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 .
Claim Status
The amendment of 04/01/2025 has been entered. Claims 7 and 9-26 are pending (claim set as filed on 04/01/2025). Claims 9-18 are withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected invention, there being no allowable generic or linking claim. Claims 19-20 and 23-26 are withdrawn as being drawn to nonelected species.
Claims 7 and 21-22 are currently under examination and were examined on their merits.
Withdrawn Objections/Rejections
All rejections of claims set forth in the previous Office action filed on 01/02/2025 are withdrawn in light of the amendment filed on 04/01/2025.
Claim Interpretation
Claim 7 recites the limitation “wherein a wild-type phenylalanine dehydrogenase exists prior to said mutation”. Therefore, the modified phenylalanine dehydrogenase cited in the instant claims is considered a product-by-process limitation, since the modified enzyme is derived from a wild-type phenylalanine dehydrogenase. The MPEP states "[E]ven though product-by-process claims are limited by and defined by the process, determination of patentability is based on the product itself. The patentability of a product does not depend on its method of production. If the product in the product-by-process claim is the same as or obvious from a product of the prior art, the claim is unpatentable even though the prior product was made by a different process." In re Thorpe, 777 F.2d 695, 698, 227 USPQ 964, 966 (Fed. Cir. 1985)” (see MPEP 2113 (I)).
As such, the claimed modified phenylalanine dehydrogenase will be interpreted by its structural features and properties described in the instant specification and claims.
Claim 22 recites ‘[t]he modified phenylalanine dehydrogenase according to claim 7, wherein said modified phenylalanine dehydrogenase further has an improved characteristic relative to a phenylalanine dehydrogenase consisting of the amino acid sequence of SEQ ID NO: 1’. The Examiner notes that any improved characteristic of the modified phenylalanine dehydrogenase according to claim 7 results from the structure of said modified phenylalanine dehydrogenase, and therefore, the in claim 22 recited ‘improved characteristic’ of the modified phenylalanine dehydrogenase according to claim 7 is considered an inherent property of the modified phenylalanine dehydrogenase in claim 7.
Claim Rejections - 35 USC § 103
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.
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 factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
Determining the scope and contents of the prior art.
Ascertaining the differences between the prior art and the claims at issue.
Resolving the level of ordinary skill in the pertinent art.
Considering objective evidence present in the application indicating obviousness or nonobviousness.
Claims 7 and 21-22 are newly rejected as necessitated by amendment under 35 U.S.C. 103 as being unpatentable over Ayyub et al. (US 2016/0168613 A1, published on 06/16/2016), hereinafter ‘Ayyub’, in view of Seah et al. (“Kinetic analysis of phenylalanine dehydrogenase mutants designed for aliphatic amino acid dehydrogenase activity with guidance from homology-based modelling”, published on 11/28/2003, Eur. J. Biochem., Vol. 270, pages 4628–4634), hereinafter ‘Seah’.
Ayyub’s general disclosure relates to “a biosensor capable of measuring
the total concentration of one or a plurality of amino acids with the use of a reagentless system comprising an electrode modified by hydrogel that comprises at least one enzyme that oxidizes at least one substrate that is at least one amino acid.” (see entire document, including abstract)
Regarding claims 7 and 22, please note the interpretation of claim 22 above under Claim Interpretation. Pertaining to a modified phenylalanine dehydrogenase, Ayyub teaches a modified phenylalanine dehydrogenase, comprising a mutation (“Metabolic enzymes that can be used individually or in combination with the biosensor, system or test strip disclosed herein include: any bacterial clone of phenylalanine dehydrogenase,”, “In some embodiments, the at least one enzyme is phenyalanine dehydrogenase or a functional fragment thereof obtained from a thermophilic bacterial cell”; “Enzyme activity will be improved, if necessary, by site directed mutagenesis to increase the affinity for the substrates or the stability”; paragraphs [0019], [0123], [0171]);
wherein a wild-type phenylalanine dehydrogenase exists prior to said mutation (“the at least one enzyme or functional fragment thereof is derived from a thermophillic bacterial species and is immobilized in the hydrogel. In some embodiments, the at least one enzyme or functional fragment thereof comprises at least about 70% sequence identity to … SEQ ID NO:2.”; paragraph [0018]; see Table on page 15; note, SEQ ID NO: 2 corresponds to phenylalanine dehydrogenase from Thermoactinomyces intermedium), and
wherein the wild-type phenylalanine dehydrogenase comprises
(A) the amino acid sequence of instant SEQ ID NO: 1 (“the at least one enzyme or functional fragment thereof comprises at least about 70% sequence identity to … SEQ ID NO: 2.”; paragraph [0018]; see Table on page 15 and SEQ ID NO: 2 on pages 15 and 29). The Examiner notes that Ayyub’s SEQ ID NO: 2 has 100 % identity with instant SEQ ID NO: 1 (see sequence alignment attached to this Office action),
wherein said modified phenylalanine dehydrogenase has a phenylalanine dehydrogenase activity (“the at least one enzyme or functional fragment thereof comprises at least about 70% sequence identity to SEQ ID NO:2”, “"functional fragment" means any portion of a polypeptide that is of a sufficient length to retain at least partial biological function that is similar to or substantially similar to the wild-type polypeptide function; paragraph [0018]). As discussed above, Ayyub’s SEQ ID NO: 2 corresponds to a phenylalanine dehydrogenase from Thermoactinomyces intermedius (see Ayyub, Table on page 15). Ayyub further teaches wherein the substrate of a phenylalanine dehydrogenase is phenylalanine (see Fig. 13), and that “[e]nzyme activity will be improved, if necessary, by site directed mutagenesis to increase the affinity for the substrates or the stability”; paragraph [0171]), thereby indicating that a modified phenylalanine dehydrogenase has phenylalanine dehydrogenase activity, which is improved compared to the non-mutated phenylalanine dehydrogenase.
Regarding claim 21, pertaining to the wild-type phenylalanine dehydrogenase, Ayyub teaches wherein the wild-type phenylalanine dehydrogenase comprises the amino acid sequence of instant SEQ ID NO: 1 (paragraph [0123], see Table and SEQ ID NO: 2 on page 15 and 29).
Additionally, Ayyub teaches wherein “[m]etabolic enzymes include those amino acid sequences wherein conservative substitutions have been introduced by
modification of polynucleotides encoding polypeptides of the invention”, wherein “[e]xemplary conservative substitutions are set out in Table A” (paragraph [0068]). Ayyub’s Table A shows that amino acid asparagine (N) can be substituted with aspartic acid (D) (see Table A, bottom row). Ayyub further teaches the use of phenylalanine dehydrogenase for detecting elevated phenylalanine in whole blood of patients (paragraph [0009]), and that phenylalanine is an amino acid marker for the metabolic disease PKU (“Phenylketonuria (PKU, phenylalanine hydroxylase)”; paragraph [0082]; see Table 1 on page 9).
Ayyub does not teach
wherein said modified phenylalanine hydrogenase comprises a mutation of N290D (instant claim 7).
wherein said modified phenylalanine dehydrogenase has an improved substrate specificity to phenylalanine relative to a phenylalanine dehydrogenase consisting of the amino acid sequence of SEQ ID NO:1 (instant claim 7).
Seah’s general disclosure relates to the kinetic analysis of phenylalanine dehydrogenase mutants (“A fuller kinetic characterization of the single- and double-mutant enzymes now reveals that the extent of the shift in specificity was underestimated in the earlier study”; see entire document, including abstract).
Regarding claims 7 and 22, pertaining to the mutation N290D, Seah teaches that the amino acid residue asparagine N290 in instant SEQ ID NO: 1 is highly conserved in amino acid dehydrogenases including phenylalanine dehydrogenases (see sequence comparison in Fig. 1B). It is noted that the sequence shown for the phenylalanine dehydrogenase of Thermoactinomyces intermedius (“PheDH (T. int.)”; see Fig. 1 B)) is identical with amino acids 274-311 of instant SEQ ID NO: 1. The Examiner notes, that the high degree of conservation of asparagine (N) in position 290 of instant SEQ ID NO:1 among amino acid dehydrogenases indicates that said asparagine (N) residue might be critical to the catalytic function of phenylalanine dehydrogenases shown in Fig. 1B.
While Ayubb does not teach wherein said modified phenylalanine hydrogenase comprises a mutation of N290D (instant claim 7 and 22), wherein said modified phenylalanine dehydrogenase has an improved substrate specificity to phenylalanine relative to a phenyl alanine dehydrogenase consisting of the amino acid sequence of SEQ ID NO:1 (instant claim 7), it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have combined Ayyub’s modified phenylalanine dehydrogenase with Ayubb’s teachings on site-directed mutagenesis for improving enzyme activity including the affinity of an enzyme for its substrate, with Ayyub’s teachings on conservative amino acid substitutions, and with Seah’s teachings on highly conserved amino acid residues in phenylalanine dehydrogenases (see above), in order to have created a modified phenylalanine dehydrogenase comprising a mutation of N290D, wherein said modified phenylalanine dehydrogenase has an improved substrate specificity to phenylalanine relative to a phenylalanine dehydrogenase consisting of the amino acid sequence of instant SEQ ID NO:1. A skilled artisan would have reasonably expected success in the combination of Ayyub’s and Seah’s teachings for the benefit of creating a superior phenylalanine dehydrogenase with increased phenylalanine dehydrogenase substrate specificity for phenylalanine that is suitable for medical diagnostics (see Ayubb above), since both references are directed to altering phenylalanine dehydrogenase activity by mutating the amino acid sequence (see Ayyub and Seah above).
Since Ayyub in view of Seah teaches the modified phenylalanine dehydrogenase according to claim 7, modified Ayyub intrinsically teaches wherein said modified phenylalanine dehydrogenase further has an improved characteristic relative to a
phenylalanine dehydrogenase consisting of the amino acid sequence of SEQ ID NO:1, wherein said characteristic is selected from the group consisting of: i) solubility,
ii) phenylalanine dehydrogenase activity, and iii) combinations thereof (instant claim 22). As discussed above under Claim Interpretation, the in claim 22 recited ‘improved characteristic’ of the modified phenylalanine dehydrogenase according to claim 7, is an inherent property of said modified phenylalanine dehydrogenase.
Response to Arguments
Applicant has traversed the previous rejection of claims 1-8 under 35 U.S.C. 103 as being unpatentable over Lu et al. (CN108795893A, published on 11/13/2018), hereinafter ‘Lu’, in view of Yousefi et al. (“Increase of Bacillus badius Phenylalanine dehydrogenase specificity towards phenylalanine substrate by site-directed mutagenesis”, published on 10/16/2017, Archives of Biochemistry and Biophysics, Vol. 635, pages 44–51), hereinafter ‘Yousefi’, Seah and Betts et al. (“Amino acid properties and consequences of substitutions”, published in Feb 2003, Bioinformatics for Geneticists, pages 289-316), hereinafter ‘Betts’, as evidenced by Takada et al. (“Thermostable Phenylalanine Dehydrogenase of Thermoactinomyces intermedius: Cloning, Expression, and Sequencing of Its Gene”, published 03/1991, J. Biochem., Vol. 109, pages 371-376), hereinafter ‘Takada’ (remarks, pages 11-15).
As discussed above, all rejections have been withdrawn and new rejections presented due to Applicant’s amendment of 04/01/2025. Lu’s, Yousefi’s, and Bett’s teachings are not relied upon in the above-presented rejection. As such, Applicant’s arguments regarding Lu’s, Yousefi’s, and Bett’s teachings are moot. Applicant’s arguments regarding Seah’s teachings and the instantly claimed increased substrate specificity for phenylalanine due to the N290D mutation have been fully considered but they are not persuasive.
In Applicant’s reply, Applicant states that “The fact that the asparagine residue corresponding to N290 in PheDH from Thermoactinomyces intermedius is highly conserved not only in PheDH but also in other amino acid dehydrogenases such as GluDH, LeuDH and ValDH, as shown in Figure 1 suggests that the N290 mutation cannot be responsible for substrate specificity for amino acids. Therefore, Fig. 1 in Yousefi effectively teaches away from the claimed invention, since the subject matter of
the claims require that the N290 mutation changes substrate specificity.” (remarks, page 13). The Examiner responds that Seah teaches that “[t]he key difference between the substrate binding sites of PheDH and LeuDH seems to lie in the residues corresponding
to positions V377 and A163 of C. symbiosum GluDH [10]. These are unchanged in LeuDH, but in PheDH leucine replaces valine in the first position, and the replacement of an alanine residue with glycine in the second position, on the opposite side of the binding pocket, is thought to create a deeper binding pocket for access of
substrates with large aromatic side-chains.” (see page 4629, left column, paragraph 3, and Fig. 1). It is noted, that V377 in C. symbiosum glutamate dehydrogenase corresponds to L294 in the T. intermedius phenylalanine dehydrogenase (see Fig. 1B), which is closely located to conserved N290. Therefore, conserved N290 might be of importance for the substrate binding pocket of amino acid dehydrogenases, and a conservative amino acid substitution might lead to a smaller shift in substrate specificity, while maintaining the overall substrate specificity of phenylalanine dehydrogenases for amino acids with aromatic side-chains, or of leucine dehydrogenases for amino acids with aliphatic side chains (“Leucine dehydrogenases (LeuDHs) have somewhat wider specificity [4,5] and react with various nonpolar aliphatic amino acids but not aromatic amino acids. Phenylalanine dehydrogenases (Phe-DHs) (EC 1.4.1.20), on the other hand, use aromatic amino acids as preferred substrates, although they also exhibit some activity with nonpolar aliphatic amino acids.”; Seah, page 4628, right column, paragraph 1).
Applicant describes that "N290D has approximately 5.3-fold improved substrate specificity for phenylalanine compared to non-modified or wild-type.” (remarks, page 11, paragraph 5). The Examiner responds that according to the instant specification (Example 4, paragraphs [000332]-[000333]), “the results of Tables 8 and 9 were calculated from an average value when an experience was conducted twice for the same sample.”, and “[f]or the value of WT, an average value of results of the wild-type PheDH prepared and subjected to substrate specificity evaluation 16 times was used.”. However, the significance of the improvement of substrate specificity of the N290D mutant for phenylalanine based on the data presented in Table 8 is unclear since the variability of individual data points is not shown (see Table 8, wild-type and single mutation-introduced PheDH). The MPEP states that “[t]he evidence relied upon should establish "that the differences in results are in fact unexpected and unobvious and of both statistical and practical significance." Ex parte Gelles, 22 USPQ2d 1318, 1319 (Bd. Pat. App. & Inter. 1992)” (see MPEP 716.02(b)(I)). As such, proper interpretation of the presented data regarding the effect of N290D on substrate specificity is not possible.
Applicant states that “the subject matters of the claims, each taken as a whole, would not have been obvious to one of ordinary skill in the art at the time of Applicant's invention” (remarks page 14, paragraph 2). In response to Applicant’s argument that there is no teaching, suggestion, or motivation to combine the references, the Examiner recognizes that obviousness may be established by combining or modifying the teachings of the prior art to produce the claimed invention where there is some teaching, suggestion, or motivation to do so found either in the references themselves or in the knowledge generally available to one of ordinary skill in the art. See In re Fine, 837 F.2d 1071, 5 USPQ2d 1596 (Fed. Cir. 1988), In re Jones, 958 F.2d 347, 21 USPQ2d 1941 (Fed. Cir. 1992), and KSR International Co. v. Teleflex, Inc., 550 U.S. 398, 82 USPQ2d 1385 (2007). In this case, as discussed above, Ayyub provides a modified phenylalanine dehydrogenase with improved phenylalanine dehydrogenase activity, site-directed mutagenesis to improve enzyme activity, and conservative amino acid substitutions, while Seah provides teachings on highly conserved amino acid residues (including N290) in phenylalanine dehydrogenases, that suggest involvement of said residues in the catalytic function of the phenylalanine dehydrogenase. As such, claims 7 and 21-22 are rendered obvious over the teachings of Ayyub and Seah.
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.
Correspondence Information
Any inquiry concerning this communication or earlier communications from the examiner should be directed to SANDRA ZINGARELLI whose telephone number is (703)756-1799. The examiner can normally be reached M-F 9-5.
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/SANDRA ZINGARELLI/Examiner, Art Unit 1653
/SHARMILA G LANDAU/Supervisory Patent Examiner, Art Unit 1653
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