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 .
Amendments Received
Amendments to the claims were received and entered on March 09, 2026.
Status of Claims
Claims 4 and 8 are cancelled, and claim 9 is newly added.
Claims 1-3, 5-7 and 9 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.
Withdrawn Objections
In view of Applicant’s amendments, objections to claims 4-8 are hereby withdrawn.
Withdrawn Rejections
In view of Applicant’s cancellation of claims 4 and 8, all rejections of claims 4 and 8 are now moot and are hereby withdrawn.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 1-3 and 5-6 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 a previous office action) and Sinkunas et al. (Cas3 is a single-stranded DNA nuclease and ATP-dependent helicase in the CRISPR/Cas immune system, EMBO J. 30:1335–1347, cited in the IDS), as evidenced by CytoScientific (“Protein Purification: Techniques, Steps, and Importance”, available at https://cytoscientific.com/protein-purification-techniques-steps-and-importance/, cited in PTO-892), Blaber, M. (“Protein Purification”, Supplemental Modules (Biochemistry), LibreTexts, Section 4.1., cited in PTO-892), and ProMega Corporation (“Protein Purification Methods”, available at https://www.promega.com/resources/guides/protein-analysis/protein-purification-methods/, cited in PTO-892).
Regarding claims 1 and 3, 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 (pg. 55). Nettleship et al. further 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”). Nettleship et al. further teaches the use of a phosphate-containing buffer during purification of recombinant proteins expressed in Sf9 insect cells. Specifically, Nettleship et al. discloses that “pelleted cells were lysed in 50 mM NaH2PO4, pH 8.0, containing 300 mM NaCl, 10 mM imidazole, 1% Tween 20” and centrifuged at 10,000 g for 10 minutes (Fig. 3). Protein purification is understood in the art as a multi-step workflow and is not limited to a single isolated chromatography step. As evidenced by conventional protein purification guidance (see below), protein purification workflows may include cell lysis, centrifugation, fractionation, protein capture, washing, elution, and chromatographic separation. Therefore, because protein purification is conventionally understood as a multi-step workflow that includes cell lysis, the phosphate-containing lysis buffer disclosed by Nettleship et al. constitutes use of a phosphate-containing buffer during protein purification.
CytoScientific, “Protein Purification: Techniques, Steps, and Importance”, available at https://cytoscientific.com/protein-purification-techniques-steps-and-importance/
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Blaber, M., “Protein Purification”, Supplemental Modules (Biochemistry), LibreTexts, Section 4.1.
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ProMega Corporation, “Protein Purification Methods”, available at https://www.promega.com/resources/guides/protein-analysis/protein-purification-methods/
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Nettleship et al. does not teach a Cas3 gene introduced into an insect cell or expressing a Cas3 protein.
Sinkunas et al. discloses cloning, expressing, and purifying the Cas3 protein, a large, multi-domain bacterial enzyme comprising both a nuclease and helicase. Sinkunas et al. further discloses Cas3 expression, specifically, “the cas3 gene from S. thermophilus DGCC7710 was cloned into the pBAD24-C-His₆ plasmid…and the Cas3 protein purified from the crude cell extracts” (pg. 1336, right column). Sinkunas et al. further explains that “the recombinant plasmid was expressed in the E. coli strain ER2267”, thereby teaching the production of a Cas3 protein from a cloned Cas3 gene (pg. 1336, left column). Sinkunas et al. further teaches expression of Cas3 under reduced temperature conditions, disclosing that E. coli cells expressing Cas3 to an OD600 of 0.5, after which “the growth temperature was decreased to 16°C and expression induced”. Thus, Sinkunas et al. teaches that cultivation temperature was recognized in the art as a parameter affecting recombinant Cas3 production.
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 a Cas3 gene into the insect-cell expression system of Nettleship et al. Given that Nettleship et al. discloses the insect cell system as a desirable alternative (to bacterial expression) for expression of certain proteins, because there are proteins not amenable to bacterial expression, a skilled artisan would have been motivated to use the baculovirus–Sf9 insect-cell system disclosed by Nettleship et al. to express Cas3, a large multi-domain protein, which may otherwise exhibit expression, solubility, or folding limitations in bacterial systems. Additionally, substituting one known expression host for another known host, for production of the same recombinant protein, constitutes routine optimization within the ordinary skill in the art and would be obvious to try among a finite number of identified predictable options. Moreover, optimization of routine expression parameters, including cultivation temperature, would have been well within the ordinary skill in the art and would have constituted no more than routine experimentation to obtain suitable recombinant protein expression. There is a reasonable expectation of success, because Nettleship et al. discloses that insect cell expression systems have been successful in recombinant protein production and is a “well-established method” for recombinant protein production. Therefore, 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.
Regarding claim 2, Sinkunas et al. teaches the expression of Cas3 derived from an S. thermophilus strain, in E. coli – “We have cloned and expressed the cas3 gene of S. thermophilus DGCC7710 in E. coli ER2267, and purified the Cas3 protein…” (pg. 1336, left column). However, Sinkunas et al. further discloses the CRISPR/Cas system of E. coli K-12, including domain architecture of the Cas3 protein from E. coli K-12 (Fig. 1). Sinkunas et al. therefore teaches both S. thermophilus Cas3 and E. coli Cas3 as known Cas3 orthologs of the same helicase-nuclease enzyme family. A person of ordinary skill in the art would have recognized that the E. coli Cas3 disclosed in Sinkunas et al. is a known functional equivalent of the S. thermophilus Cas3. Therefore, it would be obvious for a person of ordinary skill in the art, to make these substitutions, to express the E. coli derived Cas3 using the insect cell system of Nettleship et al., and expect predictable results (see MPEP 2144.06, “Substituting equivalents known for the same purpose”).
Regarding claim 5, Nettleship et al. teaches that recombinant proteins produced in Sf9 systems are routinely purified following expression and describes Ni (metal) affinity purification for His-tagged proteins in insect cells (pg. 58, right column, para 2). Additionally, Sinkunas et al. teaches purification of Cas3 protein from crude cell extracts following expression, using a His-tag and a sequence of chromatography steps. Specifically, “cells were disrupted by sonication… the supernatant was loaded onto the Ni²⁺-charged 5 ml HiTrap chelating HP column…fractions containing Cas3 were pooled” (pg. 1341, left column, 1st para).
Regarding claim 6, as described above, both Nettleship et al. and Sinkunas et al. use a His tag for protein purification, which is one member of the finite set of well-known affinity tags ( e.g., His, FLAG, GST, HN, etc.) routinely used and substituted for one another depending on vector design and purification preference. Selecting an HN tag from the small set of conventional, functionally equivalent affinity tags would have been a predictable design choice well within the skill in the art, and the substitution of one known tag for another is considered an obvious modification. Given, Nettleship et al.’s disclosure of using metal affinity chromatography and HN tags belonging to the same general class of metal-binding affinity tags, substituting the His tag used in Nettleship et al. (and Sinkunas et al.) with an HN tag, would have been obvious to a person of ordinary skill in the art (see MPEP 2144.06, “Substituting equivalents known for the same purpose”).
Thus, claims 1-3 and 5-6 are rejected under 35 U.S.C. 103 as being unpatentable over Nettleship et al. and Sinkunas et al., as evidenced by CytoScientific, Blaber and Promega.
Claim 7 is rejected under 35 U.S.C. 103 as being unpatentable over Nettleship et al. and Sinkunas et al., as applied to claims 1 and 4-6 above, and further in view of Peter et al. (WO2019241452, cited in PTO-892).
The teachings of Nettleship et al. and Sinkunas et al. as they apply to claims 1 and 4-6 have already been discussed above. Briefly, Nettleship et al. teaches expressing recombinant proteins in insect cells using a baculovirus-Sf9 expression system, and Sinkunas et al. teaches expression and purification of Cas3 using affinity chromatography. Neither Nettleship et al. nor Sinkunas et al. expressly teach further purifying the Cas3 protein by gel filtration chromatography.
Peter et al. teaches that Cas and Cascade component purified using immobilized metal affinity chromatography followed by size-exclusion chromatography (i.e., gel filtration chromatography) (Specification, para 586). More specifically, Peter et al. discloses “the nickel affinity eluate was further purified by size exclusion chromatography (SEC). The nickel affinity eluate was concentrated to a final volume of 0.5 mL by ultrafiltration at l2°C using an Amicon® (MilliporeSigma, Billerica, MA) ultrafiltration spin concentrator with an ETltracel®-50 (MilliporeSigma, Hayward, CA) membrane. The concentrated sample was filtered using a 0.22 mM ETltrafree-MC GV (MilliporeSigma, Hayward, CA) centrifugal filter before being further purified by separation at 4°C with a flow rate of 0.5 mL/minute on a HiPrep™ 16/60 Sephacryl® S-300 (GE Healthcare, Uppsala, Sweden) column equilibrated with SEC buffer composed of 50 mM Tris pH 7.5, 500 mM NaCl, 5% glycerol, 0.1 mM EDTA, and 1 mM TCEP. Proteins were eluted with SEC buffer and 1 ml fractions were collected” (para 590).
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 incorporate gel-filtration chromatography into the purification workflow of Sinkunas et al. and Nettleship et al., in order to obtain a more highly purified preparation of the Cas3 protein (see MPEP 2144.06 I., “Combining equivalents known for the same purpose”). There is a reasonable expectation of success, because Peter et al. expressly teaches adding size-exclusion (i.e., gel-filtration) chromatography following affinity chromatography, establishing such a workflow as routine and conventional in protein purification. Therefore, 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.
Thus, claim 7 is rejected under 35 U.S.C. 103 as being unpatentable over Nettleship et al. and Sinkunas et al., and further in view of Peter et al.
Claim 9 is rejected under 35 U.S.C. 103 as being unpatentable over Nettleship et al. and Sinkunas et al., as applied to claims 1 and 5 above, and further in view of 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 PTO-892).
The teachings of Nettleship et al. and Sinkunas et al. as they apply to claims 1 and 5 have already been discussed above. Briefly, Nettleship et al. teaches expression of recombinant proteins in Sf9 insect cells and purification of recombinant proteins produced therein, while Sinkunas et al. teaches expression and purification of Cas3 protein using affinity purification techniques. Neither Nettleship et al. nor Sinkunas et al. expressly teaches purifying Cas3 protein by gel filtration chromatography using a phosphate buffer as a mobile phase buffer.
Hong et al. teaches size-exclusion chromatography (i.e., gel filtration chromatography) of proteins using a mobile phase consisting of 100 mM sodium phosphate buffer at pH 6.8 (Abstract, Fig. 2, 4 and 6). Hong et al. therefore 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 employ a phosphate buffer as the mobile phase buffer in the gel filtration chromatography purification step of the Cas3 purification workflow suggested by Nettleship et al. and Sinkunas et al. Hong et al. teaches that sodium phosphate buffer was a known and conventional mobile phase for protein size-exclusion chromatography. Selecting a known phosphate-buffer mobile phase for use in a known gel filtration chromatography purification step would have constituted no more than routine optimization of chromatographic operating conditions and the use of known techniques for their established purpose. There would have been a reasonable expectation of success because Hong et al. expressly demonstrates successful protein separation using phosphate-buffer mobile phases in size-exclusion chromatography. Therefore, 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.
Thus, claim 9 is rejected under 35 U.S.C. 103 as being unpatentable over Nettleship et al. and Sinkunas et al., and further in view of Hong et al.
Response to Arguments for Prior Art Rejections
In the responses filed on March 9 and March 12, 2026, Applicant argues that the cited references do not teach or suggest the presently claimed method because the claims require culturing insect cells at 20–24°C and purifying the Cas3 protein using a phosphate buffer. Applicant further argues that Nettleship et al. only discloses washing cultured cells with PBS and does not disclose purification of Cas3 protein using a phosphate buffer. Applicant also argues that use of phosphate buffer produced unexpected improvements in thermal stability and yield relative to HEPES buffer, and that culturing at 20–24°C provided improved soluble expression and activity. Applicant’s arguments have been considered in full and have been found to be not persuasive.
With respect to the phosphate buffer limitation, Applicant’s argument that Nettleship et al. merely discloses washing cells with PBS is not accurate. Nettleship et al. expressly discloses processing recombinant proteins expressed in Sf9 insect cells using a phosphate-containing buffer. Specifically, Nettleship et al. teaches that “pelleted cells were lysed in 50 mM NaH2PO4, pH 8.0, containing 300 mM NaCl, 10 mM imidazole, 1% Tween 20” and subsequently centrifuged (Fig. 3). Thus, Nettleship et al. teaches use of a phosphate-containing buffer during recovery and processing of recombinant proteins expressed in insect cells. Furthermore, claim 1 broadly recites “purification of the Cas3 protein using a phosphate buffer” and does not limit the phosphate buffer to gel filtration chromatography, a mobile phase buffer, an affinity purification step, or any other particular purification operation. As discussed above, evidence of record demonstrates that protein purification was understood in the art as a multi-step workflow that may include cell lysis, centrifugation, fractionation, protein capture, washing, elution, and chromatographic separation. Accordingly, a person of ordinary skill in the art would have understood the broad claim language to encompass the use of phosphate-containing buffers during various stages of a purification workflow.
Applicant further argues that use of phosphate buffer unexpectedly improved thermal stability and yield relative to HEPES buffer. Examiner acknowledges Applicant’s evidence regarding the specific purification protocol described in Example 1. However, Applicant’s evidence is directed to a specific purification protocol involving affinity purification and gel filtration chromatography performed using particular buffer systems. In contrast, claim 1 broadly recites purification using a phosphate buffer and does not require affinity purification, gel filtration chromatography, use of phosphate buffer as a mobile phase buffer, or any other specific purification protocol. Therefore, the evidence is not commensurate in scope with the full breadth of claim 1 and is insufficient to outweigh the prima facie case of obviousness.
With respect to the culturing temperature, Applicant argues that culturing at 20–24°C provides improved soluble expression and activity. Examiner acknowledges Applicant’s evidence. However, Figure 2 demonstrates detectable recombinant EcoCas3 expression at multiple temperatures, including 20°C, 24°C, and 28°C. The evidence therefore does not persuasively establish that the claimed range of 20–24°C constitutes a critical range producing unexpected results relative to neighboring temperature conditions. Rather, the evidence indicates that recombinant EcoCas3 expression varies as a function of cultivation temperature, consistent with temperature being a result-effective variable that may be optimized through routine experimentation. Nettleship et al. teaches recombinant protein expression in Sf9 insect cells at 27°C, while Sinkunas et al. teaches reduced-temperature recombinant Cas3 expression at 16°C. Accordingly, optimization of expression temperature to obtain desired expression characteristics would have been within the routine skill of the ordinary artisan.
For at least these reasons, Applicant’s arguments are not persuasive, and the rejections are maintained.
Conclusion
No claim is in condition for allowance.
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 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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