DETAILED ACTION
Applicant’s amendment and response received on 6/10/26. Claims 330-333 and 345 have been canceled. Claims 329, 334-344, and 346-348 are currently pending and under examination in the instant application. An action on the merits follows.
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA . Those sections of Title 35, US code, not included in this action can be found in previous office action.
Sequence Disclosures
Applicant’s submission of a sequence listing on 6/10/26 is acknowledged. While applicant as added the sequence recited in claim 335 to the sequence listing and amended claim 335 to include the appropriate SEQ ID NO for this sequence. However, it has been determined that this application continues to contain sequence disclosures that are encompassed by the definitions for nucleotide and/or amino acid sequences set forth in 37 CFR 1.821(a)(1) and (a)(2); however, this application fails to comply with the requirements of 37 CFR 1.821 through 1.825 for the reason(s) set forth on the Notice To Comply With Requirements For Patent Applications Containing Nucleotide Sequence And/Or Amino Acid Sequence Disclosures which is attached to this communication. Figure 14 provides a comparison of several amino acid sequences which meet the definitions for amino acid sequence set forth in 37 CF 1.821()(1) and (a)(2). Neither the sequences in Figure 14, nor the brief description of Figure 14 in the instant specification identifies these sequences by SEQ ID NOS. If these amino acid sequences are present within the latest filed sequence listing and have been accorded SEQ ID NOS, compliance may be achieved by amending either Figure 14, or the brief description of Figure 14 in the specification to include the appropriate SEQ ID NOS for these sequences. If the latest filed sequence listing does not contain these sequences, a new sequence listing in paper form and CRF, along with the required statement, must be filed-see the attached PTO-2301 for details.
APPLICANT IS GIVEN A THREE MONTH EXTENDABLE PERIOD WITHIN WHICH TO COMPLY WITH THE SEQUENCE RULES, 37 CFR 1.821-1.825. Failure to comply with these requirements will result in ABANDONMENT of this application under 37 CFR 1.821 (g). Extension of time may be obtained by filing a petition accompanied by the extension fee under the provisions of 37 CFR 1.136. In no case may an applicant extend the period for response beyond the six-month statutory period. Applicant is requested to return a copy of the attached Notice To Comply with the response.
Claim Rejections - 35 USC § 101
The rejection of previously pending claims 329-334, 340-342, and 346-348 under 35 U.S.C. 101 because the claimed invention is directed to a judicial exception which is a product of nature without significantly more, is withdrawn in view of applicant’s cancellation of the claims or amendments to the claims which now recite a water forming NADH oxidase polypeptide with five specific amino acids at five specific positions relative to SEQ ID NO:1. The evidence of record does not support that naturally occurring water forming NADH oxidases or naturally occurring water forming NADPH oxidases comprise all five of these specific amino acids at the required positions relative to SEQ ID NO:1.
Claim Rejections - 35 USC § 102
The rejection of previously pending claims 329-332, 334, and 340-343 under 35 U.S.C. 102(a)(1) as being anticipated by Petschacher et al. (2014) Computational and Structural Biotechnology Journal, Vol. 9(14),e201402005, https://doi.org/10.5936/csbj.201402005, pages 1-11, is withdrawn in view of applicant’s cancellation of the claims or amendments to the claims which now recite a water forming NADH oxidase polypeptide with five specific amino acids at five specific positions relative to SEQ ID NO:1
Claim Rejections - 35 USC § 103
The rejection of claims 331-332 under 35 U.S.C. 103 as being unpatentable over Petschacher et al. (2014) Computational and Structural Biotechnology Journal, Vol. 9(14),e201402005, https://doi.org/10.5936/csbj.201402005, pages 1-11, is withdrawn in view of applicant’s cancellation of the claims or amendments to the claims which now recite a water forming NADH oxidase polypeptide with five specific amino acids at five specific positions relative to SEQ ID NO:1.
Claim Rejections - 35 USC § 112
The rejection of previously pending claims 329-348 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, is withdrawn over canceled claims 330-333 and 345, and maintained over amended claims 329, 334-344, and 346-348. Applicant’s amendments to the claims and arguments have been fully considered but have not been found persuasive in overcoming the rejection for reasons of record as discussed in detail below.
The applicant argues that the specification provides sufficient description for a water forming NADH comprising the 5 specific substitutions recited in the claims. The applicant argues that the written description requirement has been met as water forming NADH oxidases were known in the prior art and the specification further teaches over 200 examples of water forming NADH oxidases (SEQ ID NOS:1-273). In addition, the applicant argues that the claims concern the use of a known genus as part of a different invention which is the generation of polypeptides that exhibit selective water-forming NADPH oxidase activity and that therefore the well known genus need not be further described in the specification, citing the Hybritech decision and the Teva Pharmaceuticals decision – see pages 7-8 of applicant’s response for the full citations of these cases. According to applicant, in these circumstances where the genus is not the invention, a single representative species may demonstrate possession.
In response, applicant’s claims as amended recite a genus of polypeptides comprising an amino acid sequence of a water-forming NADH oxidase comprising 5 specific amino acid substitutions corresponding to 5 specific positions of SEQ ID NO:1, where the polypeptide exhibits water-forming NADPH oxidase activity. Note that the polypeptide is not limited to mutants of SEQ ID NO:1 which only differ by the 5 specific mutations, or to polypeptides with any homology to SEQ ID NO:1 and which further exhibits water-forming NADPH oxidase activity. Thus, the genus of polypeptides as currently claimed continues to encompass an extremely large number of polypeptides which are not limited to polypeptides based on any known polypeptide sequence or even any known sequence of a water-forming NADH oxidase.
Applicant’s arguments that the claimed genus of polypeptides which have the 5 recited mutations at positions relative to SEQ ID NO:1 and which have the functional activity of exhibiting water-forming NADPH oxidase activity only requires the disclosure of 1 species because the genus of water-forming NADH oxidases were known is not persuasive. Contrary to applicant’s arguments, the instant written description rejection is based on the lack of written description of a genus of water-forming NADH oxidase polypeptide sequences which have acquired water-forming NADPH oxidase activity and which have the five specific substitutions at the identified positions which correspond to particular positions in SEQ ID NO:1 based on sequence alignment. This genus is not a known genus and constitutes the actual invention claimed. The claims are not method claims which seek to use a known genus to generate a genus of modified polypeptides. The instant claims are to a genus of polypeptides with water-forming NADPH oxidase activity with at least the 5 specified amino acids but further encompassing any additional polypeptide sequence without any additional recited structural features in terms of sequence or functional domains responsible for the water-forming NADPH oxidase activity. Applicant is correct that the prior art does teach water-forming NADH oxidases, however, both the prior and the specification are limited to the description of bacterial water-forming NADH oxidases having a Rossmann fold domain having a dinucleotide-binding motif defined by the peptide G-x-G-x-x-G/A, and a cofactor specificity loop. Further, the instant claimed genus of polypeptides with water-forming NADPH activity is not limited to polypeptides derived from known bacterial NADH oxidase sequences, or to any particular polypeptide sequence based on known bacterial NADH oxidases with or without any mutations. Also note that the prior art does not teach that most bacterial NADH oxidases have any NADPH activity or exhibit a preference for NADPH over NADH oxidation. The specification, as discussed in the rejection of record, provides a description of a number of what appear to be naturally occurring water-forming NADH oxidases from a number of different bacteria, including different strains of the same bacteria (SEQ ID NOS 1-273), and sequences with 85% sequence identity or more with any one of these sequences. The specification also generally discloses sequences based on these bacterial NADH with the specific functional properties of selective oxidization of NADPH over NADH, a kcat/KM for NADPH that is increased by 10-fold or more relative to the kcat/Km exhibited for NADH, Km values for NADH and O2 of no more than about 100 pM and 20 pM, respectively, and/or the production of less than about 2% by mole of H2O2 compared to H2O production during the catalytic cycle of the polypeptide. Please note that the specification does not disclose a single polypeptide which is not a bacterial water-forming NADH oxidase which has water-forming NADH oxidase activity, NADPH oxidase activity, or any other claimed activity. Further, of all of the generally disclosed bacterial NADH oxidizing polypeptides, the specification only provides a detailed description of the functional properties of SEQ ID NO:1, a water forming NADH oxidase from Lactobacillus brevis (LbNox), and specific mutants of SEQ ID NO:1 including mutants of SEQ ID NO:1 with 5 specific substitutions- G158A, D176A, A177R, M178S, and P183R, relative to SEQ ID NO: 1. The specification does not disclose the ability of any other water forming NADH oxidases to oxidize NADPH, the Km or kcat/Km of any of these enzymes for NADH versus NADPH, or the Km values of these NADH oxidases for O2, or the percent production of H2O2 versus H2O. See claims 340-342 for the recitations of limitations regarding these properties. In addition, the specification does not teach the effects of any mutations on any bacterial water forming NADH oxidase other than LbNox.
Turning to the working examples, it is noted that the working examples are solely focused on the properties of LbNox (SEQ ID NO:1) and mutants of LbNox. The working examples disclose the sequence and the crystal structure of LbNox, and the functional properties of wild type LbNox which include high selectivity for NADH over NADPH, negligible H2O2 production relative to H2O, a very low Km of O2. The working examples also describe the production of a single mutant of LbNox (SEQ ID NO:329), a sequence based on SEQ ID NO:1 which contains all five of the specific amino acid substitutions recited in claim 329. This mutant, referred to as TPNOX in the specification, was designed based on the crystal structure of LbNOX and previous modifications to other bacterial NADH oxidases in the prior art, and exhibits a kcat of 268/s and a Km of 22 uM for NADPH, whereas the kcat for NADH was virtually non-reactive. Neither the working examples nor the remainder of the specification provides any further description of the activity of an LbNox polypeptide with the 5 specified amino acid substitutions, or any LbNox polypeptide with any additional amino acid substitutions, deletions, or insertions. Neither the specification nor the working examples provide any further description of any of the functional properties of any of polypeptides encoded by SEQ ID NOS 2-273, such as the Km or kcat/Km of any of these enzymes for NADH versus NADPH, or the Km values of these NADH oxidases for O2, or the percent production of H2O2 versus H2O, or any description of the effects of the five claimed amino acid substitutions on the Km or kcat/Km of any of these enzymes for NADH versus NADPH, or the Km values of these NADH oxidases for O2, or the percent production of H2O2 versus H2O.
Thus, of the universe of polypeptide sequences encompassed by the claims, including non-bacterial water-forming NADH oxidase polypeptides which comprise a Rossmann fold domain having a dinucleotide-binding motif defined by the peptide G-x-G-x-x-G/A, and a cofactor specificity loop, with or without the 5 specified amino acid residues that exhibit water forming NADH and/or NAPDH oxidase activity, bacterial water forming NADH oxidase polypeptides-wild type or mutated with NAPDH oxidase activity- and sequences with 85% sequence homology to SEQ ID NO:1 with specific NADH oxidase and/or NADPH oxidase activity, the specification only provides a specific description of the activity of two specific polypeptides, LbNox (SEQ ID NO:1) and TPNOX (SEQ ID NO:329) towards NADH and NADPH respectively which meet the claimed functional limitations.
Furthermore, the disclosure of a single mutant sequence which meets the claim limitations does not adequately describe the claimed genus based solely on the known genus of bacterial NADH oxidases as evidenced by the prior art. The prior art teaches that despite crystal structure guidance and comparison to other bacterial NADH and NADPH oxidases, the identification of mutations which convert an NADH oxidase to an NADPH oxidase cannot be determine a priori, and mutants with high levels of NADPH oxidation as compared to NADH may not be found for all bacterial water forming NADH oxidases. Petschacher et al. shows that mutant S. mutans NADH oxidases with amino acid substitutions in the cofactor specificity sequence did not generally result in enzymes with higher activity of NADPH over NADH (Petschacher et al., Table 2). Further, in rebuttal to applicant’s argument, Lountos et al. teaches the crystal structure of a water forming NADH oxidase from Lactobacillus sanfrancisensis (L.san) and a related homology model for a water forming NADH from Lactococcus lactis (L.lac) (Lountos et al. (2006) Biochemistry, Vol. 45, 9648-9659). Lountos et al. teaches that whereas L.lac is specific for NADH, L.san bound to both NADH and NADPH equally (Lountos et al., page 9648). Lountos et al. further teaches that based on crystal structure analysis, the amino acids involved in NADH versus NADPH binding are different in the two oxidases (Lountos et al., page 9656). It is also noted that the specific amino acids noted by Lountos et al. as important in the NADH/NADPH binding site are not the same residues identified by the specification as important for switching specificity from NADH to NADPH. It is also noted that the prior art is silent as to effects of mutations outside of the cofactor specificity sequence, and in particular mutations in the GxGXXG/A dinucleotide binding motif required for enzyme activity. Thus, the prior art fails to provide the necessary guidance missing from the instant specification for the genus of polypeptides now claimed, and further clearly demonstrates that knowledge of the genus of bacterial NADH oxidases provided in the prior art does not contribute to an adequate description of the claimed genus as the prior art shows that different species of bacterial NADH oxidases have different crystal structures and utilize different residues in NADH/NADPH binding.
As such, it is maintained that the specification does not provide sufficient description or a reduction to practice of the genus of mutant polypeptides with NADPH activity as claimed other than SEQ ID NO:329. Based on the applicant's specification and the state of the prior art at the time of filing, the skilled artisan cannot envision which additional changes can be made in the sequence of SEQ ID NO: 329 without affecting the functional properties of the polypeptide, and further cannot envision the sequence of a water-forming NADH oxidase which is not SEQ ID NO:1 in which the claimed 5 substitutions can result in water-forming NAPH oxidase activity, such that the detailed chemical structure of the genus of polypeptides encompassed by the claims as a whole cannot be envisioned. Therefore, conception is not achieved until reduction to practice has occurred, regardless of the complexity or simplicity of the method of isolation. See Fiers v. Revel, 25 USPQ2d 1602 at 1606 (CAFC 1993) and Amgen Inc. V. Chugai Pharmaceutical Co. Ltd., 18 USPQ2d 1016. Thus, for the reasons outlined above and in previous office action, it is maintained that amended claims do not meet the requirements for written description under 35 U.S.C. 112, first paragraph.
Double Patenting
The rejection of previously pending claims 329-348 on the ground of nonstatutory double patenting as being unpatentable over claims 1-5 of U.S. Patent No. 11,866,736, hereafter referred to as the ‘736 patent, alone or in view of Petschacher et al. (2014) Computational and Structural Biotechnology Journal, Vol. 9(14),e201402005, https://doi.org/10.5936/csbj.201402005, pages 1-11, is withdrawn in view of the cancellation of claims 330-333 and 345 and the submission of a terminal disclaimer on 6/10/26.
The terminal disclaimer filed on 6/10/26 disclaiming the terminal portion of any patent granted on this application which would extend beyond the expiration date of U.S. Patent 11,866,736 has been reviewed and is accepted. The terminal disclaimer has been recorded.
No claims are allowed.
THIS ACTION IS MADE FINAL. 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 from the examiner should be directed to Anne Marie S. Wehbé, Ph.D., whose telephone number is (571) 272-0737. If the examiner is not available, the examiner’s supervisor, Maria Leavitt, can be reached at (571) 272-1085. For all official communications, the technology center fax number is (571) 273-8300. Please note that all official communications and responses sent by fax must be directed to the technology center fax number. For informal, non-official communications only, the examiner’s direct fax number is (571) 273-0737. For any inquiry of a general nature, please call (571) 272-0547.
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Dr. A.M.S. Wehbé
/ANNE MARIE S WEHBE/Primary Examiner, Art Unit 1634
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