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 .
Continued Examination Under 37 CFR 1.114
A request for continued examination under 37 CFR 1.114, including the fee set forth in 37 CFR 1.17(e), was filed in this application after final rejection. Since this application is eligible for continued examination under 37 CFR 1.114, and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114. Applicant's submission filed on 10/14/2025 has been entered.
Election/Restrictions
The restriction and species election requirement of 11/27/2024 was previously withdrawn following the reply filed on Jan 27, 2025.
Priority
The priority date for this application is: 3/19/2019.
Amendments
This action is in response to papers filed 10/14/2025, in which claims 1, 3, 7 – 8, and 10 were amended, no claims were canceled, and new claims 12 – 19 were added. All of the amendments have been thoroughly reviewed and entered.
Applicant has amended:
claims 1 and 3 to overcome objections; the previous objections are withdrawn. However, new claim objections have been presented in this Office Action.
claims 7 - 8 and 10 to overcome the 112(a) rejection; the previous 112(a) new matter rejection of claims 7 – 8 and the previous 112(a) written description rejection of claims 7-8 and claim 10 has been overcome; the rejection is withdrawn.
Response to Arguments
Applicant’s arguments, see Pgs. 14 onwards, filed 10/14/2025, with respect to:
rejections of claims 1-11 under 35 USC § 103 have been fully considered but are not persuasive for the reasons discussed in this office action. The 35 USC § 103 rejections are maintained. In addition, a new 35 USC § 102 rejection in view of “Sharma” which anticipates claim 1 and some dependent claims and a new 35 USC § 103 for remaining dependent claims has been presented in this office action.
Arguments applicable to newly applied rejections to amended or newly presented claims are addressed below. Arguments that are no longer relevant are not addressed.
Objections and Rejections not reiterated here are withdrawn.
Status of the Claims
Claims 1-19 are under consideration.
Claim Objections
Claims 7 - 8 and 10 are objected to because of the following informalities:
Claims 7 - 8 and 10 recite “…wherein the lysine derivative is selected from the group consisting of: alkynyloxycarbonyl lysine derivative, BOC-lysine (t-butyloxycarboryl -L-lysine) derivative, and fatty acylated lysine;”. The recitation of derivative in the group members is redundant. The following is recommended: “…wherein the lysine derivative is selected from the group consisting of: alkynyloxycarbonyl lysine, BOC-lysine (t-butyloxycarboryl -L-lysine), and fatty acylated lysine;”.
Appropriate correction is required.
Claim Interpretation
Claims 1-3 and 12-19 are directed to sequences carrying single amino acid substitutions at various positions. Shown below are the first 60 nucleotides of instant sequences showing substitutions with respect to the wild-type sequence as in SEQ ID NO: 2. The other wild-type sequence recited in the claims, SEQ ID NO: 1, differs from SEQ ID NO: 2 with a substitution at position 444 only. Numbers on left represent SEQ ID Nos.
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Regarding claim 1, claim 1 requires at a minimum:
one wild-type lysyl-tRNA synthetase sequence to be mutated – wherein the wild-type sequence is: SEQ ID NO: 1 or SEQ ID NO: 2.
The mutations comprise: either the arginine (R) at position 19 is mutated to histidine (H); or the histidine (H) at position 29 is mutated to arginine (R) or both.
Therefore, claim 1 is being given the BRI to comprise the minimum requirements; i.e., SEQ ID NO: 1 with the arginine (R) at position 19 is mutated to histidine (H) OR SEQ ID NO: 1 with the histidine (H) at position 29 is mutated to arginine (R); and may comprise both the R19H and the H29R substitutions and any additional substitutions.
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.
Claim 4 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 include all the limitations of the claim upon which it depends.
Regarding claim 4, claim 4, which is drawn to a polynucleotide, depends from claim 1, which recites “A mutant lysyl-tRNA synthetase” and is a polypeptide/protein/peptide. Polynucleotides and polypeptides are distinct and mutually exclusive molecules. Polynucleotides are composed of nucleotides and not amino acids. Thus claim 4 does not include all the limitations of the claim from which it depends.
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 § 112(a)
Withdrawn Written Description
The rejection of Claims 7-8 and 10, as failing to comply with the written description requirement including New Matter Rejection has been withdrawn.
Response to Arguments
Applicant’s arguments, with respect to rejections under 35 USC § 112(a) have been fully considered and are persuasive. The previous rejections have been withdrawn in view of amendments.
Claim Rejections - 35 USC § 102
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 the appropriate paragraphs of 35 U.S.C. 102 that form the basis for the rejections under this section made in this Office action:
A person shall be entitled to a patent unless –
(a)(1) the claimed invention was patented, described in a printed publication, or in public use, on sale, or otherwise available to the public before the effective filing date of the claimed invention.
Claim(s) 1-10, 12, and 15-17 is/are rejected under 35 U.S.C. 102(a)(1) as being clearly anticipated by Sharma (Chembiochem, 2018 Jan 4;19(1):26-30., Epub 2017 Nov 16, IDS of 11/22/2022, and Supplementary Documents).
Sharma teach that the pyrrolysyl-tRNA synthetase system is known and previously demonstrated to function in the incorporation of unnatural amino acid, e.g., Pyrrolsysine (abstract). By mutating the N-terminal domain of this enzyme, further improvements in the enzyme’s function of recognizing and binding to tRNAPyl and incorporation of Nε-(tert-butoxycarbonyl)-L-lysine up to three-fold more than the wild type was identified. See abstract.
Thus, Sharma teaches a mutant lysyl-tRNA synthetase with improved functionality.
Regarding claims 1 and 16, Sharma teach the wild-type sequence of the enzyme, pyrrolysyl-tRNA synthetase (M. mazei PylRS, supplementary materials, section 5., pg. S-5), which is an exact match with instant SEQ ID NO 1:
RESULT 1
MMAZEIPYLRS
Query Match 100.0%; Score 2336; DB 1; Length 454;
Best Local Similarity 100.0%;
Matches 454; Conservative 0; Mismatches 0; Indels 0; Gaps 0;
Qy 1 MDKKPLNTLISATGLWMSRTGTIHKIKHHEVSRSKIYIEMACGDHLVVNNSRSSRTARAL 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1 MDKKPLNTLISATGLWMSRTGTIHKIKHHEVSRSKIYIEMACGDHLVVNNSRSSRTARAL 60
Qy 61 RHHKYRKTCKRCRVSDEDLNKFLTKANEDQTSVKVKVVSAPTRTKKAMPKSVARAPKPLE 120
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 61 RHHKYRKTCKRCRVSDEDLNKFLTKANEDQTSVKVKVVSAPTRTKKAMPKSVARAPKPLE 120
Qy 121 NTEAAQAQPSGSKFSPAIPVSTQESVSVPASVSTSISSISTGATASALVKGNTNPITSMS 180
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 121 NTEAAQAQPSGSKFSPAIPVSTQESVSVPASVSTSISSISTGATASALVKGNTNPITSMS 180
Qy 181 APVQASAPALTKSQTDRLEVLLNPKDEISLNSGKPFRELESELLSRRKKDLQQIYAEERE 240
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 181 APVQASAPALTKSQTDRLEVLLNPKDEISLNSGKPFRELESELLSRRKKDLQQIYAEERE 240
Qy 241 NYLGKLEREITRFFVDRGFLEIKSPILIPLEYIERMGIDNDTELSKQIFRVDKNFCLRPM 300
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 241 NYLGKLEREITRFFVDRGFLEIKSPILIPLEYIERMGIDNDTELSKQIFRVDKNFCLRPM 300
Qy 301 LAPNLYNYLRKLDRALPDPIKIFEIGPCYRKESDGKEHLEEFTMLNFCQMGSGCTRENLE 360
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 301 LAPNLYNYLRKLDRALPDPIKIFEIGPCYRKESDGKEHLEEFTMLNFCQMGSGCTRENLE 360
Qy 361 SIITDFLNHLGIDFKIVGDSCMVYGDTLDVMHGDLELSSAVVGPIPLDREWGIDKPWIGA 420
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 361 SIITDFLNHLGIDFKIVGDSCMVYGDTLDVMHGDLELSSAVVGPIPLDREWGIDKPWIGA 420
Qy 421 GFGLERLLKVKHDFKNIKRAARSESYYNGISTNL 454
||||||||||||||||||||||||||||||||||
Db 421 GFGLERLLKVKHDFKNIKRAARSESYYNGISTNL 454
Sharma teaches that the enzyme, further comprises mutations such as R19H mutations (The identified mutations are R19H/H29R/T122S, 4th line of abstract; R1-7mm PylRS in Table S1).Thus, Sharma teaches a mutant lysyl-tRNA synthetase, wherein the amino acid sequence of the wild-type lysyl-tRNA synthetase consists of SEQ ID NO: 1 wherein the arginine (R) at position 19 is mutated to histidine (H); fulfilling the minimum requirements for claim 1. Sharma teaches other mutations in the enzyme, e.g., the histidine (H) at position 29 is mutated to arginine (R).
Regarding claim 2, Sharma teaches further mutations in the mutant lysyl-tRNA synthetase discussed in claim 1. See the list of mutations from Supplementary table of Sharma:
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Thus, Sharma teaches further mutations that comprise mutations from this group: isoleucine(I) at position 26, threonine (T) at position 122, Leucine (L) at position 309, Cysteine at position 348 (C), Tyrosine at position 384 (Y), as required by claim 2.
Regarding claims 3, 12, and 17, Sharma teaches SEQ ID NO: 3 (R2-23mm PylRS in Table S1).
All sequences recited in claim 3 have not been examined because they are recited as being optional (any one of..).
Regarding claims 4 - 5, Sharma teaches the nucleotide sequence of the wild-type and primers for mutants, (pg. S-4); i.e., the isolated polynucleotide encoding a mutant lysyl-tRNA synthetase (claim 4) and a plasmid encoding the same (6. Construction of pEVOL-R3-11 AcKRS and pEVOL-R3-11 AcdKRSa, pg. S-6); i.e., a vector (claim 5).
Regarding claim 6, Sharma teaches co-transforming E. coli Top10 cells with the vector described (pg. S-7); i.e., a host cell containing a vector that comprises a polynucleotide that encodes the mutant lysyl-t-RNA synthetase.
Regarding claim 7, Sharma teaches E. coli (a host cell) comprising:(a) a mutant lysyl-tRNA synthetase (as discussed above); and (b) in a vector wherein the vector, pEVOL-PylRS, (pEVOL-PylRS is derived from a pEVOL vector developed by Schultz et al.[10] was constructed to contain genes coding tRNAPyl, middle of para on pg. 3; and vector map, pg. S-3) (an orthogonal tRNA capable of binding to a lysine derivative in the presence of the mutant lysyl-tRNA synthetase); and optionally (c) sfGFP134X, where X is the site for BocK, AcK or AcdK, which are lysine derivatives(for e.g., sfGFP134X on pg. S-7) (a target nucleic acid sequence encoding a target protein (GFP), which includes a codon recognized by the orthogonal tRNA at a position for introducing a lysine derivative; wherein the lysine derivative is BOC-lysine (t-butyloxycarboryl -L-lysine).
Other derivatives recited in claim 7 have not been examined because they are recited as being optional (selected from the group consisting of..).
Regarding claim 8, Sharma teach an improved expression system for ncAA incorporation (see summary line, end of pg. 5). Further, the discussion for claim 7 is incorporated herein.
Claim Interpretation for claim 9, " kit " in claim 9 is being interpreted to encompass any collection of parts and reagents that includes all of the elements of the claims including a suitable container to contain the collection because the specification does not define this term. Any further interpretation of the word is considered an “intended use” and does not impart any further structural limitation of on the claimed subject matter.
Regarding claim 9, Sharma does not expressly teach a “kit”. However, as discussed above, Sharma teach all the components of claim 9. One of skill in the art knows these must be contained in a container.
Claim Interpretation for claim 10, the claim will be interpreted as a method, wherein the step is: providing the mutant lysyl-tRNA synthetase of claim 1 to a host cell. This interpretation is consistent with the specification pg. 19, where various host cells are discussed but no in vitro or any other examples are discussed.
Regarding claim 10, that recites A method for incorporating an unnatural amino acid into a target protein or for preparing a target protein containing an unnatural amino acid, comprising a step of: … is being interpreted as: a method for incorporating an unnatural amino acid into a target protein or for preparing a target protein containing an unnatural amino acid by providing the mutant lysyl-tRNA synthetase of claim 1;wherein the unnatural amino acid is a BOC-lysine (t- butyloxycarboryl -L-lysine), to a cell.
Sharma teaches a method of incorporation of several unnatural amino acids (supplementary methods) using variants of pyrrolysyl-tRNA synthetase/tRNAPyl pairs in E.coli (Summary of Invention). Sharma teaches the steps involved in a method to incorporate such unnatural amino acids into a protein as discussed for claims 7 and 8. See also citations: 11. The Expression of Ub48BocK and Ub, pg. S-8 and Fig. S6. Thus, Sharma teaches a method of using a mutant lysyl-tRNA synthetase for incorporating an unnatural amino acid into a target protein or for preparing a target protein containing an unnatural amino acid, as per the requirements of claim 10.
Regarding claim 12, see discussion for claim 3.
Regarding claim 15, Sharma teaches a clone of mutant lysyl-tRNA synthetase with mutation of R19H in the amino acid sequence corresponding to the wild-type lysyl-tRNA synthetase (R1-7mm PylRS in Table S1) as discussed for claim 1.
See SEQ ID NO: 1 as Qy below and SEQ ID NO: 7 as Db with the first 60 bases shown:
Qy 1 MDKKPLNTLISATGLWMSRTGTIHKIKHHEVSRSKIYIEMACGDHLVVNNSRSSRTARAL 60
|||||||||||||||||| |||||||||||||||||||||||||||||||||||||||||
Db 1 MDKKPLNTLISATGLWMSHTGTIHKIKHHEVSRSKIYIEMACGDHLVVNNSRSSRTARAL 60
Regarding claim 16, Sharma teaches a clone of mutant lysyl-tRNA synthetase with mutations of R19H and H29R in the amino acid sequence corresponding to the wild-type lysyl-tRNA synthetase (R2-23mm PylRS in Table S1).
See SEQ ID NO: 1 as Qy below and SEQ ID NO: 6 as Db with the first 60 bases shown:
Qy 1 MDKKPLNTLISATGLWMSRTGTIHKIKHHEVSRSKIYIEMACGDHLVVNNSRSSRTARAL 60
|||||||||||||||||| ||||||||| |||||||||||||||||||||||||||||||
Db 1 MDKKPLNTLISATGLWMSHTGTIHKIKHREVSRSKIYIEMACGDHLVVNNSRSSRTARAL 60
Regarding claim 17, see discussion for claim 3 and claim 12.
Thus, Sharma clearly anticipates claims 1-10, 12, and 15-17.
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.
Claim 11 is rejected under 35 U.S.C. 103 as being unpatentable over Sharma (Chembiochem, 2018 Jan 4;19(1):26-30., Epub 2017 Nov 16, IDS of 11/22/2022) as applied to claims 1-10, 12, and 15-17 above in view of Chin (WO 2012/038706 A1, of record).
Regarding claim 11, the teachings of Sharma as discussed above are incorporated herein. Sharma further teach the portability of lysyl-tRNA synthetase between cell types. See recitation from pg. 2:
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Sharma does not teach a host cell genome with a polynucleotide encoding a mutant lysyl-t-RNA synthetase integrated into its genome.
However, before the effective filing date of instant, Chin had taught that the lysyl-tRNA synthetase system was known and previously demonstrated to function in the incorporation of unnatural amino acids. See recitation from pg. 42 below:
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Chin had taught a host cell genome with a polynucleotide encoding a mutant lysyl-t-RNA synthetase integrated into its genome (We have tested constructs in cell culture (Drosophila S2) and subsequently used those constructs to make transgenic lines (with the constructs genomically integrated), Chin lines 20-21 of pg.51).
It would have been obvious to one of ordinary skill in the art before the time of the invention to have made a stable integrand out of the polynucleotide of claim 4 for the advantage to be gained from a stable and inheritable genetic modification, and arrive at the limitation required by instant claim 11. For one of ordinary skill in the art, such integration would merely mean a combination of prior art elements, a known polynucleotide treated in the same manner as another known polynucleotide by a known method, wherein the latter was disclosed by Chin, to make a novel yet functional host cell with an integrated polynucleotide encoding an enzyme, lysyl-tRNA synthetase. One of ordinary skill in the art would have a reasonable expectation of success in doing so since both Sharma and Chin teach orthogonal tRNA synthetases in host cells for non-canonical biological activities. See MPEP 2144 II and 2143 I.(A).
Thus, Sharma in view of Chin make obvious instant claim 11.
Claim(s) 13 and 18 are rejected under 35 U.S.C. 103 as being unpatentable over Sharma (Chembiochem, 2018 Jan 4;19(1):26-30., Epub 2017 Nov 16, IDS of 11/22/2022) as applied to claims 1-10, 12, and 15-17 above in view of Schneider (Schneider et al., Chembiochem, 2013 Nov 4;14(16):2114-8.).
Regarding claims 13 and 18, which require SEQ ID NO: 4, i.e., four mutations R19H, H29R, I122S, and C84F in SEQ ID NO: 1, Sharma disclose clones R-23, 3-7, 3-8, 3-11, 3-12, and 3-14 (Supplementary Table S1 on pg. 3) as containing at least the three mutations: R19H, H29R, and I122S in SEQ ID NO: 1. Sharma disclose vectors, pEVOL that contain at least these mutations R19H, H29R, and I122S, as discussed for claim 5.
Sharma further state about their improved results, such mutations in the N-terminal non-catalytic domain (title, first para on pg. 2), while effective in improving efficacy of lysyl-tRNA synthetases, a dramatically higher yield was not obtained. See recitation below from bottom of pg.4:
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Sharma also did not disclose any mutations in the catalytic domain, such as Y384F, as required by instant claim.
However, before the effective filing date of instant invention, Schneider had taught Y384F. See recitation from middle of pg. 1 of Schneider:
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As per this recitation of Schneider, Y384F was one of a combination of three mutations that were tested and found to be effective lysyl-tRNA synthetases especially to improve efficacy of reactive derivatives.
By incorporating R19H, H29R, and I122S as already demonstrated by Sharma to be effective in improving lysyl-tRNA synthetase function to a certain extent, one would also be motivated to incorporate at least one mutation within the catalytic domain, such as Y384F mutation, as shown by Schneider, into instant SEQ ID NO: 1 and arrive at instant SEQ ID NO: 4, for the advantage of further improved efficacy. By doing so, one would arrive at instant SEQ ID NO: 4. See alignment below:
Qy= SEQ ID NO: 1
Db= SEQ ID NO: 4
US-17-441-101-4
Query Match 99.1%; Score 2315; DB 1; Length 454;
Best Local Similarity 99.1%;
Matches 450; Conservative 2; Mismatches 2; Indels 0; Gaps 0;
Qy 1 MDKKPLNTLISATGLWMSRTGTIHKIKHHEVSRSKIYIEMACGDHLVVNNSRSSRTARAL 60
|||||||||||||||||| ||||||||| |||||||||||||||||||||||||||||||
Db 1 MDKKPLNTLISATGLWMSHTGTIHKIKHREVSRSKIYIEMACGDHLVVNNSRSSRTARAL 60
Qy 61 RHHKYRKTCKRCRVSDEDLNKFLTKANEDQTSVKVKVVSAPTRTKKAMPKSVARAPKPLE 120
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 61 RHHKYRKTCKRCRVSDEDLNKFLTKANEDQTSVKVKVVSAPTRTKKAMPKSVARAPKPLE 120
Qy 121 NTEAAQAQPSGSKFSPAIPVSTQESVSVPASVSTSISSISTGATASALVKGNTNPITSMS 180
|:||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 121 NSEAAQAQPSGSKFSPAIPVSTQESVSVPASVSTSISSISTGATASALVKGNTNPITSMS 180
Qy 181 APVQASAPALTKSQTDRLEVLLNPKDEISLNSGKPFRELESELLSRRKKDLQQIYAEERE 240
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 181 APVQASAPALTKSQTDRLEVLLNPKDEISLNSGKPFRELESELLSRRKKDLQQIYAEERE 240
Qy 241 NYLGKLEREITRFFVDRGFLEIKSPILIPLEYIERMGIDNDTELSKQIFRVDKNFCLRPM 300
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 241 NYLGKLEREITRFFVDRGFLEIKSPILIPLEYIERMGIDNDTELSKQIFRVDKNFCLRPM 300
Qy 301 LAPNLYNYLRKLDRALPDPIKIFEIGPCYRKESDGKEHLEEFTMLNFCQMGSGCTRENLE 360
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 301 LAPNLYNYLRKLDRALPDPIKIFEIGPCYRKESDGKEHLEEFTMLNFCQMGSGCTRENLE 360
Qy 361 SIITDFLNHLGIDFKIVGDSCMVYGDTLDVMHGDLELSSAVVGPIPLDREWGIDKPWIGA 420
|||||||||||||||||||||||:||||||||||||||||||||||||||||||||||||
Db 361 SIITDFLNHLGIDFKIVGDSCMVFGDTLDVMHGDLELSSAVVGPIPLDREWGIDKPWIGA 420
Qy 421 GFGLERLLKVKHDFKNIKRAARSESYYNGISTNL 454
||||||||||||||||||||||||||||||||||
Db 421 GFGLERLLKVKHDFKNIKRAARSESYYNGISTNL 454
It would have been obvious to one of ordinary skill in the art before the instant invention to modify the lysyl-tRNA synthetase of Sharma comprising a R19H, H29R, and I122S mutations known to improve efficacy of lysyl-tRNA synthetase with the further addition of Y384F also known to improve efficacy of lysyl-tRNA synthetase as shown by Schneider because it is prima facie obvious to combine known mutations into a single composition. The MPEP states:
"It is prima facie obvious to combine two compositions each of which is taught by the prior art to be useful for the same purpose, in order to form a third composition to be used for the very same purpose.... [T]he idea of combining them flows logically from their having been individually taught in the prior art." In re Kerkhoven, 626 F.2d 846, 850, 205 USPQ 1069, 1072 (CCPA 1980).
Schneider teaches three mutations that result in improved efficacy. As such, there are a finite number (three) of identified possibilities (of mutations) presented by Schneider. It would have been obvious to try all three mutations and pick one to combine with existing mutations because both Sharma and Schneider demonstrated their mutations alone result in measured and predictable outcome and by simply “trying” a combination of mutations, one would land on an optimal combination. There would have been a reasonable expectation of success in making this modification because both the mutations of Sharma and Schneider are known mutations that effectively increase efficacy of lysyl-tRNA synthetases to some extent. One would be motivated to make this combination of mutations, because Sharma’s mutations are in the non-catalytic domain, known to effect association, and Schneider’s mutations are in the catalytic domain known to improve functionality. One of skill in the art can see that the combination would improve efficacy to a greater extent than a combination of mutations restricted to one domain or the other. MPEP 2144 II and 2143 I A and E.
Thus, Sharma in view of Schneider make obvious mutations R19H, H29R, T122S, and Y384F, i.e., SEQ ID NO: 4, as required by claims 13 and 18.
Claim(s) 14 and 19 are rejected under 35 U.S.C. 103 as being unpatentable over Sharma (Chembiochem, 2018 Jan 4;19(1):26-30., Epub 2017 Nov 16, IDS of 11/22/2022) as applied to claims 1-10, 12, and 15-17 above in view of Söll (Umehara et al. / FEBS Letters 586 (2012) 729–733).
Regarding claims 14 and 19, which require SEQ ID NO: 5, i.e., mutations R19H, H29R, L309A, and C348S in SEQ ID NO: 1, Sharma disclose clones, R-23, R3-3, 3-7, 3-8, 3-10, 3-11, 3-12, and 3-14 (Supplementary Table S1 on pg. 3) and the vectors used to make them, pEVOL-R3-11 AcKRS and pEVOL-R3-11 AcdKRS, contain at least mutations R19H and H29R.
Sharma further discuss about their improved results, such mutations while effective in improving efficacy of lysyl-tRNA synthetases, a dramatically higher yield was not obtained. See recitation below from bottom of pg.4:
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Further, Sharma disclose that the vectors used to create these clones, are based off the original pEVOL vectors made by Söll. See recitation from middle of pg. 4 of Sharma:
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As per this recitation of Soll, pg.730, section 3.2, L309A and C348S, were mutations that were tested and found to be effective lysyl-tRNA synthetases:
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By incorporating R19H and H29R as already demonstrated by Sharma to be effective in improving lysyl-tRNA synthetase function to a certain extent, one would also incorporate L309A and C348S mutations within the catalytic domain, as shown by Söll, into instant SEQ ID NO: 1 and arrive at instant SEQ ID NO: 5, for the advantage of further improved efficacy. By doing so, one would arrive at instant SEQ ID NO: 5. See alignment below:
Qy= SEQ ID NO: 1
Db= SEQ ID NO: 5
US-17-441-101-5
Query Match 98.8%; Score 2308; DB 1; Length 454;
Best Local Similarity 99.1%;
Matches 450; Conservative 0; Mismatches 4; Indels 0; Gaps 0;
Qy 1 MDKKPLNTLISATGLWMSRTGTIHKIKHHEVSRSKIYIEMACGDHLVVNNSRSSRTARAL 60
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Db 1 MDKKPLNTLISATGLWMSHTGTIHKIKHREVSRSKIYIEMACGDHLVVNNSRSSRTARAL 60
Qy 61 RHHKYRKTCKRCRVSDEDLNKFLTKANEDQTSVKVKVVSAPTRTKKAMPKSVARAPKPLE 120
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Db 61 RHHKYRKTCKRCRVSDEDLNKFLTKANEDQTSVKVKVVSAPTRTKKAMPKSVARAPKPLE 120
Qy 121 NTEAAQAQPSGSKFSPAIPVSTQESVSVPASVSTSISSISTGATASALVKGNTNPITSMS 180
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Db 121 NTEAAQAQPSGSKFSPAIPVSTQESVSVPASVSTSISSISTGATASALVKGNTNPITSMS 180
Qy 181 APVQASAPALTKSQTDRLEVLLNPKDEISLNSGKPFRELESELLSRRKKDLQQIYAEERE 240
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Db 181 APVQASAPALTKSQTDRLEVLLNPKDEISLNSGKPFRELESELLSRRKKDLQQIYAEERE 240
Qy 241 NYLGKLEREITRFFVDRGFLEIKSPILIPLEYIERMGIDNDTELSKQIFRVDKNFCLRPM 300
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Db 241 NYLGKLEREITRFFVDRGFLEIKSPILIPLEYIERMGIDNDTELSKQIFRVDKNFCLRPM 300
Qy 301 LAPNLYNYLRKLDRALPDPIKIFEIGPCYRKESDGKEHLEEFTMLNFCQMGSGCTRENLE 360
|||||||| |||||||||||||||||||||||||||||||||||||| ||||||||||||
Db 301 LAPNLYNYARKLDRALPDPIKIFEIGPCYRKESDGKEHLEEFTMLNFSQMGSGCTRENLE 360
Qy 361 SIITDFLNHLGIDFKIVGDSCMVYGDTLDVMHGDLELSSAVVGPIPLDREWGIDKPWIGA 420
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Db 361 SIITDFLNHLGIDFKIVGDSCMVYGDTLDVMHGDLELSSAVVGPIPLDREWGIDKPWIGA 420
Qy 421 GFGLERLLKVKHDFKNIKRAARSESYYNGISTNL 454
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Db 421 GFGLERLLKVKHDFKNIKRAARSESYYNGISTNL 454
It would have been obvious to one of ordinary skill in the art before the instant invention to modify the lysyl-tRNA synthetase of Sharma comprising a R19H mutation with H29R mutation known to improve efficacy of lysyl-tRNA synthetase with the further addition of L309A and C348S also known to improve efficacy of lysyl-tRNA synthetase as shown by Söll because it is prima facie obvious to combine known mutations into a single composition. The MPEP states:
"It is prima facie obvious to combine two compositions each of which is taught by the prior art to be useful for the same purpose, in order to form a third composition to be used for the very same purpose.... [T]he idea of combining them flows logically from their having been individually taught in the prior art." In re Kerkhoven, 626 F.2d 846, 850, 205 USPQ 1069, 1072 (CCPA 1980)
There would have been a reasonable expectation of success in making this modification because both the mutations of Sharma and Söll are known mutations that effectively increase efficacy of lysyl-tRNA synthetases. One would be motivated to make this combination of mutations, because Sharma’s mutations are in the non-catalytic domain, known to effect association, and Söll’s mutations are in the catalytic domain known to improve functionality. One of skill in the art can see that the combination would improve efficacy to a greater extent than a combination of mutations restricted to one domain or the other. MPEP 2144 II and 2143 I A.
Thus, Sharma in view of Söll make obvious mutations R19H, H29R, L309A, and C348S, i.e., SEQ ID NO: 5, as required by claims 14 and 19.
Therefore the invention as a whole would have been prima facie obvious to one ordinary skill in the art before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains.
III. Maintained 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 1-11 are rejected under 35 U.S.C. 103 as being unpatentable over Chin (WO 2012/038706 A1) in view of Neumann (Neumann et al., Nature chemical biology, 2008-04, Vol.4 (4), p.232-234) and Wikipedia (Wikipedia webpage, Retrieved from the Internet on 2025-03-19: https://en.wikipedia.org/wiki/Amino_acid, cited from a 2015 reference, 29 pages).
Claims 1-3 are directed to sequences carrying single amino acid substitutions at various positions. Shown below are the first 60 nucleotides of instant sequences showing substitutions with respect to the wild-type sequence as in SEQ ID NO: 2. The other wild-type sequence recited in the claims, SEQ ID NO: 1, differs from SEQ ID NO: 2 with a substitution at position 444 only.
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Chin teach that the lysyl-tRNA synthetase system is known and previously demonstrated to function in the incorporation of unnatural amino acids. See recitation from pg. 42 below:
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Further, the reference of Neumann cited by Chin in the recitation above (13) teaches that the structure of the active site of M. mazei lysyl-tRNA synthetase (PylRS bound to pyrrolysine, Fig. 2) is known and further, the active site residues of this enzyme are conserved between species. Neumann create a library of 108 mutants to decipher functional mutants of lysyl-tRNA synthetase such as those that can incorporate Nε-acetyllysine into proteins and from these studies determine that the active site residues lie within the hydrophobic binding pocket of lysyl-tRNA synthetase (pg. 233, left column). Finally, with mutations in the hydrophobic pocket, Neumann are enabled to carry out incorporation of Nε-acetyllysine, with high translational fidelity and efficiency, into proteins expressed in E. coli.
Regarding claims 1 and 2, Chin and Neumann teach that the enzyme, lysyl-tRNA synthetase, relies on a finite number of amino acids for its synthetase function. As such, there are finite number of identified amino acids that fall outside of the functional site as evidenced by the sequence of lysyl-tRNA synthetase taught by Chin (such as SEQ ID NO: 18).
Neither Chin nor Neumann teach the two specific single amino acid mutations as required by claim 1 or the various single amino acid mutations required by claim 2.
However, well before the effective filing date of instant application, the physico-chemical properties of amino acids were well-characterized. With respect to the amino acids arginine and histidine that are recited in claim 1, it was known that these two amino acids are similar. See below, taken from pg. 2 of Wikipedia.
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It would have been obvious to one of ordinary skill in the art before the time of the invention to have made a conservative substitution of histidine ↔ arginine in the regions outside of the hydrophobic binding pocket and arrive at the sequence required by instant claim 1. One of skill in the art would be motivated to make: 1. such a conservative substitution outside of the hydrophobic binding pockets for the advantage of having a novel sequence, not naturally found in nature, and still retain the function of incorporating a wide variety of unnatural amino acids into proteins with site specificity, because the active site of the enzyme, lysyl-tRNA synthetase, is unaltered. Simultaneously, one of ordinary skill in the art would 2. pursue substitutions at specific residues within the hydrophobic pocket for the advantage of changing the amino acid specificity of the enzyme in order to recognize an unnatural amino acid. For one of ordinary skill in the art, such substitutions would merely mean pursuing a finite number of possible solutions such as substituting within SEQ ID NO: 18 disclosed by Chin, to make a novel yet functional enzyme, lysyl-tRNA synthetase, because Chin and Neumann demonstrates where the function of this enzyme lies. One of ordinary skill in the art would have a reasonable expectation of success in doing so since both Neumann and Chin teach orthogonal tRNA synthetases for non-canonical biological activities further indicating where the active site of the enzyme lies. See MPEP 2144 II and 2143 I.(A) and (E).
Thus, Chin in view of Neumann and Wikipedia make obvious mutations at position 19 arginine and/or and 29 histidine, as required by claim 1 and further mutations at isoleucine(I) at position 26, threonine (T) at position 122, Leucine (L) at position 309, Cysteine at position 348 (C), Tyrosine at position 384 (Y), as required by claim 2.
Regarding claim 3, since claim 3 recites SEQ ID NO: 7, and SEQ ID NO: 7 is SEQ ID NO: 1 wherein arginine (R) at position 19 is mutated to histidine (H), the rejection of claim 1 applies to the rejection of claim 3.
Other sequences recited in claim 3 have not been examined because they are recited as being optional (any one of..).
Regarding claims 4 and 8, Chin teaches the isolated polynucleotide encoding a mutant lysyl-tRNA synthetase (claim 4) and an expression system comprising: (a) the mutant lysyl-tRNA synthetase of claim 1; and (b) an orthogonal tRNA capable of binding to a lysine derivative in the presence of the mutant lysyl-tRNA synthetase; and optionally (c) a target nucleic acid sequence encoding a target protein, which includes a codon recognized by the orthogonal tRNA at a position for introducing a lysine derivative wherein…BOC-lysine (claim 8) (An expression system comprising a nucleic acid according to any of claims 1 to 11; said system further comprising a nucleotide sequence encoding a PylRS capable of aminoacylating the tRNAPyl, Chin claim 12; BOC-lysine incorporation, Figure 14).
Regarding claim 5, Chin teaches a vector containing a polynucleotide that encodes a mutant lysyl-t-RNA synthetase (tRNA synthetase were used to amplify fragments from the codon-optimized ATP-PylS template, assembled by overlap PCR and recloned into the pMbPylRS vector, Chin last line of pg. 28).
Regarding claim 6, Chin teaches a host cell containing a vector that comprises a polynucleotide that encodes a mutant lysyl-t-RNA synthetase. (A eukaryotic cell comprising a nucleic acid according to any of claims 1 to 11 or an expression system according to claim 12 or claim 13, Chin claim 14).
Regarding claim 7, Chin teaches a host cell comprising:(a) a mutant lysyl-tRNA synthetase; and (b) an orthogonal tRNA capable of binding to a lysine derivative in the presence of the mutant lysyl-tRNA synthetase; and optionally (c) a target nucleic acid sequence encoding a target protein, which includes a codon recognized by the orthogonal tRNA at a position for introducing a lysine derivative; wherein… (A eukaryotic cell comprising a nucleic acid according to any of claims 1 to 11 or an expression system according to claim 12 or claim 13, Chin claim 14; BOC-lysine incorporation in Drosophila, Figure 14)).
Regarding claim 9, Chin teaches a kit comprising (a) a container, and (b) a mutant lysyl-tRNA synthetase or a polynucleotide encoding the mutant lysyl-tRNA synthetase or a vector containing the polynucleotide located in the container (Chin lines bridging pgs.18-19).
Regarding claim 10, that recites A method for incorporating an unnatural amino acid into a target protein or for preparing a target protein containing an unnatural amino acid, comprising a step of: … is being interpreted as: a method for incorporating an unnatural amino acid into a target protein or for preparing a target protein containing an unnatural amino acid.
Chin teaches a method of incorporation of several unnatural amino acids (Fig. 1A) using variants of pyrrolysyl-tRNA synthetase/tRNAPyl pairs in yeast (Summary of Invention). Chin teaches the steps involved in a method to incorporate such unnatural amino acids into a protein (pg. 9, lines 14 till end of page). See recitation from Chin pg. 9, lines 29-32: wherein the unnatural amino acid to be incorporated is an alkyne-containing amino acid or a posttranslationally modified amino acid or an amino acid containing bio-orthogonal chemical handles or a photo-caged amino acid or a photo-crosslinking amino acid. Further, Chin teaches nucleic acid sequences encoding a tRNA and encoding tRNA synthetase orthogonal to the yeast cell, said nucleotide sequence operably linked to a promoter capable of directing transcription by eukaryotic RNA polymerase III (pg. 19, 1st 3 lines). Thus, Chin teaches a method of using a mutant lysyl-tRNA synthetase for incorporating an unnatural amino acid into a target protein or for preparing a target protein containing an unnatural amino acid, as per the requirements of claim 10.
Regarding claim 11, Chin teaches a host cell genome with a polynucleotide encoding a mutant lysyl-t-RNA synthetase integrated into its genome (We have tested constructs in cell culture (Drosophila S2) and subsequently used those constructs to make transgenic lines (with the constructs genomically integrated), Chin lines 20-21 of pg.51).
Thus, Chin in view of Neumann and Wikipedia make obvious instant claims 1-11.
Therefore the invention as a whole would have been prima facie obvious to one ordinary skill in the art before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains.
Response to Arguments
Applicant's arguments filed 14th Oct 2025 to claim rejections under 35 USC § 103 have been fully considered but are not persuasive. Applicants argue:
1. Pgs. 9-11: the Prior Art Fails to Teach ALL the Claimed Mutations (Chin, Neuman, and Wikipedia), and the Office Action does not explain why a PHOSITA would have had some apparent reason to modify.
and
2. Pgs. 12-14: Unexpected Technical Effects are shown.
Applicants arguments are not persuasive for the reasons discussed below.
Regarding 1), Applicant’s arguments that the references do not teach the exact mutations of the invention, for e.g., R19H and H29R, is not persuasive because:
i. Current version of MPEP, MPEP § 2144.05(II)(B) has been revised to state that “after KSR, the presence of a known result-effective variable would be one, but not the only, motivation for a person of ordinary skill in the art to experiment to reach another workable product or process.” Therefore, the MPEP does not require that every limitation be taught in the prior art or a reference explicitly teach or suggest how to arrive at such a limitation. The knowledge in the art is sufficient for one of skill in the art to modify an available sequence, such as the sequence taught by Chin. Further the practice of making point mutations is a mature field and KSR foreclosed the argument that a specific teaching is required.
ii. In the instant case, the secondary reference of Neumann indicated the active site of the t-RNA synthetase, and specifically the amino acids within this site. Therefore, changing such sites would alter the function of the enzyme. Neumann did not teach away from mutating sites outside the active site; such as instant R19H and H29R. Therefore, and like, the prior Office Action had pointed out that “there are a finite number of identified, predictable solutions.” to pursue, MPEP 2143 I E. Further, Applicants concede on Remarks, middle of pg.9, there are 30 arginines and 10 histidines to pursue, as a finite number of possibilities to try.
iii. Also See MPEP 2144.05 II and In re Williams, 36 F.2d 436, 438, 4 USPQ 237 (CCPA 1929) ("It is a settled principle of law that a mere carrying forward of an original patented conception involving only change of form, proportions, or degree, or the substitution of equivalents doing the same thing as the original invention, by substantially the same means, is not such an invention as will sustain a patent, even though the changes of the kind may produce better results than prior inventions.").
Regarding 2), Arguments about Unexpected results is not found persuasive. As MPEP states, any differences between the claimed invention and the prior art may be expected to result in some differences in properties. The issue is whether the properties differ to such an extent that the difference is really unexpected." See MPEP §716.02. In the instant case, Applicants have created point mutations and shown that such change has resulted in a (small) change in activity. This is expected.
See In re Klosak, 455 F.2d 1077, 1080 (CCPA 1972): "It is not enough to show that results are obtained which differ from those obtained in the prior art: that difference must be shown to be an unexpected difference".
See In re Baxter Tavenol Labs., 952 F.2d 388,392 (Fed. Cir. 1991): "When unexpected results are used as evidence of nonobviousness, the results must be shown to be unexpected compared with the closest prior art."
In view of the foregoing, and because Applicants have not provided any evidence showing there was no reasonable expectation of success, Applicants arguments are not dispositive. The rejection is maintained.
Conclusion
No claims are allowed.
Correspondence
Any inquiry concerning this communication or earlier communications from the examiner should be directed to SHABANA MEYERING, Ph.D. whose telephone number is (703)756-4603. The examiner can normally be reached M - F: 9am to 5pm EST.
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/SHABANA S MEYERING/Examiner, Art Unit 1635
/CATHERINE KONOPKA/Primary Examiner, Art Unit 1635