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
This application is a 371 of PCT/US18/17581 filed 02/09/2018 which claims benefit of provisional application 62/457,353 filed 02/10/2017. Applicant’s claim for the benefit of a prior-filed application under 35 U.S.C. 119(e) or under 35 U.S.C. 120, 121, 365(c), or 386(c) is acknowledged.
Status of the Claims
Claims 1-8, 10-14, 19, 21, 22, 26, 29 and 33-35 are pending. Claims 1, 5, 21 and 34 are amended. Claim 17 is cancelled. Claims 26, 29, 33 and 35 are withdrawn.
Claims 1-8, 10-14, 19, 21, 22 and 34 (claim set filed 05/05/2026) are examined on the merits herein.
Withdrawal of Rejections
The response and amendment filed on 05/05/2026 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The previous claims 5, 21 and 34 objections have been withdrawn necessitated by amendment of claims 5, 21 and 34.
The previous claims 21 and 34 rejections under 35 U.S.C. 112(b) have been withdrawn necessitated by amendment of claims 21 and 34. Claims 21 and 34 were amended to identify the sequence of ClpS for V65I mutant as E.coli ClpS. The sequence E.coli ClpS is shown on Figure 4A and described in the specification as sequence with SEQ ID NO: 149.
Amendment of Claims (1.121)
"Amendments to a claim must be made by rewriting the entire claim with all changes (e.g., additions and deletions) as indicated in this subsection, except when the claim is being canceled. Each amendment document that includes a change to an existing claim, cancellation of an existing claim or addition of a new claim, must include a complete listing of all claims ever presented, including the text of all pending and withdrawn claims, in the application. The claim listing, including the text of the claims, in the amendment document will serve to replace all prior versions of the claims, in the application. In the claim listing, the status of every claim must be indicated after its claim number by using one of the following identifiers in a parenthetical expression: (Original), (Currently amended), (Canceled), (Withdrawn), (Previously presented), (New), and (Not entered)." (MPEP 1.121).
The Applicant has amended claim 21, however claim 21 is identified as (Original).
The claim 21 is not-compliant, however, in the interest of compact prosecution the Examiner will proceed with the examination of the application.
Claim Objections
Claims 21 and 34 are objected to because of the following informalities:
Claims 21 and 34 recite: “CplS_V65I mutant of E. coli ClpS protein”. Although the specification describes E. coli ClpS protein to have sequence with SEQ ID NO: 149 and the same sequence is show on Fig. 4A, Applicant is suggested to include reference to SEQ ID NO:149 for clarity in claims 21 and 34.
Additionally, Applicant is suggested to replace recitation of “E. coli” in claim 21 with “Escherichia coli” and refer to “E.coli” in claim 34.
Appropriate correction is required.
Claim Rejections - 35 USC § 112
The following is a quotation of 35 U.S.C. 112(d):
(d) REFERENCE IN DEPENDENT FORMS.—Subject to subsection (e), a claim in dependent form shall contain a reference to a claim previously set forth and then specify a further limitation of the subject matter claimed. A claim in dependent form shall be construed to incorporate by reference all the limitations of the claim to which it refers.
The following is a quotation of pre-AIA 35 U.S.C. 112, fourth paragraph:
Subject to the following paragraph [i.e., the fifth paragraph of pre-AIA 35 U.S.C. 112], a claim in dependent form shall contain a reference to a claim previously set forth and then specify a further limitation of the subject matter claimed. A claim in dependent form shall be construed to incorporate by reference all the limitations of the claim to which it refers.
Claims 3 is rejected under 35 U.S.C. 112(d) or pre-AIA 35 U.S.C. 112, 4th paragraph, as being of improper dependent form for failing to further limit the subject matter of the claim upon which it depends, or for failing to include all the limitations of the claim upon which it depends.
Claim 3, dependent on claim 1, recites the limitation for the removable protecting group to be an enzyme cleavable protective group. Claim 3 does not further limit claim 1 reciting the cell to express “an enzyme that cleaves the removable protecting group”. Claim 3 does not recite any limitation that further limits claim 1.
Applicant may cancel the claim(s), amend the claim(s) to place the claim(s) in proper dependent form, rewrite the claim(s) in independent form, or present a sufficient showing that the dependent claim(s) complies with the statutory requirements.
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 1-6, 11-14, 19 and 21 are rejected under 35 U.S.C. 103 as being unpatentable over Kwon (US 20050287639 A1 on record in IDS) in view of Piatkov (Piatkov et al. PLOS One, 2013, 8, e67952, 1-7).
Regarding claim 1, Kwon teaches a method of making a target polypeptide incorporating unnatural amino acids directly into protein in vivo (paragraphs 0125, 0127) using pair of orthogonal tRNA and aminoacyl-tRNA synthetase (paragraph 0008). Kwon describes that the host cells are genetically engineered to incorporate the NSAA via tRNA/aminoacyl-tRNA synthetase pair (paragraph 0510). Kwon provides an example of using a pair of genetically engineered tRNA and cognate aminoacyl-tRNA synthetase to express murine dihydrofolate reductase incorporating NSAA, (2-naphthyl)alanine, in Escherichia coli (paragraph 0526). Kwon mentions that the desired NSAA is incorporated in the target location at an efficiency of at least about 50% (paragraph 0068) indicating that natural amino acid or undesired NSAA can be non-selectively added to the target polypeptide.
Kwon does not teach the removable protective group attached to the target polypeptide adjacent to the amino acid target location and does not teach a protease for degrading the target polypeptide when the N-end amino acid is a standard or an undesired NSAA.
Piatkov teaches protein degradation by the N-end rule in prokaryotes and eukaryotes. Piatkov describes that proteins are degraded based on destabilizing N-terminal residue of a protein (p. 2, left column, 2nd paragraph). Piatkov discloses that bacteria such as Escherichia coli and Vibrio vulnificus contain N-end rule pathway including ClpAP protease. N-end rule substrates bind ClpS that delivers them to ClpAP protease for degradation (p. 2, left column, 2nd paragraph). Piatkov mentions the N-terminal amino acids recognized in bacteria as destabilizing are Leu, Trp, Phe or Tyr (p. 3, Figure 1).
Piatkov describes ubiquitin fusion technique to generate any desired N-terminal amino acid residue by fusion of ubiquitin to the desired N-terminal residue of a polypeptide (p. 1, left column, 3rd paragraph). Piatkov describes that, since bacteria lack ubiquitin system, Ubp1 deubiquitylase of the yeast Saccharomyces cerevisiae is coexpressed with the fusion polypeptide (Abstract). Ubp1 cleaves the ubiquitin thus generating N-end amino acid. Piatkov mentions that nascent proteins are produced with methionine on N-terminal that is removed by amino peptidases only if the second residue is a small residue. The ubiquitin fusion technique allows to overcome that problem and provides the desired N-terminal residue (p. 1, right column, 1st paragraph). Piatkov describes that when Ubp1 and fusion of DHFR-Ub to β-galactosidase with various N-terminal residue (DHFR-Ub-X-βgal) are coexpressed in E. coli, β-galactosidase with destabilizing N-terminal residue such as leucine is degraded in comparison with residues not recognized by the N-end rule such as valine or aspartate (p. 5, Figure 2).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to combine teachings of Kwon and Piatkov and add ubiquitin as removable protective group as described by Piatkov to the target polypeptide containing NSAA disclosed by Kwon and coexpress it with the UBP1 protease in E. coli based on Piatkov teaching for removal of ubiquitin and to expose the N-end amino acid which would enrich the target polypeptide with NSAA since the polypeptide with certain standard amino acids will be degraded by ClpAP protease present in E. coli. One would have been motivated to do that since Piatkov describes ubiquitin fusion technique to obtain polypeptide with the desired N-terminal amino acid and provides example of specific degradation of a protein obtained in E. coli depending on the amino acid residue on its generated N-end. A skilled artisan would have reasonably expected success in this combination since Kwon provided method of production of a target polypeptide containing NSAA and Piatkov describes technique allowing to degrade polypeptides with certain standard and undesired amino acids on N-end thus increasing selectivity of production. Thus, Kwon and Piatkov teachings render claim 1 obvious.
Regarding claim 2-5, Piatkov teaches cleavable protecting group, ubiquitin, attached to target polypeptide which is expressed in E. coli (Abstract). Ubiquitin is a protein which is orthogonal to E.coli since bacteria lack ubiquitin system (p. 2, left column, 2nd paragraph). Piatkov describes expression of Ubp1 as enzyme cleaving ubiquitin (p. 2, left column, 3rd paragraph).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to combine teachings of Kwon and Piatkov and add enzyme cleavable orthogonal protein protective group, i.e. ubiquitin, cleaved by UBP1 as described by Piatkov to the target polypeptide containing NSAA disclosed by Kwon. One would have been motivated to do that with reasonably expected success since ubiquitin fusion technique describes by Piatkov provides polypeptide with desired N-terminal residue. Thus, Kwon and Piatkov teachings render claims 2-5 obvious.
Regarding claim 6, Kwon teaches that genetic modification of the cell includes mutagenized nucleic acid encoding a polypeptide comprising one or more NSAA (paragraph 0207). Piatkov teaches removable protecting protein, ubiquitin, attached to the target polypeptide (Abstract).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to combine teachings of Kwon and Piatkov and genetically modify the cell by including nucleic acid encoding the target polypeptide including NSAA as taught by Kwon with attached removable protecting group as described by Piatkov. One would have been motivated to make this combination with reasonably expected success since Kwon provided method of production of a target polypeptide containing NSAA and Piatkov described attachment of cleavable ubiquitin to the target polypeptide to provide polypeptide with desired N-terminal residue. Thus, Kwon and Piatkov teachings render claim 6 obvious.
Regarding claim 11, Kwon teaches providing the foreign nucleic acids for modified tRNA, modified amino-acyl tRNA, nucleic acid for the target protein and the NSAA, that allows the genetically modified cell to express the target polypeptide incorporating the NSAA at the specified position (paragraph 0025). Kwon provides an example of expressing a pair of genetically engineered tRNA and cognate aminoacyl-tRNA synthetase to produce murine dihydrofolate reductase incorporating NSAA, (2- naphthyl)alanine (paragraph 0526). Thus, Kwon and Piatkov teachings render claim 11 obvious.
Regarding claim 12, Kwon teaches that the nucleic acids introduced into the cell can be introduced in any suitable vectors capable of expressing tRNA and proteins in the cell and that the sequences can be introduced with an appropriate promoter which can be inducible to control the expression of sequences (paragraph 0152).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have Ubp1 cleaving the protective ubiquitin attached to the target polypeptide as described by Piatkov under the control of the inducible promoter as taught by Kwon for the proteins expressed in the cell for production of the target polypeptide with NSAA. One would have been motivated to do that with reasonably expected success to control the expression of Ubp1 in order to start cleavage after obtaining sufficient amount of expressed target protein to stabilize the target protein during expression. Thus, Kwon and Piatkov teachings render claim 12 obvious.
Regarding claim 13 and 21, Piatkov teaches that bacteria such as Escherichia coli and Vibrio vulnificus contain N-end rule pathway including ClpAP protease. Bacterial N-end substrates are recognized by ClpS that binds to bulky hydrophobic N-terminal residues and delivers targeted N-end substrates to the protease ClpAP for degradation thus serving as an adapter protein (p. 2, left column, 2nd paragraph). Piatkov mentions the N-terminal amino acids recognized in bacteria as destabilizing are Leu, Trp, Phe or Tyr (p. 3, Figure 1). Piatkov describes ubiquitin fusion technique to generate any desired N-terminal amino acid residue by fusion of ubiquitin to the desired N-terminal residue of a polypeptide (p. 1, left column, 3rd paragraph). Piatkov describes coexpression of Ubp1 deubiquitylase of the yeast Saccharomyces cerevisiae with the fusion polypeptide (Abstract). Ubp1 cleaves the ubiquitin thus generating N-end amino acid. Piatkov describes that when Ubp1 and fusion of DHFR-Ub to β-galactosidase with various N-terminal residue (DHFR-Ub-X-βgal) are coexpressed in E. coli β-galactosidase with destabilizing N-terminal residue such as leucine is degraded in comparison with residues not recognized by the N-end rule such as valine or aspartate (p. 5, Figure 2).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to express the UBP1 protease and the ClpS-ClpAP protease system from Piatkov teaching in the genetically modified cell producing the target polypeptide with NSAA based on Kwon teaching. One would have been motivated to do that since Piatkov describes that UBP1 can remove ubiquitin attached to the N-end of the polypeptide and hence generate the open N-end and ClpS adaptor protein mediates degradation of the target protein by ClpAP protease when the N-end amino acid is a certain standard amino acid and that will remove unwanted proteins and enrich the target polypeptide with NSAA within the cell. A skilled artisan would have reasonably expected success in this combination since Kwon provided method of production of a target polypeptide containing NSAA and Piatkov described method to introduce desired amino acid residue on the N-terminus and degrade polypeptides based on N-end to increase selectively of production. Thus, Kwon and Piatkov teachings render claims 13 and 21 obvious.
Regarding claims 14 and 19, Kwon teaches that additional nucleic acid constructs encoding one or more proteins required for biosynthesis of NSAA can be introduced (paragraph 0049). Kwon discloses that the additional nucleic constricts are operably linked to and subject to the control of an inducible promoter (paragraph 0050). Piatkov teaches the adaptor protein, ClpS that coordinates with ClpAP protease that degrades the target polypeptide when the N-end amino acid is a standard amino acid, Leu, Trp, Phe or Tyr (p. 2, left column, 2nd paragraph and Figure 1). Piatkov teaches expression UBP1 protease cleaving ubiquitin serving as protective group on the N-end of the polypeptide (Abstract).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to express the UBP1 protease from Piatkov teaching and express the adaptor protein ClpS from Piatkov teaching under the influence of an inducible promoter as taught by Kwon for the proteins expressed in the cell for production of the target polypeptide with NSAA in the genetically modified cell producing the target polypeptide with NSAA. One would have been motivated to do that with reasonably expected success since Piatkov describes that UBP1 can remove ubiquitin attached to the N-end of the polypeptide and hence generate the open N-end and that adaptor protein coordinates the protease degrading the target polypeptide when the N-end amino acid is a certain standard amino acid and that will enrich the target polypeptide with NSAA and inducible promoter will provide controlled degradation of unwanted proteins. Thus, Kwon and Piatkov teachings render claims 14 and 19 obvious.
Claims 7, 8, 10 and 22 are rejected under 35 U.S.C. 103 as being unpatentable over Kwon (US 20050287639 A1 on record in IDS) in view of Piatkov (Piatkov et al. PLOS One, 2013, 8, e67952, 1-7) as applied to claim 1 above, and further in view of Palmer (Palmer and Freeman Comp. Funct. Genom., 2004, 5, 342-353).
The teachings of Kwon and Piatkov have been set forth above.
Kwon and Piatkov do not teach a detectable moiety attached to the C-end of the polypeptide and being fluorescent moiety or a reporter protein.
Regarding claims 7 and 8, Palmer teaches investigation of the use of C- and N-terminal GFP fusion proteins for subcellular localization (Abstract). Palmer mentions that GFP has the advantage of high stability and can be visualized via standard confocal or fluorescent microscopy (p. 343, left column, 1st paragraph). Palmer describes preparation of fusion of 16 proteins with GFP tags. All C-terminal fusion proteins localized to cellular compartments in accordance with the previous studies or predictions, however, only half of N-terminal fusion proteins localized correctly (Abstract). Palmer suggested that GFP present of N-terminal can effect folding of the protein of interest since GFP is 238 amino acid long and will fold first disrupting further folding. Palmer concluded that the C-terminal tagging with GFP is superior to N-terminal and is expected to maintain functional characteristics of native protein of interest (p. 352, left column, 2nd paragraph).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to follow Palmer teaching and add GFP protein as detectable fluorescent moiety to the C-end of the target protein produced based on teachings of Kwon and Piatkov. One would have been motivated to make this combination to detect production of the target polypeptide with NSAA since Palmer teaches that GFP-tagged protein can be easily visualized via standard confocal or fluorescent microscopy and that C-terminal fusion of GFP with different proteins is expected to maintain functional characteristics of native proteins. A skilled artisan would have reasonably expected success in this combination since Kwon provided method of production of a target polypeptide containing NSAA, Piatkov describes technique allowing to degrade polypeptides with certain standard and undesired amino acid on N-end thus increase selectivity of production and Palmer provides protein modification for detection of the produced target polypeptide. Thus, Kwon, Piatkov and Palmer teachings render claims 7 and 8 obvious.
Regarding claim 10, Kwon teaches that genetic modification of the cell includes mutagenized nucleic acid encoding a polypeptide comprising one or more NSAA (paragraph 0207). Piatkov teaches removable protecting protein, ubiquitin, attached to the target polypeptide (Abstract). Palmer teaches detectable moiety attached to the C-end of the target polypeptide (Abstract). Kwon discloses that the NSAA can be encoded by the sense codon and provides example of the sense UUU codon which normally encodes phenylalanine. However, in the presence of engineered tRNA and aminoacyl-tRNA synthetase with relaxed substrate specificity, the NSAA analog of phenylalanine, i.e. L-3- 2-naphthyl)alanine (NaI), is incorporated into the target protein (paragraphs 0515-0519).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to combine teachings of Kwon, Piatkov and Palmer and genetically modify the cell by including nucleic acid encoding the target polypeptide including NSAA encoded by a corresponding sense codon as taught by Kwon with attached removable protecting group as described by Piatkov and detectable moiety as taught by Palmer. One would have been motivated to make this combination with reasonably expected success since Kwon provided method of production of a target polypeptide containing NSAA, Piatkov described attachment of cleavable ubiquitin to the target polypeptide to generate the desired N-end amino acid residue after ubiquitin cleavage and technique allowing to degrade polypeptides with certain standard and undesired amino acid on N-end thus increase selectivity of production and Palmer provided target protein modification for detection of the produced target polypeptide. Thus, Kwon, Piatkov and Palmer teachings render claim 10 obvious.
Regarding claim 22, Piatkov teaches removal of protective group, ubiquitin, from the N-end catalyzed by the ubiquitin-cleaving enzyme, UBP1 protease (Abstract). Piatkov teaches the adaptor protein, ClpS that coordinates with ClpAP protease that degrades the target polypeptide when the N-end amino acid is a standard amino acid, Leu, Trp, Phe or Tyr (Figure 1). Palmer teaches investigation of the use of C- and N-terminal GFP fusion proteins for subcellular localization (Abstract). Palmer mentions that GFP has the advantage of high stability and can be visualized via standard confocal or fluorescent microscopy (p. 343, left column, 1st paragraph). Palmer describes preparation of fusion of 16 proteins with GFP tags. All C-terminal fusion proteins localized to cellular compartments in accordance with the previous studies or predictions, however, only half of N-terminal fusion proteins localized correctly (Abstract). Palmer suggested that GFP present of N-terminal can effect folding of the protein of interest since GFP is 238 amino acid long and will fold first disrupting further folding. Palmer concluded that the C-terminal tagging with GFP is superior to N-terminal and is expected to maintain functional characteristic of native protein of interest (p. 352, left column, 2nd paragraph).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to combine teaching of Kwon, Piatkov and Palmer and prepare genetically modified cell comprising the target polypeptide with incorporated NSAA as taught by Kwon, add GFP protein as detectable fluorescent moiety to the C-end of the target polypeptide to measure produced protein and express the UBP1 protease and the adaptor protein ClpS with ClpAP protease from Piatkov teaching to remove unwanted polypeptides. One would have been motivated to make this combination since Piatkov describes that UBP1 can remove ubiquitin attached to the N-end of the polypeptide and hence generate the open N-end and that adaptor protein coordinates the protease degrading the target polypeptide when the N-end amino acid is a certain standard amino acid and that will enrich the target polypeptide with NSAAA and Palmer teaches that GFP-tagging of the target polypeptide on C-end is expected to maintain functional characteristic of the polypeptide and provides detection by fluorescence allowing to measure the amount of produced target polypeptide. A skilled artisan would have reasonably expected success in this combination since Kwon provides method of production of a target polypeptide containing NSAA, Piatkov describes protein components increasing selectivity of the target polypeptide with NSAA production by removing the unwanted proteins and Palmer provides detection of the target polypeptide with NSAA. Thus, Kwon, Piatkov and Palmer teachings render claim 22 obvious.
Claim 34 is rejected under 35 U.S.C. 103 as being unpatentable over Kwon (US 20050287639 A1 on record in IDS) in view of Piatkov (Piatkov et al. PLOS One, 2013, 8, e67952, 1-7) as applied to claims 1 and 21 above, and further in view of Varshavsky (Varshavsky Protein Science, 2011, 20, 1298-1345).
The teachings of Kwon and Piatkov have been set forth above.
Kwon and Piatkov do not teach ClpS_V65I mutant of E. coli ClpS.
Regarding claim 34, Varshavsky teaches the N-end rule pathway and regulation by proteolysis (Title). Varshavsky compares the N-end pathway in different organisms and shows difference in recognition of destabilizing and promoting degradation N-terminal amino acid residues in bacteria and eukaryotes (Figure 4). Varshavsky describes that N-terminal amino acid is recognized by ClpS N-recognin in bacteria and E3 Ub ligases in eukaryotes such as Ubr1 (p. 1300, right column, last paragraph, Figures 7 and 8). Varshavsky discloses the sequence alignment of bacterial ClpS and eukaryotic Ubr1 on Figure 6. Figure 6 identifies several positions of crystallographically determined contacts between the ClpS and the N-end rule peptide. As can be seen, certain positions have the same conserved amino acid residues while others differ and can reflect the difference in specificity for bacterial ClpS and eukaryotic Ubr1 towards the N-terminal amino acid. One of the positions is Val65 in E. coli ClpS that is isoleucine in all shown eukaryotic Ubr1 (Figure 6D).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to follow teaching of Varshavsky and try to create and evaluate mutant ClpS including V65I mutant for the specificity of ClpS recognition of the N-terminal residue and to apply it to making of a target polypeptide including NSAA based on Kwon and Piatkov teachings. One would have been motivated to do that since Varshavsky teaches difference is recognition of N-terminal residue between bacteria and eukaryotes, provides sequence alignment of proteins binding to the N-terminal residue in procaryotes and eukaryotes and points to critical residues binding N-end peptide based on crystallography and mutation of ClpS residues involved in binding can change the specificity of recognition of N-terminal residues and promote degradation of standard amino acids or undesired NSAA. A skilled artisan would have reasonably expected success in this combination since Kwon provided method of production of a target polypeptide containing NSAA and Piatkov described method to introduce desired amino acid residue on the N-terminus and degrade polypeptides based on N-end to increase selectively of production and Varshavsky provides information of recognition of N-end peptide by bacteria and eukaryotes. Thus, Kwon, Piatkov and Varshavsky teachings render claim 34 obvious.
Response to Arguments
Applicant's arguments filed 05/05/2026 have been fully considered but they are not persuasive.
Applicant’s arguments have been considered but are moot because the new ground of rejection does not rely on references of Plucienniczak and Graciet applied in the prior rejection of record for any teaching or matter specifically challenged in the argument. Current rejection is based on combination of prior art of Kwon and Piatkov as described in the rejection above. The pertinent arguments regarding Kwon will be addressed.
Applicant argues(addressing p. 13-15 of the Remarks) that Kwon “provides no suggestion for enriching within the cell of the target polypeptide with the desired NSAA or even how it can be accomplished” and provides no teaching, suggestion or motivation to degrade proteins within the cell. These arguments are not persuasive because:
In response to applicant's arguments against the references individually, one cannot show nonobviousness by attacking references individually where the rejections are based on combinations of references. See In re Keller, 642 F.2d 413, 208 USPQ 871 (CCPA 1981); In re Merck & Co., 800 F.2d 1091, 231 USPQ 375 (Fed. Cir. 1986). Current rejection is based on combination of prior art of Kwon and Piatkov as described in the rejection above wherein Kwon provides method of generation of the target polypeptide containing NSAA and Piatkov teaches ubiquitin fusion technique to obtain polypeptide with the desired N-terminal amino acid including attachment of ubiquitin to the N-terminal of the polypeptide and coexpression the fusion polypeptide with Ubp1 protease cleaving ubiquitin and Piatkov teaches mechanism of specific degradation of a protein obtained in E. coli depending on the amino acid residue on its generated N-end by ClpS-ClpAP protease system providing motivation to add ubiquitin as removable protective group to the target polypeptide containing NSAA disclosed by Kwon and coexpress it with the UBP1 protease in E. coli for removal of ubiquitin and to expose the N-end amino acid which would enrich the target polypeptide with NSAA since the polypeptide with certain standard amino acids will be degraded by ClpAP protease of E. coli.
Double Patenting
The nonstatutory double patenting rejection is based on a judicially created doctrine grounded in public policy (a policy reflected in the statute) so as to prevent the unjustified or improper timewise extension of the “right to exclude” granted by a patent and to prevent possible harassment by multiple assignees. A nonstatutory double patenting rejection is appropriate where the conflicting claims are not identical, but at least one examined application claim is not patentably distinct from the reference claim(s) because the examined application claim is either anticipated by, or would have been obvious over, the reference claim(s). See, e.g., In re Berg, 140 F.3d 1428, 46 USPQ2d 1226 (Fed. Cir. 1998); In re Goodman, 11 F.3d 1046, 29 USPQ2d 2010 (Fed. Cir. 1993); In re Longi, 759 F.2d 887, 225 USPQ 645 (Fed. Cir. 1985); In re Van Ornum, 686 F.2d 937, 214 USPQ 761 (CCPA 1982); In re Vogel, 422 F.2d 438, 164 USPQ 619 (CCPA 1970); In re Thorington, 418 F.2d 528, 163 USPQ 644 (CCPA 1969).
A timely filed terminal disclaimer in compliance with 37 CFR 1.321(c) or 1.321(d) may be used to overcome an actual or provisional rejection based on nonstatutory double patenting provided the reference application or patent either is shown to be commonly owned with the examined application, or claims an invention made as a result of activities undertaken within the scope of a joint research agreement. See MPEP § 717.02 for applications subject to examination under the first inventor to file provisions of the AIA as explained in MPEP § 2159. See MPEP § 2146 et seq. for applications not subject to examination under the first inventor to file provisions of the AIA . A terminal disclaimer must be signed in compliance with 37 CFR 1.321(b).
The filing of a terminal disclaimer by itself is not a complete reply to a nonstatutory double patenting (NSDP) rejection. A complete reply requires that the terminal disclaimer be accompanied by a reply requesting reconsideration of the prior Office action. Even where the NSDP rejection is provisional the reply must be complete. See MPEP § 804, subsection I.B.1. For a reply to a non-final Office action, see 37 CFR 1.111(a). For a reply to final Office action, see 37 CFR 1.113(c). A request for reconsideration while not provided for in 37 CFR 1.113(c) may be filed after final for consideration. See MPEP §§ 706.07(e) and 714.13.
The USPTO Internet website contains terminal disclaimer forms which may be used. Please visit www.uspto.gov/patent/patents-forms. The actual filing date of the application in which the form is filed determines what form (e.g., PTO/SB/25, PTO/SB/26, PTO/AIA /25, or PTO/AIA /26) should be used. A web-based eTerminal Disclaimer may be filled out completely online using web-screens. An eTerminal Disclaimer that meets all requirements is auto-processed and approved immediately upon submission. For more information about eTerminal Disclaimers, refer to www.uspto.gov/patents/apply/applying-online/eterminal-disclaimer.
Claims 1-8, 10, 12-14, 19, 21 and 22 are rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1-8, 10, 11, 13 and 14 of U.S. Patent No. 11649450 in view of Piatkov (Piatkov et al. PLOS One, 2013, 8, e67952, 1-7).
Claim 1 of instant application is drawn to a method of making a target polypeptide including NSAA comprising genetically modifying the cell to express the target polypeptide with NSAA using engineered amino-acyl tRNA and tRNA pair corresponding to the NSAA wherein the cell expresses the target polypeptide including a standard amino acid or undesired NSAA and wherein a removable protecting group is attached to the target polypeptide such that when it is removed, the N-end amino acid is exposed and wherein the cell expresses an enzyme cleaving the removable protective group and a protease that degrades the target polypeptide when the N-end amino acid is a standard amino acid or undesired NSAA.
Regarding claim 1, claim 1 of reference application teaches a method of degrading polypeptides expressed in a cell from a first nucleic acid encoding a polypeptide with NSAA and a removable protecting group at the N-terminal of the polypeptide, wherein the cell includes the second nucleic acid encoding engineered amino-acyl tRNA synthetase and tRNA pair corresponding to the NSAA, comprising expressing nucleic acids and producing polypeptide with target NSAA, nontarget NSAA or standard amino acid and removing the removable protecting group to expose N-end and degrade polypeptide with nontarget NSAA and standard amino acid at target location with ClpS-ClpAP protease system wherein ClpS has recited sequence with recited mutations.
Thus, claim 1 of reference application teaches the steps and components of instant claim 1, including production of the target polypeptide with NSAA, with undesired NSAA and with standard amino acid at the target location, genetic modification of the cell incorporating foreign nucleic acid encoding engineered amino-acyl tRNA synthetase and tRNA and the target polypeptide with protective group removal of which exposes N-end of the polypeptide and degradation of polypeptides with undesired NSAA or standard amino acid with protease.
Reference claim 1 teaches removing the protective group but does not teach expressing the enzyme cleaving the removable protective group.
Piatkov teaches ubiquitin fusion technique to generate any desired N-terminal amino acid residue by fusion of ubiquitin to the desired N-terminal residue of a polypeptide (p. 1, left column, 3rd paragraph). Piatkov describes expression of Ubp1 deubiquitylase of the yeast Saccharomyces cerevisiae that cleaves the ubiquitin thus generating N-end amino acid.
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to add Piatkov teaching to reference claims and express the UBP1 from Piatkov teaching in the genetically modified cell producing the target polypeptide with NSAA based on reference claim 1. One would have been motivated to do that since Piatkov describes that UBP1 can remove ubiquitin attached to the N-end of the polypeptide and hence generate the open N-end providing desired amino acid on the N-end and ClpS-ClpAP protease system of reference claim 1 degrades the target protein when the N-end amino acid is a standard amino acid or undesired NSAA. A skilled artisan would have reasonably expected success in this combination since reference claim 1 provides method of production of a target polypeptide containing NSAA and removable protective group and Piatkov provides protease to cleave the protective group. Thus, claim 1 of reference application and Piatkov teaching render instant claim 1 obvious.
Claim 2 of instant application is drawn to removable protective group being orthogonal within the cell. Claim 3 of reference application teaches removable protecting group being ubiquitin. Reference claim 13 teaches that the cell is a bacterium and reference claim 14 is drawn to the cell being genetically modified E. coli. Piatkov also teaches ubiquitin as removable protective group for polypeptide expressed in E. coli and mentions that bacteria lack ubiquitin system (p. 2, left column, 2nd paragraph). Therefore the ubiquitin is a protein which is orthogonal to E.coli.
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to add orthogonal to the host cell removable protective group such as ubiquitin to the target polypeptide and express it in bacterial cells such as E. coli as taught by reference claims and Piatkov. One would have been motivated to do that with reasonably expected success since Piatkov describes that bacteria lack the ubiquitin system and addition of ubiquitin to the target polypeptide as protective group allows to control its cleavage with the expressed protease. Thus, reference claims 3, 13 and 14 and Piatkov teaching render instant claim 2 obvious.
Claim 3 of instant application is drawn to the removable protecting group being an enzyme cleavable protecting group. That correspond to reference claim 2 teaching the removable protecting group that is cleavable by a corresponding enzyme. Thus, reference claim 2 and Piatkov teaching render instant claim 3 obvious.
Claim 4 of instant application is drawn to the removable protecting group being a protein that is cleavable by a corresponding enzyme. That correspond to reference claim 2 teaching the removable protecting group being a protein that is cleavable by a corresponding enzyme. Thus, reference claim 2 and Piatkov teaching render instant claim 4 obvious.
Claim 5 of instant application is drawn to the removable protecting group being ubiquitin cleavable by Ubp1. That correspond to claim 3 of reference application. Thus, reference claim 3 and Piatkov teaching render instant claim 5 obvious.
Claim 6 of instant application is directed to genetic modification of the cell by incorporation of foreign nucleic acid encoding the target polypeptide with NSAA and removable protective group attached to the target polypeptide. These features are recited in reference claim 1 as described above and hence reference claim 1 and Piatkov teaching render instant claim 6 obvious.
Claim 7 of instant application is drawn to the detectable moiety attached to the C-end of the target polypeptide. That correspond to claim 4 of reference application. Thus, reference claim 4 and Piatkov teaching render instant claim 7 obvious.
Claim 8 of instant application is drawn to the detectable moiety attached to the C-end of the target polypeptide and being a fluorescent moiety or a reporter protein. That correspond to claim 5 of reference application directed to the detectable moiety attached to the C-end of the polypeptides and being a fluorescent moiety and claim 6 teaching detectable moiety as a reporter protein. Thus, reference claims 5 and 6 and Piatkov teaching render instant claim 8 obvious.
Claim 10 of instant application is directed to genetic modification of the cell by incorporation of foreign nucleic acid encoding the target polypeptide with NSAA and removable protective group attached to the target polypeptide and detectable moiety attached to the C-end of the target polypeptide wherein the NSAA is encoded by a corresponding nonsense or sense codon. Genetic modification of the cell by incorporation of foreign nucleic acid encoding the target polypeptide with NSAA and removable protective group is recited in reference claim 1 as described above. Reference claim 4 is drawn to a detectable moiety attached to the C-end of the target polypeptide. Reference claim 7 recites the NSAA to be encoded by a corresponding nonsense or sense codon. Thus, reference claims 1, 4 and 7 and Piatkov teaching render instant claim 10 obvious.
Claim 12 of instant application is drawn to a foreign nucleic acid encoding an enzyme for cleaving the removable protecting group under the influence of a constitutive or an inducible promoter. That corresponds to reference claim 8. Thus, reference claim 8 and Piatkov teaching render instant claim 12 obvious.
Claim 13 of instant application is directed to adaptor protein coordinating with protease for degrading the target polypeptide with standard amino acid or undesired NSAA on N-end.
Reference claim 1 teaches ClpS-ClpAP protease system to degrade the polypeptide with standard amino acid or undesired NSAA on N-end. Piatkov teaches that ClpS binds to bulky hydrophobic N-terminal residues and delivers targeted N-end substrates to the protease ClpAP for degradation thus serving as an adapter protein (p. 2, left column, 2nd paragraph).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention that ClpS of reference claim 1 is an adaptor protein coordinating with the ClpAP protease for degradation the target polypeptide with standard amino acid or undesired NSAA. One would have been motivated to expect that since Piatkov describes ClpS to bind destabilizing bulky hydrophobic amino acids and delivers targeted N-end substrates to the protease ClpAP for degradation Thus, reference claim 1 and Piatkov teaching render claim 13 obvious.
Claim 14 of instant application is directed to adaptor protein coordinating with protease for degrading the target polypeptide with standard amino acid or undesired NSAA on N-end wherein the adaptor protein is under influence of a constitutive or inducible promoter. Limitation of adaptor protein coordinating with protease for degrading the target polypeptide with standard amino acid or undesired NSAA on N-end was addressed as described for claim 13 above. Additionally, reference claim 10 teaches ClpS being under influence of a constitutive or inducible promoter. Therefore, reference claims 1 and 10 and Piatkov teaching render claim 14 obvious.
Claim 19 of instant application is drawn to the method comprising cell expressing an enzyme cleaving the removable protective group and adaptor protein coordinating with protease for degrading the target polypeptide with standard amino acid or undesired NSAA on N-end wherein the adaptor protein is under influence of a constitutive or inducible promoter.
Reference claim 1 teaches ClpS-ClpAP protease system to degrade the polypeptide with standard amino acid or undesired NSAA on N-end. Piatkov teaches that ClpS binds to bulky hydrophobic N-terminal residues and delivers targeted N-end substrates to the protease ClpAP for degradation thus serving as an adapter protein (p. 2, left column, 2nd paragraph). Reference claim 10 teaches ClpS being under influence of a constitutive or inducible promoter.
Piatkov teaches ubiquitin fusion technique to generate any desired N-terminal amino acid residue by fusion of ubiquitin to the desired N-terminal residue of a polypeptide (p. 1, left column, 3rd paragraph). Piatkov describes expression of Ubp1 deubiquitylase of the yeast Saccharomyces cerevisiae that cleaves the ubiquitin thus generating N-end amino acid.
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to add Piatkov teaching to reference claims and express the UBP1 from Piatkov teaching in the genetically modified cell producing the target polypeptide with NSAA based on reference claim 1. One would have been motivated to do that since Piatkov describes that UBP1 can remove ubiquitin attached to the N-end of the polypeptide and hence generate the open N-end providing desired amino acid on the N-end and ClpS-ClpAP protease system of reference claim 1 degrades the target protein when the N-end amino acid is a standard amino acid or undesired NSAA.
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention that ClpS of reference claim 1 is an adaptor protein coordinating with the ClpAP protease for degradation the target polypeptide with standard amino acid or undesired NSAA. One would have been motivated to expect that since Piatkov describes ClpS to bind destabilizing bulky hydrophobic amino acids and delivers targeted N-end substrates to the protease ClpAP for degradation A skilled artisan would have reasonably expected success in this combination since reference claim 1 provides method of production of a target polypeptide containing NSAA and removable protective group, Piatkov provides protease to cleave the protective group and both reference claim 1 and Piatkov teach ClpS-ClpAP protease system. Thus, claims 1 and 10 of reference application and Piatkov teaching render instant claim 19 obvious.
Claim 21 of instant application is drawn to the method comprising the cell expressing an enzyme cleaving the removable protective group and ClpS-ClpAP protease system that degrades the target polypeptide with standard amino acid or undesired NSAA on N-end.
Reference claim 1 teaches ClpS-ClpAP protease system to degrade the polypeptide with standard amino acid or undesired NSAA on N-end. Piatkov teaches ubiquitin fusion technique to generate any desired N-terminal amino acid residue by fusion of ubiquitin to the desired N-terminal residue of a polypeptide (p. 1, left column, 3rd paragraph). Piatkov describes expression of Ubp1 deubiquitylase of the yeast Saccharomyces cerevisiae that cleaves the ubiquitin thus generating N-end amino acid.
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to add Piatkov teaching to reference claims and express the UBP1 from Piatkov teaching in the genetically modified cell producing the target polypeptide with NSAA based on reference claim 1. One would have been motivated to do that with reasonably expected success since Piatkov describes that UBP1 can remove ubiquitin attached to the N-end of the polypeptide and hence generate the open N-end providing desired amino acid on the N-end and ClpS-ClpAP protease system of reference claim 1 degrades the target protein when the N-end amino acid is a standard amino acid or undesired NSAA. Thus, claim 1 of reference application and Piatkov teaching render instant claim 21 obvious.
Claim 22 of instant application is drawn to the method of claim 1 wherein a detectable moiety is attached to the C-end of the target polypeptide and comprising the cell expressing an enzyme cleaving the removable protective group and adaptor protein for protease for degrading the target polypeptide with standard amino acid or undesired NSAA on N-end and detecting the detectable moiety as a measure of the amount of the target polypeptide with NSAA.
Reference claim 1 teaches ClpS-ClpAP protease system to degrade the polypeptide with standard amino acid or undesired NSAA on N-end. Piatkov teaches that ClpS binds to bulky hydrophobic N-terminal residues and delivers targeted N-end substrates to the protease ClpAP for degradation thus serving as an adapter protein (p. 2, left column, 2nd paragraph). Piatkov teaches ubiquitin fusion technique to generate any desired N-terminal amino acid residue by fusion of ubiquitin to the desired N-terminal residue of a polypeptide (p. 1, left column, 3rd paragraph). Piatkov describes expression of Ubp1 deubiquitylase of the yeast Saccharomyces cerevisiae that cleaves the ubiquitin thus generating N-end amino acid. Reference claim 11 teaches a detectable moiety attached to the C-end of the target polypeptide and detecting the detectable moiety as a measure of the amount of the target polypeptide with NSAA.
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to add Piatkov teaching to reference claims and express the UBP1 from Piatkov teaching in the genetically modified cell producing the target polypeptide with NSAA based on reference claim 1. One would have been motivated to do that since Piatkov describes that UBP1 can remove ubiquitin attached to the N-end of the polypeptide and hence generate the open N-end providing desired amino acid on the N-end and ClpS-ClpAP protease system of reference claim 1 degrades the target protein when the N-end amino acid is a standard amino acid or undesired NSAA.
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention that ClpS of reference claim 1 is an adaptor protein coordinating with the ClpAP protease for degradation the target polypeptide with standard amino acid or undesired NSAA. One would have been motivated to expect that since Piatkov describes ClpS to bind destabilizing bulky hydrophobic amino acids and delivers targeted N-end substrates to the protease ClpAP for degradation A skilled artisan would have reasonably expected success in this combination since reference claim 1 provides method of production of a target polypeptide containing NSAA and removable protective group, Piatkov provides protease to cleave the protective group and both reference claim 1 and Piatkov teach ClpS-ClpAP protease system. Thus, claims 1 and 11 of reference application and Piatkov teaching render instant claim 22 obvious.
Therefore, since instant claims 1-8, 10, 12-14, 19, 21 and 22 encompass the subject matter of the reference claims 1-8, 10, 11, 13 and 14 they are rejected under obviousness double patenting.
Response to Arguments
Applicant's arguments filed 05/05/2026 have been fully considered but they are not persuasive.
Applicant argues that amendment of claim 1 by including subject matter of claim 17 obviates the rejection. These arguments are not persuasive because the double patenting rejection was reconsidered and modified by incorporation of the secondary reference as described above.
Conclusion
No claims are allowed.
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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/L.G.K./Examiner, Art Unit 1653
/SHARMILA G LANDAU/Supervisory Patent Examiner, Art Unit 1653
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