DETAILED ACTION
Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Priority
Acknowledgment is made of applicant’s claim for foreign priority under 35 U.S.C. 119 (a)-(d) based on TW111107819, filed on 0303/2022. Receipt is acknowledged of certified copies of papers required by 37 CFR 1.55.
Status of the Claims
Applicant’s amendment filed on 06/09/2025 is acknowledged.
Claim 1 is amended. Claims 2-11 and 15 are cancelled. Claims 1, 12-14 and 16-20 are pending. Claims 14 and 16-20 are withdrawn.
Claims 1, 12 and 13 (claims set filed 06/09/2025) and are examined on the merits herein.
Withdrawal of Rejections
The response and amendment filed on 06/09/2025 are acknowledged. All of the amendment and arguments have been thoroughly reviewed and considered.
For the purposes of clarity of the record, the reasons for the Examiner's withdrawal and/or maintaining if applicable, of the substantive or essential claim rejections are detailed directly below and/or in the Examiner's response to arguments section.
The previous claims 1, 5-9 and 11-13 rejection under 35 U.S.C. 112(b) has been withdrawn necessitated by amendment of claim 1 and cancellation of claims 5-9 and 11.
The previous claims 1, 2, 5, 12 and 13 rejection under 35 U.S.C. 112(a) has been withdrawn necessitated by amendment of claim 1 and cancellation of claims 2 and 5.
The previous claim 8 rejection under 35 U.S.C. 112(d) has been withdrawn necessitated by cancellation of claim 8.
Claim Rejections - 35 USC § 112
The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
Claims 1, 12 and 13 are rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
Claim 1 recites in lines 21-28: “ … the amino acid residue 402 of the mutant FMO is formed by mutating valine residue 402 of the wild-type FMO to an arginine or a methionine residue, the amino acid residue 185 of the mutant FMO is formed by mutating methionine residue 185 of the wild-type FMO to a leucine residue, and the amino acid residue 402 of the mutant FMO is formed by mutating valine residue 402 of the wild-type FMO to an alanine residue, or the amino acid residue 185 of the mutant FMO is formed by mutating methionine residue 185 of the wild-type FMO to a valine residue, and the amino acid residue 402 of the mutant FMO is formed by mutating valine residue 402 of the wild-type FMO to a methionine residue”.
It is not clear what the alternatives are. It is not clear if alternatives are: (i) V402R or V402M, M185L and V402A and (ii) M185V and V402M or alternatives are: (i) V402R or V402M; (ii) double mutant M185L/V402A; or (iii) double mutant M185V/V402M. The scope and boundaries of claim 1 are not certain making claim 1 indefinite. The examiner suggests clearly defining the alternatives using Markush language, i.e. from the group consisting of.
Claims 12 and 13 do not solve the issues mentioned above and are rejected.
Claim 1 is interpreted as including limitation for mutant FMO as alternatives: : (i) V402R or V402M; (ii) double mutant M185L/V402A; or (iii) double mutant M185V/V402M.
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 13 are rejected under 35 U.S.C. 103 as being unpatentable over Jung (Jung et al. J. Life Sciences, 2018, 28, 656-662) in view of Loncar (Loncar et al. Int. J. Mol. Sciences, 2019, 20, 6148), Ameria (Ameria et al. Biotechnol. Lett., 2015, 37, 1637-1644), Choi (Choi et al. Biochem. Biophys. Res. Commun., 2003, 306, 930-936) and Han (Han et al. J. Biotechnol., 2012, 164, 179-187).
It should be noted that amendment of claim 1 reintroduced limitation of mutant FMO with valine residue 402 of the wild-type FMO mutated to an arginine which was covered by the prior art as described in the Office Action of 09/13/2024.
Regarding claim 1, Jung teaches protein engineering of flavin-containing monooxygenase from Corynebacterium glutamicum (cFMO) for improved production of indigo and indirubin (Title). Jung discloses mutagenesis of wild-type cFMO to enhance indigoid production: “Flavin-containing monooxygenases from Corynebacterium (cFMOs) were mutagenized based on homolog modeling to develop variants with an enhanced indigoid production capability.” (Abstract). Jung describes expression of mutant cFMOs in Escherichia coli in the presence of tryptophan that results in increased production of indigoid compound including indirubin: “When indigoid production was carried out in LB broth with 2.5 g/l of tryptophan, Escherichia coli expressing cFMO produced 684 mg/l of indigo and 104 mg/l of indirubin, while cells harboring T326S produced 1,040 mg/l of indigo and 112 mg/l of indirubin.” (Abstract). Jung teaches mutant cFMOs containing single mutation site: “The four mutants, F170Y, A210G, A210S, and T326S, which fused to a maltose-binding protein (MBP), were constructed, and their biochemical properties were characterized.” (Abstract).
Jung teaches mutating three amino acid residues from cFMO, i.e. F170, A210 and T326 to increase indigoid production by cFMO. The residues are selected based on homology modeling with flavin-containing monooxygenase from Methylophaga aminisulfidivorans (mFMO). mFMO was shown previously to produce indigoid compounds in bacterial cells: “… high titers of indigo and indirubin were achieved by recombinant cells expressing bacterial FMOs from M. aminisulfidivorans and optimization of culture media” (p. 656, right column). The modeling is based on known crystal structure of mFMO and 56% sequence identity between cFMO and mFMO: “… we could build a reliable 3D structure model by homology modeling because its sequence has a quite high identity with mFMO (56%), of which structure data were published (PDB ID: 2XVE)” (p. 658, right column, 1st paragraph). The selected residues were found to be located close to the active site of mFMO: “… three residues which are located closely enough to cofactors and substrates at the catalytic site were chosen” (p. 658, right column, 1st paragraph). Two of these residues were substituted with the corresponding residues of mFMO, i.e. in A210S and T326S mutants. Both mutant cFMOs showed increase in indole oxygenase activity and mutant T326S cFMO demonstrated significant increase in indigoid production (Abstract).
Jung does not teach mutation of cFMO on amino acid residue 185 or 402 and does not teach bacterial culture to comprise cysteine.
Loncar teaches structure-based redesign of mFMO to increase indigo production (Title). Loncar discloses several amino acid residues in the active site of mFMO based on crystal structure of mFMO: “The residues that form the entrance and shape of this cavity are highlighted in Figure 1 and listed in Table 1).” (p. 2, last paragraph, Figure 1, Table 1). One of these residues is Trp400 that corresponds to Trp401 of cFMO in instant application based of sequence alignment.
Ameria teaches characterization of cFMO and its application to indigoid compounds production (Title). Ameria presents results of sequence alignment of cFMO with FMO from different sources, including mFMO (Fig. 3). The Trp residue in the active site of mFMO, i.e. Trp400 in Loncar teaching equal to Trp405 in Ameria teaching is conserved between FMO from different sources. The residue next to the conserved Trp is a positively charged Lys residue in mFMO sequence as well as in another two bacterial FMO sequences. At the same time C. glutamicum FMO sequence has Val in this position that corresponds to Val402 of instant application.
Choi teaches and indigo synthesis in Escherichia coli by mFMO. Choi presents sequence alignment of mFMO with several eukaryotic FMOs (Fig. 2). From these eukaryotic sequences sequence of Arabidopsis thaliana has conserved Trp residue and positively charged residue, Arg, next to it, i.e. Arg359 (Fig. 2). Even though sequences of human and porcine FMO do not have conserved tryptophan, they do have neighboring homologous positively charged amino acid residue, Lys for human and His for porcine FMO sequences.
Hwan teaches enhanced production of indirubin in recombinant Escherichia coli harboring a flavin-containing monooxygenase from Methylophaga aminisulfidivorans by cysteine supplementation (Abstract). Hwan describes that production of indirubin was greatly increased reaching 223.6 mg/L when cysteine was added to culture medium containing tryptophan from (Abstract). Hwan mentions that indirubin plays important role in the treatments of leukemia and Alzheimer disease (p. 179, left column, 1st paragraph).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify Jung teaching on method to increase indigoid compound production by cFMO by mutating amino acid residues in the active site of cFMO based on guidance provided by Loncar, Ameria and Choi and substitute Val402 in cFMO of instant application with positively charged residue, Lys or Arg. One would have been motivated to do so since (i) Val402 is close to the active site of FMO based on Loncar teaching of position of neighboring conserved Trp in the active site of mFMO and (ii) sequence alignment in Ameria and Choi teachings of FMOs from several procaryotic and eucaryotic species, including mFMO shown to produce indigoid compounds by Ameria, demonstrated positively charged residue, Lys or Arg, in position of Val402 of instant cFMO. A skilled artisan would have reasonably expected success in the modification because Jung described method of indigoid production by mutant FMO expressed in Escherichia coli, including two successful substitutions of two cFMO residues with mFMO residues, Loncar provided guidance to the active site residues of FMO and America and Choi provided sequence alignments of cFMO with FMO from different sources showing positively charged residues in Val402 position and all the references are teaching FMOs producing indigoid compounds.
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to add cysteine as described by Han to the culture medium for production of indigoid compound by mutant FMO based on prior art of Jung, Loncar, Ameria and Choi. One would have been motivated to do so since Han showed increase in production of indirubin by cysteine supplementation and indicated important role of indirubin in the treatments of leukemia and Alzheimer disease. A skilled artisan would have reasonably expected success in the combination because Jung, Ameria, Choi and Han teach production of indigoid compounds including indirubin by FMO expressed in Escherichia coli.
Thus, teachings of Jung, Loncar, Ameria, Choi and Han render claim 1 obvious.
Regarding claim 13, Jung teaches production of indigo and indirubin in Escherichia coli expressing mutant cFMO for 48h: “Production of indigo and indirubin was performed in LB medium containing 2.5 g/l of tryptophan with IPTG induction in a shaking incubator at 32℃ for 48 hr using recombinant E. coli W3110 expressing mutant cFMOs.” (p. 658, left column, last paragraph). Thus, Jung teaching in combination with Loncar, Ameria, Choi and Han teachings renders claim 13 obvious.
Claim 12 is rejected under 35 U.S.C. 103 as being unpatentable over Jung (Jung et al. J. Life Sciences, 2018, 28, 656-662) in view of Loncar (Loncar et al. Int. J. Mol. Sciences, 2019, 20, 6148), Ameria (Ameria et al. Biotechnol. Lett., 2015, 37, 1637-1644), Choi (Choi et al. Biochem. Biophys. Res. Commun., 2003, 306, 930-936) and Han (Han et al. J. Biotechnol., 2012, 164, 179-187) as applied to claim 1 above, and further in view of Bocola (WO 2020126625 A1).
The teaching of Jung, Loncar, Ameria, Choi and Han has been set forth above.
Jung, Loncar, Ameria, Choi and Han do not teach Escherichia coli XL-1 blue for expression of mutant FMO.
Bocola teaches cloning and expression of P450 monooxygenase in bacterial cells: “… a nucleic acid encoding a P450 monooxygenase enzyme of the invention can be introduced into a suitable host cell(s) to produce the respective protein by recombinant means. ” (p. 25, lines 25-27). Bocola mentions a variety of Escherichia coli strains which can be used as host cells for P450 monooxygenase expression, including XL-1 Blue strain: “A variety of E.coli bacterial host cells are known to a person skilled in the art and include, but are not limited to strains such as BL21 (DE3), DH5-alpha, HB101 , MV1190, JM109, JM101 , or XL-1 blue…” (p. 25, lines 17-19).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to combine the teachings of Jung, Loncar, Ameria, Choi and Han on the method for increased indigoid compound production by mutant cFMO and teaching of Bocola and try several Escherichia coli strains for expression of mutant cFMO. One would have been motivated to select Escherichia coli strain with highest efficiency in recombinant protein production depending on application and conditions used. A skilled artisan would have reasonably expected success in the combination because Jung described method of indigoid production by mutant FMO expressed in Escherichia coli, Han described method to increase indirubin production by cysteine supplementation, Loncar, Ameria and Choi provided guidance on mutation of cFMO and Bocola described several strains of Escherichia coli suitable for expression of another monooxygenase. Thus, Bocola teaching in combination with Jung, Loncar, Ameria, Choi and Han teachings renders claim 12 obvious.
Response to Arguments
Applicant’s arguments, see p. 1-2 of the Remarks filed 06/09/2025 with respect to 35 U.S.C. 112(a) enablement rejection have been fully considered and are persuasive. The 35 U.S.C. 112(b) of claims 1, 2, 5, 12 and 13 rejection has been withdrawn necessitated by amendment of claim 1.
The 35 U.S.C. 103 rejection was applied necessitated by amendment of claim 1 that introduced limitation of V402R mutant FMO which is rendered obvious by the prior art as described in the rejection above.
Conclusion
No claims are allowed.
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 LIOUBOV G KOROTCHKINA whose telephone number is (571)270-0911. The examiner can normally be reached Monday-Friday: 8:00-5:30.
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If attempts to reach the examiner by telephone are unsuccessful, the examiner’s supervisor, Sharmila G Landau can be reached at (571)272-0614. The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300.
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/L.G.K./ Examiner, Art Unit 1653
/SHARMILA G LANDAU/ Supervisory Patent Examiner, Art Unit 1653