH1, it would have been predictable to have similarly used CRISPR-Cas9 to knockout the cell wall proteins of Weinhandl via the targeting of nucleotide sequences that encode the proteins. And because Liao teaches that the knockout efficiency was near 100%, one would have been motivated to do so.
It is noted that the claims require the knockout of the claimed CCW14, EMW1, and PUN1 cell wall proteins. However, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have arrived at the requirements of the claimed knockout of the CCW14, EMW1, and PUN1 genes because it would have merely amounted to simple substitution of known P. pastoris cell wall proteins and nucleotide sequences encoding the proteins for another. Since Weinhandl teaches that cell wall proteins can be advantageously knocked out in P. pastoris in order to improve the permeability of the cell wall, increase the growth rate, and decrease the tendency to form cellular clusters, then it would have been predictable to have knocked out other known P. pastoris cell wall proteins in order to achieve the same effect. And because Weinhandl teaches that the knockout of the cell wall genes is advantageous in large cell bioreactors, one would have been motived to do so.
It further would have been obvious to have overexpressed both the claimed EXG1 and SPR1 genes in the P. pastoris cell of Weinhandl because it would have merely amounted to a simple combination of prior art elements according to known methods to yield predictable results. Since Jiang teaches a similar modification of yeast cells when compared to Weinhandl, namely the modification of genes that alter the glucan content in a yeast’s cell wall in order to achieve a desired phenotype, then it would have been predictable to have similarly overexpressed the claimed EXG1 and SPR1 genes in the yeast cell of Weinhandl in order to increase the secretory capacity of the cell through the reduction in the total amount of glucans in the cell’s cell wall. Further, both the prior art and the instant specification teach that the claimed genes produce proteins that have identical enzymatic activities and would therefore be expected to produce enzymes that enact identical modifications on a yeast cell’s cell wall. And because Jiang further teaches that the overexpression of the gene resulted in favorite phenotypes, including toxin resistance, one would have been motivated to do so.
Regarding claim 4, Weinhandl teaches that the recombinant BCAT protein may be fused to an alpha mating factor secretion signal from S. cerevisiae in the recombinant P. pastoris (i.e., the recombinant P. pastoris may further comprise a signal peptide sequence) in order to promote secretion (pg. 85, 88).
Claim(s) 5 is/are rejected under 35 U.S.C. 103 as being unpatentable over Weinhandl ("Pichia pastoris mutants as host strains for efficient secretion of recombinant branched chain aminotransferase (BCAT)." Journal of biotechnology 235 (2016): 84-91) in view of Zhu ("High level expression and purification of recombinant human serum albumin in Pichia pastoris." Protein Expression and Purification 147 (2018): 61-68), Liao ("A versatile toolbox for CRISPR-based genome engineering in Pichia pastoris." Applied microbiology and biotechnology 105.24 (2021): 9211-9218), “A0A1G4KPI2” (Uniprot Accession No. A0A1G4KPI2; published 18 January 2017), “A0A1G4KPG0” (Uniprot Accession No. A0A1G4KPG0; published 18 January 2017), “F2QLI3” (Uniprot Accession No. F2QLI3; published 31 May 2011), “XM_002490597.1” (NCBI Reference sequence XM_002490597.1; published 17 February 2023), “XM_002490426.1” (NCBI Reference sequence XM_002490426.1; published 17 February 2023), “XM_002490236.1” (NCBI Reference sequence XM_002490236.1; published 17 February 2023), Jiang ("Regulation of cell wall beta-glucan assembly: PTC1 negatively affects PBS2." (1995)), “A0A1B2JCC4” (Uniprot Accession No. A0A1B2JCC4; published 2 November 2016), and “A0A1G4KPG0” (Uniprot Accession No. A0A1G4KPG0; published 2 November 2016) as applied to claims 1 and 3-4 above, and further in view of Weninger (Journal of cellular biochemistry 119.4 (2018): 3183-3198). This rejection is necessitated by amendment.
Regarding claim 5, Weinhandl in view of Zhu, Liao, AOA1G4KPI2, A0A1G4KPG0, F2QLI3, XM_002490597.1, XM_002490426.1, XM_002490236.1, Jiang, A0A1B2JCC4, and A0A1G4KPG0 renders obvious claims 1 and 3-4 as described above.
Liao further teaches the use of a Cas9 and gRNA co-expression plasmid (pg. 9212).
Weinhandl in view of Zhu, Liao, AOA1G4KPI2, A0A1G4KPG0, F2QLI3, XM_002490597.1, XM_002490426.1, XM_002490236.1, Jiang, A0A1B2JCC4, and A0A1G4KPG0 does not teach or suggest the preparation of a donor DNA (Claim 5).
Weninger is directed towards a study concerned with expanding the CRISPR/Cas9 toolkit for editing in P. pastoris through the integration of marker-less donor cassettes (i.e., donor DNA) (Abstract). Weninger teaches that the marker-less donor cassettes can be utilized to knockout target genes of interest within P. pastoris (pg. 3184). Weninger teaches that utilizing the marker-less donor cassettes are especially valuable for allows for the efficient replacement or removal of DNA sequences, where artifacts such as scars generated by site specific recombinases or selection marker cassettes might influence their functionality (pg. 3195).
Therefore, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have arrived at the requirements of the claimed utilization of a donor DNA because it would have merely amounted to simple substitution of one known gene knockout method for another. Since Weninger teaches the use of donor DNA for a similar purpose as Weinhandl, Zhu, Liao, AOA1G4KPI2, A0A1G4KPG0, F2QLI3, XM_002490597.1, XM_002490426.1, XM_002490236.1, Jiang, A0A1B2JCC4, and A0A1G4KPG0, namely the knockout of a target gene of interest, then it would have been predicable to have similarly used the marker-less donor DNA described in Weninger for the knockout of the claimed CCW14, EMW1, and PUN1 genes. And because Weninger teaches that using the marker-less donor cassettes in gene knockout does not leave undesirable artifacts, one would have been motivated to do so.
Claim(s) 6 is/are rejected under 35 U.S.C. 103 as being unpatentable over Weinhandl ("Pichia pastoris mutants as host strains for efficient secretion of recombinant branched chain aminotransferase (BCAT)." Journal of biotechnology 235 (2016): 84-91) in view of Zhu ("High level expression and purification of recombinant human serum albumin in Pichia pastoris." Protein Expression and Purification 147 (2018): 61-68), Liao ("A versatile toolbox for CRISPR-based genome engineering in Pichia pastoris." Applied microbiology and biotechnology 105.24 (2021): 9211-9218), “A0A1G4KPI2” (Uniprot Accession No. A0A1G4KPI2; published 18 January 2017), “A0A1G4KPG0” (Uniprot Accession No. A0A1G4KPG0; published 18 January 2017), “F2QLI3” (Uniprot Accession No. F2QLI3; published 31 May 2011), “XM_002490597.1” (NCBI Reference sequence XM_002490597.1; published 17 February 2023), “XM_002490426.1” (NCBI Reference sequence XM_002490426.1; published 17 February 2023), “XM_002490236.1” (NCBI Reference sequence XM_002490236.1; published 17 February 2023), Jiang ("Regulation of cell wall beta-glucan assembly: PTC1 negatively affects PBS2." (1995)), “A0A1B2JCC4” (Uniprot Accession No. A0A1B2JCC4; published 2 November 2016), and “A0A1G4KPG0” (Uniprot Accession No. A0A1G4KPG0; published 2 November 2016) as applied to claims 1 and 3-4 above, and further in view of Liu ("CRISPR–Cas9-mediated genomic multiloci integration in Pichia pastoris." Microbial cell factories 18.1 (2019): 144). This rejection is necessitated by amendment.
Regarding claim 6, Weinhandl in view of Zhu, Liao, AOA1G4KPI2, A0A1G4KPG0, F2QLI3, XM_002490597.1, XM_002490426.1, XM_002490236.1, Jiang, A0A1B2JCC4, and A0A1G4KPG0 renders obvious claims 1 and 3-4 as described above.
Weinhandl in view of Zhu, Liao, AOA1G4KPI2, A0A1G4KPG0, F2QLI3, XM_002490597.1, XM_002490426.1, XM_002490236.1, Jiang, A0A1B2JCC4, and A0A1G4KPG0 does not teach or suggest that a recombinant expression cassette encoding each of the EXG1 and SPR1 genes is located on a nucleic acid construct (Claim 6).
Liu is drawn towards a study concerned with CRISPR-Cas9 mediated genomic multiloci integration of transgenes in P. pastoris (Abstract). Liu teaches the use of a single vector (i.e., a single nucleic acid) that can comprise 3 different expression cassettes, each encoding a different GFP, mCherry, and BFP transgene that can be subsequently inserted into the genome of a P. pastoris strain in order to express the transgenes (pg. 5; see Fig. 2). Liu teaches that Cas9 guide RNAs can be designed and tested in order to confirm suitable single-locus integration sites for each of the expression cassettes (pg. 3). Liu teaches that the engineered P. pastoris was able to successfully express each of the different inserted transgenes (pg. 1). Liu teaches that the multiloci integration can be used to rapidly assemble pathways without the requirement of selective markers, which may extend the use of P. pastoris for synthesizing complicated pharmaceuticals and chemicals (pg. 8).
Therefore, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have arrived at the requirements of the claimed nucleic acid comprising the recombinant expression cassettes because it would have merely amounted to a simple combination of prior art elements according to known methods to yield predictable results. Because Liu teaches the modification of a P. pastoris cell for a similar purpose as Weinhandl, Zhu, Liao, AOA1G4KPI2, A0A1G4KPG0, F2QLI3, XM_002490597.1, XM_002490426.1, XM_002490236.1, Jiang, A0A1B2JCC4, and A0A1G4KPG0, namely the expression of recombinant proteins of interest within the cell that can be used to effect cellular pathways, then one would have had a reasonable expectation of success in using the knock-in method described in Liu as a method for expressing the claimed EXG1 and SPR1 genes in the P. pastoris cells. And because Liu teaches that using the knock-in method is advantageous in the claimed P. pastoris because it allows for the synthesis of complicated chemicals, one would have been motived to do so.
Claim(s) 7 is/are rejected under 35 U.S.C. 103 as being unpatentable over Weinhandl ("Pichia pastoris mutants as host strains for efficient secretion of recombinant branched chain aminotransferase (BCAT)." Journal of biotechnology 235 (2016): 84-91) in view of Zhu ("High level expression and purification of recombinant human serum albumin in Pichia pastoris." Protein Expression and Purification 147 (2018): 61-68), Liao ("A versatile toolbox for CRISPR-based genome engineering in Pichia pastoris." Applied microbiology and biotechnology 105.24 (2021): 9211-9218), “A0A1G4KPI2” (Uniprot Accession No. A0A1G4KPI2; published 18 January 2017), “A0A1G4KPG0” (Uniprot Accession No. A0A1G4KPG0; published 18 January 2017), “F2QLI3” (Uniprot Accession No. F2QLI3; published 31 May 2011), “XM_002490597.1” (NCBI Reference sequence XM_002490597.1; published 17 February 2023), “XM_002490426.1” (NCBI Reference sequence XM_002490426.1; published 17 February 2023), “XM_002490236.1” (NCBI Reference sequence XM_002490236.1; published 17 February 2023), Jiang ("Regulation of cell wall beta-glucan assembly: PTC1 negatively affects PBS2." (1995)), “A0A1B2JCC4” (Uniprot Accession No. A0A1B2JCC4; published 2 November 2016), and “A0A1G4KPG0” (Uniprot Accession No. A0A1G4KPG0; published 2 November 2016) as applied to claims 1 and 3-4 above, and further in view of Herpe (bioRxiv (2022): 2022-12). This rejection is necessitated by amendment.
Regarding claim 7, Weinhandl in view of Zhu, Liao, AOA1G4KPI2, A0A1G4KPG0, F2QLI3, XM_002490597.1, XM_002490426.1, XM_002490236.1, Jiang, A0A1B2JCC4, and A0A1G4KPG0 renders obvious claims 1, 4, and 6 as described above.
Weinhandl in view of Zhu, Liao, AOA1G4KPI2, A0A1G4KPG0, F2QLI3, XM_002490597.1, XM_002490426.1, XM_002490236.1, Jiang, A0A1B2JCC4, and A0A1G4KPG0 does not teach or suggest that the recombinant engineering bacterium is formed by modifying K. phaffii CBS7435 (Claim 7).
Herpe is directed towards a study concerned with K. phaffii protein expression toolkits (Abstract). Herpe teaches that K. phaffii CBS7435 is a known strain of K. phaffii that is publicly available (pg. 4).
Therefore, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have arrived at the requirements of the claimed engineering bacterium because it would have merely amounted to a simple substitution of one known strain of K. phaffii for another. Since Weinhandl in view of Zhu, Liao, AOA1G4KPI2, A0A1G4KPG0, F2QLI3, XM_002490597.1, XM_002490426.1, XM_002490236.1, Jiang, A0A1B2JCC4, and A0A1G4KPG0 teaches the genetic modification of a P. pastoris strain (i.e., a K. phaffii strain), it would have been predictable to have similarly modified the K. phaffii CBS7435 strain in order to achieve the same results of an increased production of human albumin.
Response to Arguments
Insofar as Applicant’s arguments pertain to the newly cited references, Applicant's arguments filed 24 March 2026 have been fully considered but they are not persuasive.
Applicant alleges that the particular combination of the claimed recombinant microorganism is non-obvious because Applicant has provided evidence of the surprising result that the knockout of CCW14, EMW1, and PUN1 genes, combined with the concurrent overexpression of EXG1 and SPR1 genes, exerts a synergistic effect that significantly boosts the secretion yield of recombinant human albumin (Remarks; pg. 10). Applicant alleges that overexpression of SPR1 gene on the basis of the aforementioned triple knockout (bacterium 8) leads to a 111% increase in the secretion yield of recombinant human albumin (Remarks; pg. 10). Applicant alleges that concurrent overexpression of both SPR1 and EXG1 genes on the basis of the triple knockout (bacterium 9) achieves a 138% increase in the secretion yield of recombinant human albumin (Remarks; pg. 10).
With regard to the alleged unexpected results as seen in bacterium 9, as discussed above, Weinhandl teaches that combining OCH1 deletion with the deletion in a cell wall protein (CWP1) deletion resulted in a significantly increased growth rate and a diminished tendency to form cell clusters that would be advantageous for simplified cultivation on larger scale in bioreactors (pg. 90-91). Weinhandl teaches that the combined knockout of OCH1 and either CWP1 or SCW10 resulted in over 100% increases in the amount of BCAT activity (i.e., BCAT secretion) detected when compared to a wildtype strain that did not have the combined knockout of OCH1 and either CWP1 or SCW10 (pg. 87; see Fig. 3). Weinhandl further teaches that SCW10 participates in building the glucan scaffold of the inner part of the cell wall (i.e., Weinhandl teaches that reducing the glucan content of the cell’s inner cell wall, through the knockout of a gene that builds the glucan scaffold of the wall, combined with a deletion of a cell wall protein resulted in an increased secretion capacity) (pg. 85).
Further, as discussed above, Jiang teaches that it was known in the prior art that the claimed EXG1 and SPR1 genes were known glucosidases that specifically reduced the amount of glucan present in a yeast cell’s cell wall (i.e., a phenotype that, as taught by Weinhandl, results in an increased secretion capacity of a host yeast cell).
Accordingly, the prior art teaches that the deletion of genes encoding cell wall proteins and genes responsible for the building of glucan content in the cell wall results in a synergistic effect that boosts the production of a recombinant protein in a recombinant bacterium by over 100%. Further, the prior art demonstrates that the claimed EXG1 and SPR1 genes encoded known proteins that reduced the glucan content of a yeast cell wall. Therefore, one of ordinary skill in the art would have expected the combination of deletion of cell wall proteins alongside the overexpression of genes encoding proteins that further reduced the amount of glucan content within a cell wall to synergistically weaken the cell wall and allow for higher secretion capacity through the reduction of the amount of proteins and glucans within the cell wall.
Therefore, the alleged surprising result claimed by Applicant is expected in view of the closest prior art.
Conclusion
Applicant's amendment necessitated the new ground(s) of rejection presented in this Office action. Accordingly, THIS ACTION IS MADE FINAL. See MPEP § 706.07(a). Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action. In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any nonprovisional extension fee (37 CFR 1.17(a)) pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action. In no event, however, will the statutory period for reply expire later than SIX MONTHS from the mailing date of this final action.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to KYLE T REGA whose telephone number is (571)272-2073. The examiner can normally be reached M-R 8:30-4:30, every other F 8:30-4:30 (EDT/EST).
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/KYLE T REGA/ Examiner, Art Unit 1636
/NEIL P HAMMELL/ Supervisory Patent Examiner, Art Unit 1636
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