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 .
Continued Examination Under 37 CFR 1.114
A request for continued examination under 37 CFR 1.114, including the fee set forth in 37 CFR 1.17(e), was filed in this application after final rejection. Since this application is eligible for continued examination under 37 CFR 1.114, and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114. Applicant's submission filed on 08/26/2026 has been entered.
Amendments Received
Amendments to the claims were received and entered on 08/26/2026.
Status of Claims
Claims 2, 5-7 and 9 have been cancelled.
Claims 1 and 3 are currently pending and under consideration.
Priority
The present application claims status as a 371 (National Stage) of PCT/JP2022/007821 filed on 02/25/2022 and claims priority to Japanese application JP2021-031907 filed on 03/01/2021. Acknowledgment is made of applicant' s claim for foreign priority and papers submitted under 35 U.S.C. 119 (a)-(d). Please note that the Japanese application is in a foreign language and therefore cannot be reviewed. In future actions, the effective filing date may change due to amendments or further review of priority documents.
Information Disclosure Statement
The information disclosure statement (IDS) submitted on 08/26/2026 is in compliance with the provisions of 37 CFR 1.97. Accordingly, the information disclosure statement has been considered by the examiner.
Withdrawn Rejections
In view of Applicant’s cancellation of claims 2, 5-7 and 9, all rejections of claims 2, 5-7 and 9 are now moot and are hereby withdrawn.
In view of Applicant’s amendments and remarks filed on 08/26/2026, rejections of claims 1 and 3 under 35 USC § 103 over Nettleship et al. and Sinkunas et al., as evidenced by CytoScientific, Blaber and ProMega, are hereby withdrawn.
Maintained/Modified Rejections Necessitated by Amendment
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.
Claims 1 and 3 are rejected under 35 U.S.C. 103 as being unpatentable over Nettleship et al. (Recent advances in the production of proteins in insect and mammalian cells for structural biology, Journal of Structural Biology 172 (2010) 55–65, cited in previous office action), Mulepati et al. (In Vitro Reconstitution of an Escherichia coli RNA-guided Immune System Reveals Unidirectional, ATP-dependent Degradation of DNA Target, THE JOURNAL OF BIOLOGICAL CHEMISTRY, August 2, 2013, pages 22184-22192, vol. 288, no. 31, cited in the IDS), Lee et al. (Functional efficacy of human recombinant FGF-2s tagged with (His)6 and (His-Asn)6 at the N- and C-termini in human gingival fibroblast and periodontal ligament-derived cells, Protein Expression and Purification 135 (2017) 37e44, cited in PTO-892), Hatfaludi et al. (Screening of 71 P. multocida proteins for protective efficacy in a fowl cholera infection model and characterization of the protective antigen PlpE. PLoS One. 2012;7(7):e39973, cited in PTO-892), and Hong et al. (Size-Exclusion Chromatography for the Analysis of Protein Biotherapeutics and their Aggregates, Journal of Liquid Chromatography & Related Technologies, 35:2923-2950, 2012, cited in a previous office action).
Nettleship et al. teaches using insect cells, including Sf9 insect cells, as established host cells for recombinant protein expression. Nettleship et al. teaches that the baculovirus-insect cell expression system is “a well-established method for the production of recombinant proteins” and that there are proteins not amenable to bacterial expression, e.g., membrane proteins, protein complexes, etc., specifically referring to E. coli. Nettleship et al. further teaches the use of multi-host vectors containing promoters for E. coli and baculovirus expression in insect cells, thereby enabling target genes to be evaluated in both bacterial and higher eukaryotic hosts and permitting parallel screening of bacterial and insect-cell expression systems to identify a suitable expression host (pg. 55-56). Furthermore, Nettleship et al. teaches the specific insect cell line used, noting that Autographa californica multiple nucleopolyhedrovirus infects Spodoptera frugiperda (Sf9) insect cells (pg. 56, left column). Additionally, Nettleship et al. discloses explicit culturing conditions for Sf9 insect cells, specifically culturing Sf9 insect cells “at 27°C on an orbital shaker” (pg. 58, left column, “optimizing expression”), teaches that achieving the best expression of a recombinant protein requires optimization of expression parameters (e.g., temperature) and that optimal expression parameters may be determined for a given protein before proceeding to large-scale production. Nettleship et al. further teaches purification of recombinant proteins produced in Sf9 insect cells following expression. Specifically, that expression constructs generally include a hexahistidine tag permitting purification of proteins from cell lysates using metal affinity chromatography and describes the use of Ni-NTA for capture of soluble proteins (pg. 58, right column). Nettleship et al. further discloses cell disruption and centrifugation prior to purification, stating that pelleted Sf9 cells were lysed in 50 mM NaH₂PO₄, pH 8.0, containing 300 mM NaCl, 10 mM imidazole, and 1% Tween 20 and centrifuged at 10,000 g for 10 minutes (Fig. 3). Nettleship et al. does not expressly teach expressing a Cas3 protein derived from E. coli, culturing the Cas3 gene-introduced insect cells at 20 to 24°C, or an HN tag attached to the Cas3 protein. Nettleship et al. also does not expressly teach the claimed purification sequence comprising affinity purification of HN-tagged Cas3 using a nickel column and a phosphate buffer, followed by further purification of the Cas3 protein by gel filtration chromatography using a phosphate buffer as the mobile phase buffer.
Mulepati et al. discloses expression and purification of a Cas3 protein derived from E. coli. Specifically, Mulepati et al. teaches that “E. coli Cas3 was overexpressed in the T7Express strain of E. coli,” wherein the cells were grown at 20°C, harvested, lysed, clarified by centrifugation, and loaded onto an immobilized metal affinity chromatography column. The resulting sample was subsequently loaded onto a size-exclusion chromatography column (pg. 22185, “Protein Expression and Purification”). Mulepati et al. further teaches that the E. coli Cas3 contains an N-terminal His₆-maltose-binding protein tag and that the tagged protein was purified from clarified cell lysate by nickel affinity and size-exclusion chromatographies. Furthermore, Mulepati et al. teaches that cultivation temperature directly affected soluble E. coli Cas3 production, specifically disclosing that the maximum yield of soluble Cas3 was obtained when cultures were grown at 20°C, whereas cultures grown at higher temperatures produced little or no soluble Cas3 (pg. 22186, “Overexpression and Purification of Recombinant E. coli Cas3”).
Lee et al. teaches the use of an HN tag for purification of recombinant proteins by nickel affinity chromatography. Specifically, Lee et al. teaches that although a hexahistidine (H6) tag is commonly used for immobilized metal affinity chromatography (IMAC), a hexahistidine-asparagine (HN6) tag is also efficient for purification because it is readily exposed on the surface of the protein (pg. 37 and Abstract). Lee et al. further teaches recombinant proteins comprising HN6 tags at either the N- or C-terminus and describes HN6 as comprising six repeats of histidine-asparagine (pg. 38 and 40). Lee et al. further discloses purification of the recombinant HN6-tagged proteins by immobilized metal affinity chromatography and specifically demonstrates purification of HN6-tagged recombinant proteins using Ni-NTA beads in column chromatography (pg. 40, “Large scale purification of recombinant FGF-2 proteins” and Fig. 2E). Thus, Lee et al. teaches that an HN tag was a known affinity tag suitable for purification of recombinant proteins using nickel affinity chromatography.
Hatfaludi et al. teaches purification of recombinant proteins using a phosphate buffer during nickel affinity chromatography followed by size-exclusion chromatography. Specifically, Hatfaludi et al. teaches that cells expressing recombinant proteins were resuspended in a nickel affinity buffer comprising 100 mM sodium phosphate buffer, pH 7.4, containing 150 mM NaCl and 10 mM imidazole, lysed by sonication, and the soluble and insoluble fractions separated by centrifugation (pg. 8, “Expression and Purification of Antigens”). For soluble proteins, Hatfaludi et al. further teaches that the soluble fraction was filtered and loaded onto a HisTrap FF nickel affinity column. After washing the column with the nickel affinity buffer, the recombinant proteins were eluted from the nickel affinity column with 100 mM sodium phosphate buffer, pH 7.4, containing 150 mM NaCl and 0.5 M imidazole. Hatfaludi et al. further teaches that the proteins eluted from the nickel affinity column were subsequently loaded onto a HiLoad 16/60 Superdex 200 size-exclusion chromatography column, and that fractions containing the protein of interest were collected in 100 mM sodium phosphate buffer, pH 7.4, containing 150 mM NaCl (pg. 8). Thus, Hatfaludi et al. teaches a recombinant protein purification workflow comprising cell disruption, centrifugation, collection of a soluble fraction, nickel affinity chromatography using a phosphate buffer, and subsequent size-exclusion chromatography in which the protein-containing fractions are collected in phosphate buffer.
Hong et al. teaches size-exclusion chromatography (i.e., gel filtration chromatography) of proteins using a phosphate buffer as a mobile phase buffer. Specifically, Hong et al. teaches separation of proteins by size-exclusion chromatography using a mobile phase comprising 100 mM sodium phosphate buffer at pH 6.8 (see Fig. 2). Hong et al. further demonstrates the use of a mobile phase comprising 100 mM sodium phosphate buffer, pH 6.8, in evaluating size-exclusion chromatography operating conditions (see Figs. 4 and 6). Thus, Hong et al. teaches the use of a phosphate buffer as a mobile phase buffer in gel filtration chromatography.
An invention would have been obvious to a person of ordinary skill in the art if some teaching in the prior art would have led that person to combine prior art reference teachings to arrive at the claimed invention. Before the effective filing date of the claimed invention, it would have been obvious to a person of ordinary skill in the art to introduce the E. coli Cas3 gene taught by Mulepati et al. into the baculovirus-Sf9 insect-cell expression system of Nettleship et al. and to culture the Cas3 gene-introduced insect cells at a temperature within the claimed range of 20 to 24°C. Nettleship et al. teaches that baculovirus-insect cell expression is a well-established method for recombinant protein production and further teaches evaluating target genes in bacterial and insect-cell expression systems and screening such systems to identify a suitable expression host. Mulepati et al. specifically teaches production of E. coli Cas3 and demonstrates that cultivation temperature affects soluble E. coli Cas3 production, with the maximum yield of soluble Cas3 obtained at 20°C and little or no soluble Cas3 produced at higher temperatures. Thus, a person of ordinary skill in the art would have been motivated to employ Nettleship et al.’s established insect-cell expression system for production of the known E. coli Cas3 protein of Mulepati et al. and to employ the 20°C cultivation temperature taught by Mulepati et al. for soluble E. coli Cas3 production. It would further have been obvious to purify the expressed Cas3 protein using the known recombinant protein purification techniques taught by Mulepati et al., Lee et al., Hatfaludi et al., and Hong et al. Mulepati et al. teaches a purification workflow for E. coli Cas3 comprising cell disruption, centrifugation and collection of the soluble fraction, followed by nickel affinity chromatography and size-exclusion chromatography. Lee et al. teaches that an HN tag is a known metal-affinity tag suitable for purification of recombinant proteins using nickel affinity chromatography. Accordingly, substituting Lee et al.’s HN tag for the His-containing affinity tag used by Mulepati et al. would have constituted the substitution of one known metal-affinity tag for another known for the same purpose, with the predictable result of permitting purification of the tagged recombinant protein by nickel affinity chromatography. In view of Hatfaludi et al. further teaching the use of a phosphate buffer in a recombinant protein purification workflow comprising nickel affinity chromatography followed by size-exclusion chromatography, while Hong et al. teaches the use of a phosphate buffer as a mobile phase buffer in size-exclusion chromatography, it would have also been obvious to employ the phosphate-buffer purification conditions taught by Hatfaludi et al. and Hong et al. in the nickel-affinity and size-exclusion chromatography purification workflow taught by Mulepati et al. in order to purify the recombinant Cas3 protein using known chromatographic conditions for their established purposes. There would have been a reasonable expectation of success because Nettleship et al. establishes baculovirus-Sf9 insect cells as a well-established recombinant protein expression system; Mulepati et al. demonstrates successful production of soluble E. coli Cas3 at 20°C and successful purification of E. coli Cas3 by nickel affinity chromatography followed by size-exclusion chromatography; Lee et al. demonstrates successful nickel-affinity purification of HN-tagged recombinant proteins; Hatfaludi et al. demonstrates successful recombinant protein purification using phosphate-buffered nickel affinity chromatography followed by size-exclusion chromatography; and Hong et al. demonstrates successful protein size-exclusion chromatography using a phosphate-buffer mobile phase. Accordingly, the claimed method represents the combination of known recombinant protein expression and purification techniques according to their established functions, and the invention as a whole would have been prima facie obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention.
Response to Arguments for Rejections under 35 USC § 103
In the response filed on 08/26/2026, Applicant argues that the cited prior art fails to teach or suggest culturing E. coli Cas3 gene-introduced insect cells at 20 to 24°C and fails to teach or suggest purification of the Cas3 protein using a phosphate buffer during both nickel affinity chromatography and gel filtration chromatography. Applicant further argues that the claimed conditions provide unexpected results, including improved production of soluble and active Cas3 at reduced cultivation temperatures and reduced aggregation and improved thermal stability of Cas3 when phosphate buffer is used during purification. Applicant’s arguments have been fully considered but are not persuasive of patentability in view of the rejection set forth above.
As discussed above, Mulepati et al. specifically teaches production of E. coli Cas3 and reports that the maximum yield of soluble Cas3 was obtained at 20°C, with cultures grown at higher temperatures producing little or no soluble Cas3. Nettleship et al. teaches the use of Sf9 insect cells as an established recombinant protein expression system and the evaluation and optimization of expression conditions for a given recombinant protein. With respect to the claimed purification conditions, Mulepati et al. teaches purification of E. coli Cas3 by nickel affinity chromatography followed by size-exclusion chromatography; Lee et al. teaches HN-tagged recombinant proteins and their purification by nickel affinity chromatography; Hatfaludi et al. teaches the use of phosphate buffer during nickel affinity purification followed by size-exclusion chromatography in which the protein-containing fractions are collected in phosphate buffer; and Hong et al. teaches the use of phosphate buffer as a mobile phase in size-exclusion chromatography. Thus, the limitations relied upon by Applicant to distinguish amended claim 1 are taught or suggested by the prior art as combined above.
Applicant’s evidence of unexpected results has also been considered. However, the evidence does not outweigh the evidence of obviousness. With respect to temperature, the Specification demonstrates soluble expression of E. coli Cas3 at 20°C, 24°C, and 28°C, and does not establish a critical or unexpected difference throughout the claimed 20 to 24°C range relative to the prior-art conditions. Moreover, Mulepati et al. expressly teaches 20°C for maximizing soluble production of E. coli Cas3. With respect to phosphate buffer, although Applicant reports reduced aggregation and increased thermal stability relative to HEPES buffer, the prior art demonstrates that phosphate buffers were known for use in both nickel affinity and size-exclusion chromatographic purification of recombinant proteins, as discussed above. Accordingly, when the evidence of unexpected results is considered together with the prior art as a whole, it is insufficient to overcome the prima facie case of obviousness.
Conclusion
No claim is in condition for allowance.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to NAGHMEH NINA MOAZZAMI whose telephone number is (703)756-4770. The examiner can normally be reached Monday-Friday, 9:00-5:00.
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/NAGHMEH NINA MOAZZAMI/Examiner, Art Unit 1652
/ROBERT B MONDESI/Supervisory Patent Examiner, Art Unit 1652
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