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 .
Election/Restrictions
Applicant’s election of Group I, claims 1-5, in the reply filed on 12/30/2025 is acknowledged. Because applicant did not distinctly and specifically point out the supposed errors in the restriction requirement, the election has been treated as an election without traverse (MPEP § 818.01(a)).
In response to species election, applicant’s election is shown as follows, reading on claims 1-5.
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Claims 6-20 withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected invention, there being no allowable generic or linking claim. Election was made without traverse in the reply filed on 12/30/2025.
Claim Status
Claims 1-20 are pending.
Claims 6-20 are withdrawn as being directed to a non-elected invention, the election having been made on 12/30/2025.
Claims 1-5 have been examined.
Priority
This application has PRO 63/329,329 04/08/2022.
Information Disclosure Statement
The information disclosure statement (IDS) submitted on 2/22/2024 is in compliance with the provisions of 37 CFR 1.97. Accordingly, the information disclosure statement has been considered by the examiner.
Claim Objections
Claims 4-5 are objected to because of the following informalities:
The word “encodes” at line 2 in claim 4 is commonly used for a nucleic acid sequence “encoding” a gene, which is translated into a polypeptide. The examiner suggests replacement of the word “encodes” by the word “comprises” in claim 4.
The term “PTDH” in claim 5 should be spelled out as “phosphite dehydrogenase”.
Appropriate correction is required.
Claim Rejections - 35 USC § 112
The following is a quotation of the first paragraph of 35 U.S.C. 112(a):
(a) IN GENERAL.—The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor or joint inventor of carrying out the invention.
The following is a quotation of the first paragraph of pre-AIA 35 U.S.C. 112:
The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor of carrying out his invention.
Claims 1-4 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, as failing to comply with the written description requirement. The claim(s) contains subject matter which was not described in the specification in such a way as to reasonably convey to one skilled in the relevant art that the inventor or a joint inventor, or for applications subject to pre-AIA 35 U.S.C. 112, the inventor(s), at the time the application was filed, had possession of the claimed invention for the reasons as follows.
Claims 1-3 recite polypeptide with only functional languages. The analysis as follows is used to address if the claims satisfy written description requirements.
actual reduction to practice
Applicant disclosed SEQ ID Nos: 1-6 are highly homologous peptide sequences and satisfy actual reduction to practice. Furthermore, the specification disclosed screening a polypeptide library is required to identify the claimed polypeptides [0022, 0094-0095]; thus, the limited disclosure are insufficient to represent the entire genus of the known or unknown polypeptide sequences defined by the functional limitation in claims.
disclosure of drawings or structural chemical formulas:
Claims 1-3 recite polypeptide with only functional language in general. Figures 3-7 suggest a protein folding structure may be used to facilitate mutant polypeptide design before screening a polypeptide library. Fig. 6A shows results of screening a polypeptide library are
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unpredictable based on amino acid substitution in the mutant polypeptides. Furthermore, the disclosed method of screening a polypeptide library comprising amino acid substitutions does not translate to sufficient disclosure of the polypeptide sequences with 1-12 amino acid, comprise insertion, deletion, and/or substitution, compared to a reference polypeptide (a wild-type or parent polypeptide or a thermostable variant) in the claims.
(c) sufficient relevant identifying characteristics in the way of complete/partial structure or physical and/or chemical properties or functional characteristics when coupled with known or disclosed correlation with structure.
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Figures 3-7 suggest a protein folding structure may be used to facilitate mutant polypeptide design for screening a polypeptide library. A prior art, Zhao et al. (WO 2006/074194 A2), teaches the use of error-prone (EP) PCR for site-directed mutagenesis (SDM) to identify mutant with improve or desired function [00050, Fig 20] based on phosphite dehydrogenase (PTDH) pathway shown in figure 18 as follows. Zhao’s recombinant peptide SEQ ID NO: 35 (Fig 31, claim 7) is NOT 100% identity to the instant SEQ ID NO: 2, but reads on the limitations of certain claims.
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The other reference, Black et al. (Nat Chem Biol. 2020 January; 16(1): 87–94, cited in IDS), teach the use of protein structure of glucose dehydrogenase.to facilitate design a mutant polypeptide. However, a bioassay is required to prove the concept of polypeptide design due to the unpredictability of protein design according to Black’s figure (p21). Unfortunately, Black et al. dis not show the mutant glucose dehydrogenase polypeptide sequence. Thus, the prior art references known in the art are insufficient to establish correlation of structure and function to support the entire genus of the claimed polypeptide sequences as claimed due to the unpredictability of polypeptide design and screening a polypeptide library.
representative number of samples.
The limited disclosure of SEQ ID Nos of SEQ ID NO: 3-6 are highly homologous peptide sequence and insufficient to represent 1-12 amino acid mutations comprising insertion, deletion, and substitution in a reference polypeptide (a wild-type or parent polypeptide or a thermostable variant) for the entire genus of the polypeptides as claimed.
With respect to claim 4, the claim merely disclosed the reference polypeptide sequences before making the 1-12 mutations comprising insertion, deletion, and/or substitution in the reference polypeptide. As neither protein structure prediction nor screening a library is able to show the correlation between structure and function for entire genus of polypeptides as claimed, claim 4 also fails to satisfy written description requirement.
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 1-2 and 4 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 1-2 are unclear because both the mutant catalytic polypeptide sequences and an oxidized form of a noncanonical cofactor are distinct variables and applicant is using a functional limitation to define both variables simultaneous. Although the specification discloses the mutant catalytic polypeptides of SEQ ID NO:s 1-8 are dehydrogenase or oxidoreductase, but it is improper to import claim limitations from the specification. See MPEP 2111.01(II). Similarly, the oxidized form of a noncanonical cofactor is not distinctly claimed even though the specification has disclosed noncanonical cofactor compounds. However, it is improper to import claim limitations from the specification.
With respect to claim 4, it is unclear with respect to the phrase “the wild-type or parent polypeptide, or a thermostable variant thereof encodes a dehydrogenase or an oxidoreductase”. The specification disclosed a nucleic acid sequence which encodes an amino acid [0054] and a parent polynucleotide or gene encodes for a parent protein or enzyme [0057]. Claim 12 discloses genes that encode proteins. Since neither the specification not the claims explicitly define how a polypeptide encodes a protein or an enzyme, rendering the phrase in claim 4 indefinite.
The rejection may be overcome by distinctly claiming the mutant polypeptide sequence with SEQ ID Nos to specify the mutation sites for its oxidized noncanonical cofactor. Merely functional limitation of a mutant polypeptide is insufficient to define the mutation sites in the mutant polypeptide sequence or a particular oxidized noncanonical cofactor catalyzed by the mutant polypeptide in claims because it is unclear the correlation between mutation sites of a catalytic polypeptide and the function of increasing catalytic efficiency for the oxidized form of a noncanonical cofactor.
Claim Rejections - 35 USC § 102
The following is a quotation of the appropriate paragraphs of 35 U.S.C. 102 that form the basis for the rejections under this section made in this Office action:
A person shall be entitled to a patent unless –
(a)(1) the claimed invention was patented, described in a printed publication, or in public use, on sale, or otherwise available to the public before the effective filing date of the claimed invention.
Claims 1-5 are rejected under 35 U.S.C. 102(a)(1) as being anticipated by Zhao et al. (WO 2006/074194 A2).
Claim 1 is drawn to a recombinant polypeptide with improved catalytic activity to generate a reduced noncanonical cofactor from its oxidized form as follows
A recombinantly engineered polypeptide that has improved catalytic efficiency for an oxidized form of a noncanonical cofactor, wherein the recombinantly engineered polypeptide comprises from 1 to 12 amino acid mutations in comparison to the sequence of the wild-type or parent polypeptide, or a thermostable variant thereof;
wherein the amino acid mutations promote catalytic efficiency of the recombinantly engineered polypeptide in reducing the oxidized form of the noncanonical cofactor and/or disrupt electrostatic complementarity between the recombinantly engineered polypeptide and a natural cofactor, and wherein a polypeptide having the sequence of the wild-type or parent polypeptide, or a thermostable variant thereof, has minimal to very limited catalytic efficiency for the oxidized form of the noncanonical cofactor;
wherein the recombinantly engineered polypeptide is configured to in vivo recycle the nonconical cofactor when used with a second recombinantly engineered polypeptide that utilizes the reduced form of noncanonical cofactor for cell growth.
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Zhao et al. teach phosphite dehydrogenase mutant enzymes for nicotinamide cofactor regeneration (NADH and NADPH [0008, 00018]) having relaxed cofactor specificity, increased thermostability, increased activity, solubility, and expression over the wild-type enzyme [Abstract, 0009]. Zhao et al. show a recombinant peptide SEQ ID NO: 35 (Fig 31).
With respect to the limitation (a), A peptide sequence alignment of Zhao’s SEQ ID NO: 35 (Art) compared to the elected thermostable variant SEQ ID NO: 2 shows a single amino acid mismatch mutation as follows.
With respect to the limitation (b), Zhao et al. teach the recombinant phosphite dehydrogenase mutant enzymes able to produce NADPH (reduced form) from NADP+(oxidized form) and NADH from NAD+. HPLC data indicated no detectable remaining oxidized cofactor after reaction [00800], demonstrating mutations promote catalytic efficiency of the recombinantly engineered polypeptide in reducing the oxidized form of the noncanonical cofactor with very limited catalytic efficiency for the oxidized form of the noncanonical cofactor.
With respect to the limitation (c), Zhao et al. teach the Phosphite dehydrogenases disclosed herein are also useful to regenerate NADH for in vivo biocatalytic processes requiring it as a reducing equivalent, reading on configured to in vivo recycle the nonconical cofactor. It is further noted that the limitation “when used with a second recombinantly engineered polypeptide that utilizes the reduced form of noncanonical cofactor for cell growth” does not change the structure or function of Zhao’s recombinant peptide SEQ ID NO: 35.
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With respect to claim 2, Zhao et al. teach mutant enzymes exhibited improved catalytic efficiency (e.g., El75A; Al76R) having a 4-fold higher catalytic efficiency for NAD+ and an 1000-fold higher efficiency for NADP+. The cofactor specificity was changed from 100-fold in favor of NAD+ for the wild-type enzyme [00074].
With respect to claims 3 and 5, Zhao’s SEQ ID NO: 36 compared to the elected species of SEQ ID NO: 6 with 99% identity as follows. Because Zhao’s SEQ ID NO: 36 satisfies the limitation of claim 5 including the elected species SEQ ID NO: 6, Zhao’s SEQ ID NO: 36 is necessary to satisfy a noncanonical cofactor in claim 3 including the elected species of nicotinamide mononucleotide. MPEP 2112.01 (I) states “Product and apparatus claims - When the structure recited in the reference is substantially identical to that of the claims, claimed properties or functions are presumed to be inherent”.
With respect to claim 4, Zhao et al. teach the recombinant peptide SEQ ID NO: 35 as a phosphite dehydrogenase mutant providing relaxed cofactor specificity, increased thermostability, increased activity, solubility, and expression over the wild-type enzyme (Abstract).
Claims 1-4 are rejected under 35 U.S.C. 102(a)(1) as being anticipated by Black et al. (Nat Chem Biol. 2020 January; 16(1): 87–94, cited in IDS).
A person shall be entitled to a patent unless –
(a)(1) the claimed invention was patented, described in a printed publication, or in public use, on sale, or otherwise available to the public before the effective filing date of the claimed invention.
Claim 1 is drawn to a recombinant polypeptide with improved catalytic activity to generate a reduced noncanonical cofactor from its oxidized form as follows
A recombinantly engineered polypeptide that has improved catalytic efficiency for an oxidized form of a noncanonical cofactor, wherein the recombinantly engineered polypeptide comprises from 1 to 12 amino acid mutations in comparison to the sequence of the wild-type or parent polypeptide, or a thermostable variant thereof;
wherein the amino acid mutations promote catalytic efficiency of the recombinantly engineered polypeptide in reducing the oxidized form of the noncanonical cofactor and/or disrupt electrostatic complementarity between the recombinantly engineered polypeptide and a natural cofactor, and wherein a polypeptide having the sequence of the wild-type or parent polypeptide, or a thermostable variant thereof, has minimal to very limited catalytic efficiency for the oxidized form of the noncanonical cofactor;
wherein the recombinantly engineered polypeptide is configured to in vivo recycle the nonconical cofactor when used with a second recombinantly engineered polypeptide that utilizes the reduced form of noncanonical cofactor for cell growth.
Black et al. teach “Engineering a nicotinamide mononucleotide redox cofactor system for biocatalysis” (Title). Black et al. teach the key enzyme in the system is a computationally designed glucose dehydrogenase with a 107-fold cofactor specificity switch toward NMN+ over NADP+ based on apparent enzymatic activity (Abstract).
With respect to the limitation (a), Black et al. show the mutant enzymes comprising 3 or 4 point mutations and mutant enzymes have much faster catalyst rate shown as Kcat/Km (p26, Table 1).
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With respect to the limitation (b), Black et al. show the mutant enzymes in reducing the oxidized form of the noncanonical cofactor substrate of nicotinamide mononucleotide, NMN+, (p27, Table 2).
With respect to the limitation (c), Black et al. show XenA was present to recycle NMNH (p23, Fig 3).
With respect to claim 2, Black et al. teach the triple mutant (I195R-A93K-Y39Q, Bs GDH Triple) exhibits a kcat/Km of ~0.51 mM−1 s−1, which corresponds to a 1,000-fold increase in catalytic efficiency over the wild-type enzyme toward NMN+ (p4, para 1).
With respect to claim 3, Black et al. teach the noncanonical cofactor nicotinamide mononucleotide, NMN, (p3, para 1).
With respect to claim 4, Black et al. teach the mutant enzymes are glucose dehydrogenase (p9, last para).
Additional Reference
Beyer et al. P450BM3 fused to phosphite dehydrogenase allows phosphite-driven selective oxidations. Appl Microbiol Biotechnol (2017) 101:2319–2331.
GenBank: API65466.1 PTDH-P450BM3 fusion construct reading on several other peptide sequences in claim 5.
CN 104946706A
Conclusion
No claim is allowed.
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/J.L/Examiner, Art Unit 1658
06-March-2026
/LI N KOMATSU/ Primary Examiner, Art Unit 1658