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and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114.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 11/10/2025 has been entered.
DETAILED ACTION
Claims 1-3, 10, 14-17, and 20 are pending. Claims 10-14, 17, and 20 are withdrawn. Claims 4-9, 11-13, 18-19, and 21-22 are cancelled. Claims 1-3 are under examination on their merits.
Response to Arguments
Applicant's arguments filed 11/10/2025 have been fully considered but they are not persuasive.
Applicant argues against the rejection of claims 1-3 under 35 U.S.C. 103 as being obvious over Panbangred in view of Zhang and Ma on the grounds that the Office Action does not establish sufficient facts to support an obviousness rejection based on an obvious-to-try rationale. Namely, 1) a finding that at the time of the invention, there had been a recognized problem or need in the art, 2) a finding that there had been a finite number of identified, predictable potential solutions to the recognized need or problem; 3) a finding that one of ordinary skill in the art could have pursued the known potential solutions with a reasonable expectation of success (Arguments, paragraph 1 on page 10).
In response, Applicant has amended claim 1 to recite “A method of increasing extracellular endoxylanase activity in a Bacillus subtilis bacterium as compared to a wild type strain comprising: administering a plasmid comprising a sequence encoding B. subtilis YwmC signal peptide, and a sequence encoding an endoxylanase from Bacillus pumilus or a variant thereof.” The broadest reasonable interpretation of claim 1 is that the Bacillus subtilis bacterium to which the plasmid is administered has increased extracellular endoxylanase activity relative to any non-engineered (wild type) strain of bacteria. However, the claim does not limit the wild type strain and thus encompasses any unmodified bacteria. The claim does not recite “compared to Bacillus subtilis bacterium without the plasmid.”
With respect to each of the points to support the obviousness rationale, note that the “obvious-to-try” rationale only extends to the signal peptide selection. The modification by Ma to replace the endoxylanase gene of Panbangred with another endoxylanase (ACA00160.1) is based on a different rationale: “One of ordinary skill in the art would have been motivated by Ma’s teaching that ACA00160.1 had high stability at pH 9.2 and 60 °C, which would have been valuable for industrial applications. One of ordinary skill in the art would have had a reasonable expectation of success given that Ma’s ACA00160.1 and Panbangred’s xynA were both xylanase genes from Bacillus pumilus“ (see the rejection under 35 U.S.C. 103 below).
With respect to supporting the obvious-to-try rationale for the signal peptide, each point is addressed as follows. Panbangred teaches the extracellular and intracellular endoxylanase activity in B. subtilis in Table 1 (page 261, right column). Although the extracellular activity is higher than the intracellular endoxylanase activity, there is still some intracellular endoxylanase activity. This is a finding that there is a recognized problem or need in the art because the person of ordinary skill in the art would have been motivated to further increase extracellular activity relative to intracellular activity. Zhang teaches a finite list of B. subtilis signal peptides (Table 1), which is a finding that there had been a finite number of identified, predictable potential solutions to the recognized need or problem. Finally, the person of ordinary skill in the art could have pursued the known potential solutions with a reasonable expectation of success given that Zhang teaches that when screening for extracellular activity of a B. pumilus endoxylanase, the majority of the signal peptides result in detectable expression of the B. pumilus endoxylanase (Figure 2). Therefore, the person of ordinary skill in the art could have pursued the known potential solutions (Zhang’s signal peptides) with a reasonable expectation of success.
Applicant argues that the number of possible endoxylanases from different bacteria combined with the number of potential signal peptides is not a finite number of identified, predictable solutions (Arguments, paragraph 2 on page 11).
In response, the “obvious-to-try” rationale applies only to the signal peptide, not the endoxylanase, as discussed above. Furthermore, Zhang’s list of signal peptides is finite.
Applicant argues that the Varman declaration highlights that the data presented in Zhang is not reliable and thus, one skilled in the art would not rely on Zhang to provide the required expectation of success for the specific combination as claimed (Arguments, paragraph 2 on page 11).
In response, the Varman declaration filed on 6/6/2025 merely questions the magnitude of Zhang’s endoxylanase activity (point 5). However, this point is insufficient to establish that one skilled in the art would not rely on Zhang. For a reasonable expectation of success, the person of ordinary skill in the art only requires knowledge that expressing many of the signal peptides in combination with an endoxylanase from B. pumilus in the host B. subtilis results in detectable levels of endoxylanase. Assurance of the absolute magnitude of endoxylanase activity is not required for the obviousness rationale.
Freudl et al. is a reference cited by the examiner previously as evidence that replacing the native signal peptide of a gene with a signal peptide selected from a small or large list of signal peptides from the host in order to enhance heterologous protein expression is also routine optimization by one of ordinary skill in the art. Applicant argues that Freudl actually teaches that it is not possible so far to predict in advance which signal peptide will perform best in the context of a given target protein and a given bacterial expression host. Applicant argues that Freudl teaches that the relative performance of the signal peptides with respect to cutinase secretion was found to differ drastically when compared between B. subtilis and C. glutamicum, indicating that the optimal fitted signal peptide for a given target protein has to be identified from scratch for each expression host. Applicant argues that Freudl also teaches that an enormous influence of the nature of the signal peptide on the final yields of the investigated target proteins has been observed (Arguments, paragraph 1, page 13).
Applicant then argues that the instant application demonstrates unexpected results (Arguments, final paragraph on page 13). The results in particular that Applicant argues are unexpected are that the strain SSL26 (comprising the YwmC signal peptide and the endoxylanase SEQ ID NO: 4) provides far more xylanase activity compared to other strains (Arguments, first paragraph page 14).
In response, each of the points raised by Applicant with respect to the teachings of Freudl serves as evidence that Applicant’s results are not unexpected. Applicant asserts that it is unexpected that strain SSL26 (corresponding to the signal peptide YwmC in combination with the endoxylanase according to SEQ ID NO: 4) provides far more xylanase activity compared to other strains. As pointed out by Applicant, Freudl teaches that an enormous influence of the nature of the signal peptide on the final yields of the investigated target proteins has been observed (Freudl page 6, right column, paragraph 3). Thus, the fact that YwmC improves the yield of endoxylanase is not surprising. Applicant also points to Freudl’s teaching that it is not possible so far to predict in advance which signal peptide will perform best in the context of a given target protein and a given bacterial expression host. Again, this knowledge is not required for the person of ordinary skill in the art to have a reasonable expectation of success in picking a signal peptide from Zhang’s list in Table 1. The person of ordinary skill in the art only requires the knowledge that at least one of the signal peptides would have produced detectable activity. Regarding Applicant’s point that Freudl teaches that the relative performance of the signal peptides with respect to cutinase secretion was found to differ drastically when compared between B. subtilis and C. glutamicum, knowledge of the relative performance of signal peptides is also not required for the person of ordinary skill in the art to have a reasonable expectation of success in picking a signal peptide from Zhang’s list in Table 1. The claimed invention does not require any magnitude of extracellular activity. The only requirement is that the extracellular activity in a Bacillus subtilis bacterium is increased as compared to a wild type strain. Although not limited to any particular wild type strain, when compared to wild type B. subtilis, which does not express SEQ ID NO: 4 (endoxylanase from B. pumilus), any combination of signal peptide with the endoxylanase that results in at least some extracellular activity of the endoxylanase meets this claim limitation.
Lastly, Applicant argues that Fig. 5A and Fig. 6 of Exhibit A (Mhatre et al., Renewable Energy 193 (2022): 288-298) corroborate the unexpected results (Arguments, paragraph 2 on page 14).
In response, Fig. 5A and Fig. 6 of Exhibit A merely demonstrate the application of the recombinant B. subtilis strain in breaking down xylan to xylose. This result is due to the enhanced extracellular endoxylanase activity of the strain. As explained above, the enhanced extracellular endoxylanase activity is not surprising given that Freudl teaches that an enormous influence of the nature of the signal peptide on the final yields of the investigated target proteins has been observed (page 6, right column, paragraph 3).
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 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 are rejected under 35 U.S.C. 103 as being unpatentable over Panbangred (Applied microbiology and biotechnology 22 (1985): 259-264; cited in the Non-Final Action mailed on 3/7/2025) in view of Zhang et al. (Applied Microbiology and Biotechnology 100 (2016): 8745-8756; cited in the Non-Final Action mailed on 3/7/2025) and Ma et al. (CN104928270A; machine translation; cited in the Non-Final Action mailed on 3/7/2025), as evidenced by Kyoto Encyclopedia of Genes and Genomes (2025, website; hereafter KEGG; cited in the Non-Final Action mailed on 3/7/2025).
In claim 1, the limitation “administering a plasmid” is interpreted as transforming a Bacillus subtilis bacterium with a plasmid.
Panbangred teaches expressing xynA encoding a 1,4-β-D xylan xylanohydrolase in Bacillus subtilis by administering a plasmid comprising the xylanase gene xynA from Bacillus pumilus (Summary paragraphs 1 and 3, Introduction paragraph 1). XynA encodes a 1,4-β-D-xylan xylanohydrolase (Introduction, paragraph 1, right column), which is an endoxylanase, as evidenced by KEGG (see Name section). Panbangred includes flanking regions of xynA in the plasmid (Summary, paragraph 1). Panbangred teaches the extracellular and intracellular endoxylanase activity in B. subtilis in Table 1 (page 261, right column). Although the extracellular activity is higher than the intracellular endoxylanase activity, there is still some intracellular endoxylanase activity.
Panbangred does not teach that the plasmid further comprises a sequence encoding a signal peptide, wherein the sequence encoding the signal peptide is upstream of the sequence encoding the endoxylanase, nor does Panbangred teach that administering the plasmid increases secretion of the endoxylanase, thereby increasing extracellular endoxylanase activity of the bacterium relative to wild type.
Zhang teaches testing the effect of a finite number of different Bacillus subtilis signal peptides on the secretion of a B. pumilus xylanase expressed in a B. subtilis host (page 8746, left column, bottom paragraph and page 8750, left column, paragraph 1, lines 1-2). Zhang teaches a plasmid encoding the B. pumilus xylanase xynBYG with a signal peptide upstream of xynBYG (Fig. 1). Zhang teaches that the signal peptide is selected from Table 1 (page 8750, left column, paragraph 1 and Table 1 caption), which includes YwmC (number 79). Zhang’s YwmC is identical to SEQ ID NO: 3 (OA Appendix D of the Non-Final Action mailed on 3/7/2025).
Zhang also teaches that specific xylanases are needed for different industries. For example, in pulp bleaching, the xylanase is required to be thermophilic and alkali stable (page 8746, left column, top paragraph).
It would have been obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to try replacing the native signal peptide (included in the flanking regions of xynA) in the plasmid of Panbangred with any of the finite number of heterologous signal peptides disclosed by Zhang, including YwmC. One of ordinary skill in the art would have been motivated to replace the native signal peptide with a heterologous signal peptide in order to improve secretion of the endoxylanase (i.e. increase the extracellular endoxylanase activity relative to the intracellular endoxylanase activity). One of ordinary skill in the art would have had a reasonable expectation of success given that the majority of Zhang’s signal peptides, including YwmC, enhance secretion of xynBYG (see Zhang Table 1, Xylanase activity) relative to wild-type B. subtilis without the plasmid. A wild-type B. subtilis without the plasmid necessarily has no B. pumilus endoxylanase activity.
Neither Panbangred nor Zhang teach a plasmid comprising a sequence encoding an endoxylanase with an amino acid sequence having at least 90% identity to SEQ ID NO: 4.
Ma teaches a xylanase gene xynA from Bacillus pumilus with NCBI accession ACA00160.1 that is 98.25% identical to SEQ ID NO: 4 ([0011] on page 5, [0007] on page 4, and OA Appendix F of the Non-Final Action mailed on 3/7/2025). Ma teaches that xynA has high stability at pH 9.2 and 60°C (last line on page 4 through top line on page 5).
It would have been obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to replace xynA in the plasmid of Panbangred modified by Zhang with Ma’s ACA00160.1, which is 98.25% identical to the instant SEQ ID NO: 4. One of ordinary skill in the art would have been motivated by Ma’s teaching that ACA00160.1 has high stability at pH 9.2 and 60 °C, which would have been valuable for industrial applications, as suggested by Zhang (page 8746, left column, top paragraph). One of ordinary skill in the art would have had a reasonable expectation of success given that Ma’s ACA00160.1 and Panbangred’s xynA are both xylanase genes from Bacillus pumilus. The value of 98.25% sequence identity to SEQ ID NO: 4 is within the claimed range of at least 90% sequence identity to SEQ ID NO: 4 (claim 1) as well as within the claimed range of at least 98% sequence identity to SEQ ID NO: 4 (claim 2).
Regarding claim 3, Zhang’s YwmC is identical to SEQ ID NO: 3 (OA Appendix D of the Non-Final Action mailed on 3/7/2025).
Conclusion
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/LOUISE W HUMPHREY/Supervisory Patent Examiner, Art Unit 1657
/CANDICE LEE SWIFT/Examiner, Art Unit 1657