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 .
DETAILED ACTION
This application is a 371 of PCT/EP2021/068279.
The response filed on September 29, 2025 has been entered.
Election/Restrictions
Applicant's election with traverse of Group I with a species election of (1) SEQ ID NO:1 (MaPylRS) as the parent enzyme (parent aaRS), (2) V168C+M129L as the amino acid modification, (3) pyroolysyl-tRNA synthetase activity, specifically aminoacylation of non-canonical amino acids, and (4) NƐ-((2-azidoethoxy)carbonyl)-L-lysine (AzK) as the lysine derivative in the reply filed on September 29, 2025 is acknowledged. The traversal is on the ground(s) that Sakamoto discloses Y126A/M129L and Y126A/V168C amino acid substitutions and does not disclose the specific combination of V168C+M129L and Sakamoto does not teach or suggest the lysine derivative AzK. This is partially found persuasive. Sakamoto discloses making combinatorial variants comprising multiple amino acid substitutions to evaluate non-canonical amino acid introduction into a protein (Examples 5 and 6). Sakamoto discloses that the Y126A+M129L variant and Y126A+V168C variant showed higher amount of ZLys introduced into a protein compared to Y126A or Y126A + M129 (Example 7 and Figures 10-11). MaPylRS variants of Sakamoto catalyzes aminoacylation of azide-lysine, a lysine derivative and non-canonical amino acid, to its cognate tRNA (Example 7). Azide-lysine is synonymous with Azide-K (NƐ-((2-azidoethoxy)carbonyl)-L-lysine). Therefore, it would have been obvious to one having ordinary skill in the art before the time the claimed invention was effectively filed to make a MaPylRS variant having Y126A + M129L + V168C amino acid substitutions. See the 103 rejection below. Also, Tugel (Development of new orthogonal pairs for site-specific incorporation of non-canonical amino acids. Master’s Thesis. Graz University of Technology. Pages 1-83. October 2019 - form PTO-892) discloses a Methanomethylophilus alvus (having 100% sequence identity to SEQ ID NO:1) strain Mx1201Ca aminoacyl-tRNA synthetase (MaPylRS) variant having V168C + M129L amino acid substitutions and uses Azk (NƐ-((2-azidoethoxy)carbonyl)-L-lysine) as a substrate (abstract, Section 3.1.1. at pages 25-27, and Table 5). See the 102 rejection below. Therefore, the technical feature linking Groups I-V and species does not constitute a special technical feature as defined by PCT Rule 13.2, as it does not define a contribution over the prior art.
Claims 2, 14-18, 20, and 23-24 are withdrawn from further consideration pursuant to 37 CFR 1.142(b), as being drawn to a nonelected invention, there being no allowable generic or linking claim. Applicant timely traversed the restriction (election) requirement in the reply filed on September 29, 2005.
Status of Claims
Claims 1-18, 20-21, and 23-24 are pending.
Claims 2, 14-18, 20, and 23-24 are withdrawn.
Claims 1, 3-13, and 21 are under examination.
Claim for Foreign Priority
Receipt is acknowledged of certified copies of papers required by 37 CFR 1.55.
Information Disclosure Statement
The information disclosure statement (IDS) submitted on September 4, 2024 and December 29, 2022 are in compliance with the provisions of 37 CFR 1.97. Accordingly, the information disclosure statements are being considered by the examiner.
Claim Rejections - 35 USC § 112
The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
Claims 1 and 4 and claims 3, 5-13, and 21 depending therefrom are rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
A broad range or limitation together with a narrow range or limitation that falls within the broad range or limitation (in the same claim) may be considered indefinite if the resulting claim does not clearly set forth the metes and bounds of the patent protection desired. See MPEP § 2173.05(c). In the present instance, claims 1 and 4 recite the broad recitation “at least 70”.. but less than 100%” and “at least 81%...100%”, respectively. The limitation “at least 70%” is equivalent to a range of “70%- less than 100%” and the limitation “at least 81%” is equivalent to a range of “81-100%”. The claim(s) are considered indefinite because there is a question or doubt as to whether the feature introduced by such narrower language is (a) merely exemplary of the remainder of the claim, and therefore not required, or (b) a required feature of the claims.
Claims 1, 3-4, 7, and 9-13 and claims 5-6, 8-9, and 21 depending therefrom are rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
Regarding claims 1, 3-4, 7, and 9-13, the phrase "e.g.” renders the claims indefinite because it is unclear whether the limitation(s) following the phrase are part of the claimed invention. See MPEP § 2173.05(d).
Claims 7, 9, and 10 are rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
Claims 7, 9, and 10 recite the phrase “preferably”. The phrase renders the claims indefinite because it is unclear whether the limitation(s) following the phrase are part of the claimed invention. See MPEP § 2173.05(d).
Claim 7 is rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
Claim 7 recites the limitation “said amino acid” in line 2. There is insufficient antecedent basis for this limitation in the claim. Claim 7 depends from claim 1. However, claim 1 does not recite an “amino acid”. It is unclear if the “amino acid” refers to an amino acid within the aminoacyl-tRNA synthetase variant or if the “amino acid” refers to the amino acid that attaches to the tRNA by the aminoacyl-tRNA synthetase variant. Clarification is requested.
Claims 8-9 are rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
Claims 8-9 recites the limitation “said lysine derivative” in line 2. There is insufficient antecedent basis for this limitation in the claim. Claims 8-9 both depend from claim 1. However, claim 1 does not recite a “lysine derivative”. It is unclear if the “said lysine derivative” refers to an amino acid within the aminoacyl-tRNA synthetase variant or if the “said lysine derivative refers to the amino acid that attaches to the tRNA by the aminoacyl-tRNA synthetase variant. Clarification is requested.
Claim 8 is rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
Regarding claim 8, the phrase "such as" renders the claim indefinite because it is unclear whether the limitations following the phrase are part of the claimed invention. See MPEP § 2173.05(d).
The following is a quotation of the first paragraph of 35 U.S.C. 112(a):
(a) IN GENERAL.—The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor or joint inventor of carrying out the invention.
The following is a quotation of the first paragraph of pre-AIA 35 U.S.C. 112:
The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor of carrying out his invention.
Claims 1, 3-4, 6-13, and 21 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, as failing to comply with the written description requirement. The claim(s) contains subject matter which was not described in the specification in such a way as to reasonably convey to one skilled in the relevant art that the inventor or a joint inventor, or for pre-AIA the inventor(s), at the time the application was filed, had possession of the claimed invention.
MPEP 2111.01 states that ''[d]uring examination, the claims must be interpreted as broadly as their terms reasonably allow.'' In this case, the claims have been broadly interpreted to encompass (1) an aminoacyl-tRNA synthase variant having 70-100% sequence identity to SEQ ID NO:1, having X168C+X129L or V168C+M129L amino acid substitutions, and having aminoacyl-tRNA synthetase activity and pyrrolysyl-tRNA synthetase activity and/or is capable of catalyzing the aminoacylation of its cognate tRNA with an aromatic amino acid, aliphatic amino acid, a lysine derivative, AzK, or a non-canonical amino acid or (2) an aminoacyl-tRNA synthetase having at least 80-99% sequence identity to SEQ ID NO:2, having X168C+X129L or V168C+M129L amino acid substitutions, and having aminoacyl-tRNA synthetase activity. Therefore, the claims are drawn to a genus of polypeptides having unknown structure, except having the V168C+M129L amino acid substitutions, but having the function of having aminoacyl-tRNA synthetase activity and/or pyrrolysyl-tRNA synthetase activity and/or is capable of catalyzing the aminoacylation of its cognate tRNA with an aromatic amino acid, aliphatic amino acid, a lysine derivative, AzK, or a non-canonical amino acid.
MPEP 2163 I. states that to “satisfy the written description requirement, a patent specification must describe the claimed invention in sufficient detail that one skilled in the art can reasonably conclude that the inventor had possession of the claimed invention.
MPEP 2163. II.A.3.(a) sates that “Possession may be shown in many ways. For example, possession may be shown by describing an actual reduction to practice of the claimed invention. Possession may also be shown by a clear depiction of the invention in detailed drawings or in structural chemical formulas which permit a person skilled in the art to clearly recognize that inventor had possession of the claimed invention. An adequate written description of the invention may be shown by any description of sufficient, relevant, identifying characteristics so long as a person skilled in the art would recognize that the inventor had possession of the claimed invention.
According to MPEP 2163.II.A.3.(a).ii), “Satisfactory disclosure of a ‘representative number’ depends on whether one of skill in the art would recognize that the applicant was in possession of the necessary common attributes or features possessed by the members of the genus in view of the species disclosed. For inventions in an unpredictable art, adequate written description of a genus which embraces widely variant species cannot be achieved by disclosing only one species within the genus…Instead, the disclosure must adequately reflect the structural diversity of the claimed genus, either through the disclosure of sufficient species that are ‘representative of the full variety or scope of the genus,’ or by the establishment of ‘a reasonable structure-function correlation.’"
The recitation of “aminoacyl-tRNA synthetase”, “pyrrolysyl-tRNA synthetase”, “catalyzing the aminoacylation of its cognate tRNA with an aromatic or aliphatic amino acid”, “lysine derivative”, and “Azk” fails to provide a sufficient description of the genus of the polypeptides as it merely describes the functional features of the genus without providing any definition of the structural features of the species within the genus. The specification does not specifically define any of the species that fall within the genus. The specification does not define any structural features commonly possessed by members of the genus that distinguish them from others. One skilled in the art therefore cannot, as one can do with a fully described genus, visualize or recognize the identity of the members of the genus.
Methanomethylophilus alvus PylRS (MaPylRS) having the amino acid sequence of SEQ ID NO:1 of the instant application was known in the art, see Borrel (Unique characteristics of the pyrrolysine system in the 7th order of methanogens: implications for the evolution of a genetic code expansion cassette. Archaea. 2014 Jan 27;2014:374146 – form PTO-892 Section 2 at page 2 “CP004049.1” and see the sequence alignment below). Fladischer ((Non-canonical amino acids as building blocks: New translation systems for non-canonical amino acids. Dissertation. Technischen Universaitat Graz. February 2018 – form PTO-892) discloses that AzideK (NƐ-((2-azidoethoxy)carbonyl)-L-lysine) is barely accepted as a substrate by MaPylRS but MmPylRS accepts Azide K very well (bottom of page 196 through top of page 197). The prior art does not teach aminoacyl-tRNA synthetases having at least 70% sequence identity to SEQ ID NO:1 or having at least 80% sequence identity to SEQ ID NO:2, wherein the polypeptide has aminoacyl-tRNA synthetase activity, pyrrolysyl-tRNA synthetase activity, and is capable of catalyzing the aminoacylation of its cognate tRNA with an aromatic amino acid, aliphatic amino acid, a lysine derivative, AzK, or a non-canonical amino acid.
Fransceus (J Ind Microbiol Biotechnol. 2017 May;44(4-5):687-695. – form PTO-892) reviews protein engineering techniques, such as random mutagenesis and recombination, directed evolution and iterative or combinatory saturation “hotspots”. Fransceus states that “a recurring problem, however, is choosing which amino acid positions should be targeted. Answering this question is not an easy feat and requires substantial insight in the relationship between an enzyme’s sequence or structure and its properties.” Sanavia (Computational and Structural Biotechnology Journal, Volume 18, 2020, Pages 1968-1979. – form PTO-892) discloses challenges in the prediction of protein stability in the occurrence of multiple mutations. “Multiple-point mutations are common variations of the protein sequence that may be needed in protein engineering when a single-point mutation is not enough to yield the desired stability change. Dealing with multiple-site variations adds another level of complexity beyond the prediction of the effect of a single variant on protein stability, since it requires the learning of many types of combinatorial effects”.
The specification is limited to description of the aminoacyl-tRNA synthetase having the amino acid sequence of SEQ ID NO:2, which is the variant of SEQ ID NO:1 consisting of V168C+M129L amino acid substitutions, wherein the aminoacyl-tRNA synthetase has pyrrolysyl-tRNA synthetase activity and incorporates the lysine derivate, Azk. While MPEP 2163 acknowledges that in certain situations “one species adequately supports a genus,” it also acknowledges that “[f]or inventions in an unpredictable art, adequate written description of a genus which embraces widely variant species cannot be achieved by disclosing only one species within the genus.” In view of the widely variant species encompassed by the genus, the example described above is not enough and does not constitute a representative number of species to describe the whole genus of polynucleotides having at least 85% sequence identity to SEQ ID NO:2079 and encoding polypeptides having galactose oxidase activity. Therefore, the specification fails to describe a representative species of the claimed genus.
Further, one of skill in the art could identify polynucleotide having at least 70% sequence identity to SEQ ID NO:1 or at least 80% sequence identity to SEQ ID NO:2. However, there is no teaching regarding which 30% of the amino acids can vary from SEQ ID NO:1 or which 20% amino acids can vary from SEQ ID NO:2 and result in polypeptide having aminoacyl-tRNA synthetase activity, pyrrolysyl-tRNA synthetase activity, and is capable of catalyzing the aminoacylation of its cognate tRNA with an aromatic amino acid, aliphatic amino acid, a lysine derivative, AzK, or a non-canonical amino acid. An important consideration is that structure is not necessarily a reliable indicator of function. In the instant case, there is no disclosure relating similarity of structure to conservation of function. Conservation of structure is not necessarily a surrogate for conservation of function. Since the claimed invention is that of an enzyme, and there is no disclosure of the domains responsible for having aminoacyl-tRNA synthetase activity, pyrrolysyl-tRNA synthetase activity, and is capable of catalyzing the aminoacylation of its cognate tRNA with an aromatic amino acid, aliphatic amino acid, a lysine derivative, AzK, or a non-canonical amino acid, the absence of information may be persuasive that those of skill in the art would not take the disclosure as generic.
Given this lack of description of the representative species encompassed by the genus of the claims, the specification fails to sufficiently describe the claimed invention in such full, clear, concise, and exact terms that a skilled artisan would recognize that applicants were in possession of the inventions of claims 1, 3-4, 6-13, and 21.
Claims 1, 3-4, 6-13, and 21 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, because the specification, while being enabling for the aminoacyl-tRNA synthetase having the amino acid sequence of SEQ ID NO:2, which is the variant of SEQ ID NO:1 consisting of V168C+M129L amino acid substitutions, does not reasonably provide enablement for polypeptides having aminoacyl-tRNA synthetase activity and/or pyrrolysyl-tRNA synthetase activity and/or is capable of catalyzing the aminoacylation of its cognate tRNA with an aromatic amino acid, aliphatic amino acid, a lysine derivative, AzK, or a non-canonical amino acid. The specification does not enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the invention commensurate in scope with these claims.
Factors to be considered in determining whether undue experimentation is required are summarized in In re Wands 858 F.2d 731, 8 USPQ2nd 1400 (Fed. Cir, 1988). They include (1) the quantity of experimentation necessary, (2) the amount of direction or guidance presented, (3) the presence or absence of working examples, (4) the nature of the invention, (5) the state of the prior art, (6) the relative skill of those in the art, (7) the predictability or unpredictability of the art, and (8) the breadth of the claims.
The breadth of the claims.
MPEP 2111.01 states that ''[d]uring examination, the claims must be interpreted as broadly as their terms reasonably allow.'' In this case, the claims have been broadly interpreted to encompass (1) an aminoacyl-tRNA synthase variant having 70-100% sequence identity to SEQ ID NO:1, having X168C+X129L or V168C+M129L amino acid substitutions, and having aminoacyl-tRNA synthetase activity and pyrrolysyl-tRNA synthetase activity and/or is capable of catalyzing the aminoacylation of its cognate tRNA with an aromatic amino acid, aliphatic amino acid, a lysine derivative, AzK, or a non-canonical amino acid or (2) an aminoacyl-tRNA synthetase having at least 80-99% sequence identity to SEQ ID NO:2, having X168C+X129L or V168C+M129L amino acid substitutions, and having aminoacyl-tRNA synthetase activity. Therefore, the claims are drawn to any polypeptide having unknown structure, except having the V168C+M129L amino acid substitutions, but having the function of having aminoacyl-tRNA synthetase activity and/or pyrrolysyl-tRNA synthetase activity and/or is capable of catalyzing the aminoacylation of its cognate tRNA with an aromatic amino acid, aliphatic amino acid, a lysine derivative, AzK, or a non-canonical amino acid.
The claims are not commensurate with the enablement provided by the disclosure with regard to the extremely large number of polypeptides having aminoacyl-tRNA synthetase activity and/or pyrrolysyl-tRNA synthetase activity and/or is capable of catalyzing the aminoacylation of its cognate tRNA with an aromatic amino acid, aliphatic amino acid, a lysine derivative, AzK, or a non-canonical amino acid. In the instant case, the specification is limited to the aminoacyl-tRNA synthetase having the amino acid sequence of SEQ ID NO:2, which is the variant of SEQ ID NO:1 consisting of V168C+M129L amino acid substitutions, wherein the aminoacyl-tRNA synthetase has pyrrolysyl-tRNA synthetase activity and incorporates the lysine derivate, Azk.
The quantity of experimentation required to practice the claimed invention based on the teachings of the specification.
While enzyme isolation techniques, recombinant and mutagenesis techniques were known in the art at the time of the invention, e.g. mutagenesis, and it is routine in the art to screen for variants comprising multiple substitutions or multiple modifications as encompassed by the instant claims, the specific amino acid positions within the protein's sequence where amino acid modifications can be made with a reasonable expectation of success in obtaining the desired activity/utility are limited in any protein and the result of such modifications is unpredictable. In addition, one skilled in the art would expect any tolerance to modification for a given protein to diminish with each further and additional modification, e.g. multiple substitutions.
In the absence of: (a) rational and predictable scheme for making any polypetide having at least 70% sequence identity to SEQ ID NO:1 or having at least 80% sequence identity to SEQ ID NO:2 having aminoacyl-tRNA synthetase activity and/or pyrrolysyl-tRNA synthetase activity and/or is capable of catalyzing the aminoacylation of its cognate tRNA with an aromatic amino acid, aliphatic amino acid, a lysine derivative, AzK, or a non-canonical amino acid, and (b) a correlation between structure and the function of having aminoacyl-tRNA synthetase activity and/or pyrrolysyl-tRNA synthetase activity and/or is capable of catalyzing the aminoacylation of its cognate tRNA with an aromatic amino acid, aliphatic amino acid, a lysine derivative, AzK, or a non-canonical amino acid, the specification provides insufficient guidance as to which of the essentially infinite possible choices is likely to be successful. One of skill in the art would have to test these infinite possible polypeptides to determine which polypeptides have aminoacyl-tRNA synthetase activity and/or pyrrolysyl-tRNA synthetase activity and/or is capable of catalyzing the aminoacylation of its cognate tRNA with an aromatic amino acid, aliphatic amino acid, a lysine derivative, AzK, or a non-canonical amino acid. While enablement is not precluded by the necessity for routine screening, if a large amount of screening is required, as is the case herein, the specification must provide a reasonable amount of guidance which respect to the direction in which the experimentation should proceed so that a reasonable number of species can be selected for testing. In view of the fact that such guidance has not been provided in the instant specification, it would require undue experimentation to enable the full scope of the claims.
The state of prior art, the relative skill of those in the art, and predictability or unpredictability of the art.
Since the amino acid sequence of the mutant determines its structural and functional properties, predictability of which changes can be tolerated in a protein's amino acid sequence and obtain the desired activity requires a knowledge of and guidance with regard to which amino acids in the protein's sequence, if any, are tolerant of modification and which are conserved (i.e. expectedly intolerant to modification), and detailed knowledge of the ways in which the proteins' structure relates to its function. In the instant case, neither the specification or the art provide a correlation between structure and activity such that one of skill in the art can envision the structure of any polypeptides having aminoacyl-tRNA synthetase activity and/or pyrrolysyl-tRNA synthetase activity and/or is capable of catalyzing the aminoacylation of its cognate tRNA with an aromatic amino acid, aliphatic amino acid, a lysine derivative, AzK, or a non-canonical amino acid or predict said function of a polypeptide from its primary structure. In addition, the art does not provide any teaching or guidance as to (1) which amino acids within the polypeptide of SEQ ID NO:1 or 3 (other than V168 and M129) that can be modified and which ones are conserved such that one of skill in the art can make the recited polypeptides having aminoacyl-tRNA synthetase activity and/or pyrrolysyl-tRNA synthetase activity and/or is capable of catalyzing the aminoacylation of its cognate tRNA with an aromatic amino acid, aliphatic amino acid, a lysine derivative, AzK, or a non-canonical amino acid, (2) which segments of the polypeptide of SEQ ID NO:1 or 2 that are essential for polypeptides having aminoacyl-tRNA synthetase activity and/or pyrrolysyl-tRNA synthetase activity and/or is capable of catalyzing the aminoacylation of its cognate tRNA with an aromatic amino acid, aliphatic amino acid, a lysine derivative, AzK, or a non-canonical amino acid, and (3) the general tolerance of the polypeptide of SEQ ID NO:1 or 2 to structural modifications and the extent of such tolerance. The art clearly teaches that changes in a protein's amino acid sequence to obtain the desired activity without any guidance/knowledge as to which amino acids in a protein are required for that activity is highly unpredictable. At the time of the invention there was a high level of unpredictability associated with altering a polypeptide sequence with an expectation that the polypeptide will maintain the desired activity. For example, Studer (Residue mutations and their impact on protein structure and function: detecting beneficial and pathogenic changes. Biochem. J. (2013) 449, 581–594. – form PTO-892) teach that (1) protein engineers are frequently surprised by the range of effects caused by single mutations that they hoped would change only one specific and simple property in enzymes, (2) the often surprising results obtained by experiments where single mutations are made reveal how little is known about the rules of protein stability, and (3) the difficulties in designing de novo stable proteins with specific functions.
Methanomethylophilus alvus PylRS (MaPylRS) having the amino acid sequence of SEQ ID NO:1 of the instant application was known in the art, see Borrel (Unique characteristics of the pyrrolysine system in the 7th order of methanogens: implications for the evolution of a genetic code expansion cassette. Archaea. 2014 Jan 27;2014:374146 – form PTO-892 Section 2 at page 2 “CP004049.1” and see the sequence alignment below). Fladischer ((Non-canonical amino acids as building blocks: New translation systems for non-canonical amino acids. Dissertation. Technischen Universaitat Graz. February 2018 – form PTO-892) discloses that AzideK (NƐ-((2-azidoethoxy)carbonyl)-L-lysine) is barely accepted as a substrate by MaPylRS but MmPylRS accepts Azide K very well (bottom of page 196 through top of page 197). The prior art does not teach aminoacyl-tRNA synthetases having at least 70% sequence identity to SEQ ID NO:1 or having at least 80% sequence identity to SEQ ID NO:2, wherein the polypeptide has aminoacyl-tRNA synthetase activity, pyrrolysyl-tRNA synthetase activity, and is capable of catalyzing the aminoacylation of its cognate tRNA with an aromatic amino acid, aliphatic amino acid, a lysine derivative, AzK, or a non-canonical amino acid.
Fransceus (J Ind Microbiol Biotechnol. 2017 May;44(4-5):687-695. – form PTO-892) reviews protein engineering techniques, such as random mutagenesis and recombination, directed evolution and iterative or combinatory saturation “hotspots”. Fransceus states that “a recurring problem, however, is choosing which amino acid positions should be targeted. Answering this question is not an easy feat and requires substantial insight in the relationship between an enzyme’s sequence or structure and its properties.” Sanavia (Computational and Structural Biotechnology Journal, Volume 18, 2020, Pages 1968-1979. – form PTO-892) discloses challenges in the prediction of protein stability in the occurrence of multiple mutations. “Multiple-point mutations are common variations of the protein sequence that may be needed in protein engineering when a single-point mutation is not enough to yield the desired stability change. Dealing with multiple-site variations adds another level of complexity beyond the prediction of the effect of a single variant on protein stability, since it requires the learning of many types of combinatorial effects”.
The amount of direction or guidance presented and the existence of working examples.
The specification is limited to the aminoacyl-tRNA synthetase having the amino acid sequence of SEQ ID NO:2, which is the variant of SEQ ID NO:1 consisting of V168C+M129L amino acid substitutions, wherein the aminoacyl-tRNA synthetase has pyrrolysyl-tRNA synthetase activity and incorporates the lysine derivate, Azk. However, the speciation fails to provide any information as to (1) specific substrates associated with polypeptides having at least 70% sequence identity to SEQ ID NO: 1 or having at least 80% sequence identity to SEQ ID NO:2 or (2) structural elements required in a polypeptide having at least 70% sequence identity to SEQ ID NO:1 or having at least 80% sequence identity to SEQ ID NO:2 (other than V168 and M129) and having aminoacyl-tRNA synthetase activity and/or pyrrolysyl-tRNA synthetase activity and/or is capable of catalyzing the aminoacylation of its cognate tRNA with an aromatic amino acid, aliphatic amino acid, a lysine derivative, AzK, or a non-canonical amino acid No correlation between structure and function of having galactose oxidase activity has been presented. There is no information or guidance as to which amino acid residues in the polypeptide of SEQ ID NO:1 or 2 that can be modified and which ones are to be conserved to create a a polypeptide having aminoacyl-tRNA synthetase activity and/or pyrrolysyl-tRNA synthetase activity and/or is capable of catalyzing the aminoacylation of its cognate tRNA with an aromatic amino acid, aliphatic amino acid, a lysine derivative, AzK, or a non-canonical amino acid.
Thus, in view of the overly broad scope of the claims, the lack of guidance and working examples provided in the specification, the high level of unpredictability of the prior art in regard to structural changes and their effect on function and the lack of knowledge about a correlation between structure and function, an undue experimentation would be necessary one having ordinary skill in the art to make and use the claimed invention in a manner reasonably correlated with the scope of the claims. The scope of the claims must bear a reasonable correlation with the scope of enablement (In re Fisher, 166 USPQ 19 24 (CCPA 1970)). Without sufficient guidance, determination of polypeptides having the desired biological characteristics recited in the claims are unpredictable and the experimentation left to those skilled in the art is unnecessarily, and improperly, extensive and undue. See In re Wands 858 F.2d 731, 8 USPQ2nd 1400 (Fed. Cir, 1988).
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 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, 3-13, and 21 is/are rejected under 35 U.S.C. 102(a)(1) as being anticipated by Tugel (Development of new orthogonal pairs for site-specific
incorporation of non-canonical amino acids. Master’s Thesis. Graz University of Technology. Pages 1-83. October 2019 - form PTO-892).
Regarding claims 1, 3, and 11, Tugel discloses a Methanomethylophilus alvus strain Mx1201Ca aminoacyl-tRNA synthetase (MaPylRS) variant having V168C + M129L amino acid substitutions (abstract, Section 3.1.1. at pages 25-27, and Table 5). The MaPylRS of Tugel has 100% sequence identity to the MaPylRS of SEQ ID NO:1 of the instant application, as evidenced by reference 41 (Borrel. Unique characteristics of the pyrrolysine system in the 7th order of methanogens: implications for the evolution of a genetic code expansion cassette. Archaea. 2014 Jan 27;2014:374146 – form PTO-892, Section 2 at page 2 “CP004049.1” and see the sequence alignment below).
Regarding claims 4-5, SEQ ID NO:2 of the instant application is a variant of MaPylRS of SEQ ID NO:1 of the instant application, wherein the variant consists of V168C + M129L amino acid substitutions. Since the MaPylRS variant of Tugel consists of V168C + M129L amino acid substitutions and the parent MaPylRS of Tugel is identical to the MaPylRS of SEQ ID NO:1, The MaPylRS variant of Tugel consists of the amino acid sequence of SEQ ID NO:2 of the instant application.
Regarding claim 6-10, the MaPylRS V168C + M129L variant of Tugel catalyzes aminoacylation of Azk (NƐ-((2-azidoethoxy)carbonyl)-L-lysine), a lysine derivative and non-canonical amino acid, to its cognate tRNA (abstract and page 27).
Regarding claims 12-13, the MaPylRS has pyrrolysyl-tRNA synthetase activity and the MaPylRS V168C + M129L variant of Tugel has pyrrolysyl-tRNA synthetase activity (abstract, page 5, Section 3.1.1. at pages 25-27, Table 5, and page 56).
Regarding claim 21, Tugel discloses a composition comprising the MaPylRS V168C + M129L variant (page 26-27).
Therefore, the reference of Tugel anticipates claims 1, 3-13, and 21.
Claim Rejections - 35 USC § 103
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
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(s) 1, 3-4, 6-13, and 21 is/are rejected under 35 U.S.C. 103 as being unpatentable over Sakamoto (WO 2020/045656 – cited previously on form PTO-892 and English Translation of WO 2020/045656 retrieved on May 22, 2025 – cited previously on form PTO-892. The English Translation of WO 2020/045656 is used for specific passages of Sakamoto) and evidenced by Fladischer (Non-canonical amino acids as building blocks: New translation systems for non-canonical amino acids. Dissertation. Pages 1-334. Technischen Universaitat Graz. February 2018 – form PTO-892)
Regarding claims 1, 3, and 11, Sakamoto discloses a Methanomethylophilus alvus aminoacyl-tRNA synthetase (MaPylRS) of SEQ ID NO:5 having pyrrolysyl-tRNA synthetase activity (abstract and last paragraph at page 7). The MaPylRS of Sakamoto has 100% sequence identity to the MaPylRS of SEQ ID NO:1 of the instant application (see the sequence alignment below).
Regarding claims 1 and 3, Sakamoto discloses a MaPylRS variant having Y126, M129X/L, V168X/C or Y206 (page 9, 4th full paragraph). Sakamoto disclose double mutants of MaPylRS, Y126A+M129L and Y126A+V168C (Figure 10 and Example 7).
Regarding claim 4, SEQ ID NO:2 of instant application is a variant of MaPylRS of SEQ ID NO:1 of the instant application, wherein the variant consists of V168C + M129L amino acid substitutions. Since the MaPylRS variants of Sakamoto consists of two amino acid substitutions, said MaPylRS variants of Sakamoto have at least 80%sequence identity to SEQ ID NO:2 of the instant application.
Regarding claims 6-10, the MaPylRS variants of Sakamoto catalyzes aminoacylation of azide-lysine, a lysine derivative and non-canonical amino acid, to its cognate tRNA (Example 7). Azide-lysine is synonymous with Azide-K (NƐ-((2-azidoethoxy)carbonyl)-L-lysine), as evidenced by Fladischer (bottom of page 196).
Regarding claims 12-13, the MaPylRS of Sakamoto has pyrrolysyl-tRNA synthetase activity and the MaPylRS variants of Sakamoto has pyrrolysyl-tRNA synthetase activity (Example 7).
Regarding claim 21, Sakamoto discloses a composition comprising the MaPylRS variants (5th full paragraph at page10).
Sakamoto does not disclose a MaPylRS variant having M129L + V168C amino acid substitutions.
However, Sakamoto discloses making combinatorial variants comprising multiple amino acid substitutions to evaluate non-canonical amino acid introduction into a protein (Examples 5 and 6). Sakamoto discloses that the Y126A+M129L variant and Y126A+V168C variant showed higher amount of ZLys introduced into a protein compared to Y126A (Example 7 and Figures 10-11).
Therefore, it would have been obvious to one having ordinary skill in the art before the time the claimed invention was effectively filed to modify the MaPylRS variant of Sakamoto by combining the Y126A+M129L and Y126A+V168C, resulting in a MaPylRS variant having Y126A + M129L + V168C amino acid substitutions. One having ordinary skill in the art would have been motivated to do so in order to further improve incorporation of ZLys since the presence of each of M129L and V168C amino acid substitutions results in higher incorporation of Zlys compared to the Y126A alone. One having ordinary skill in the art would have has a reasonable expectation of success since Sakamoto discloses MaPylRS variants having higher incorporation of Zlys into proteins than the parent MaPylRS. Using the known technique of combining the double mutants of Sakamoto into a single MaPylRS would have been obvious to one of ordinary skill. The rationale supporting that the claims would have been obvious is that a method of enhancing a particular class of devices (introduction of Y126A, M129L, and V168C) has been made part of the ordinary capabilities of one skilled in the art based upon the teaching of such improvement in other situations. One of ordinary skill in the art would have been capable of applying this known method of enhancement to a “base” device (MaPylRS) in the prior art and the results would have been predictable to one of ordinary skill in the art.
Therefore, the above references render claims 1, 3-4, 6-13, and 21 prima facie obvious.
Claim(s) 1, 3-4, 6-13, and 21 is/are rejected under 35 U.S.C. 103 as being unpatentable over Fladischer (Non-canonical amino acids as building blocks: New translation systems for non-canonical amino acids. Dissertation. Technischen Universaitat Graz. Pages 1-334. February 2018 – form PTO-892).
Regarding claims 1, 3, and 11, Fladischer disclsoes a Methanomethylophilus alvus aminoacyl-tRNA synthetase (MaPylRS) having pyrrolysyl-tRNA synthetase activity (abstract and page 191 and Supporting Figure 2 at page 222). The MaPylRS of Fladischer has 100% sequence identity to the MaPylRS of SEQ ID NO:1 of the instant application, as evidenced by reference 22 at page 191 (Borrel. Comparative genomics highlights the unique biology of Methanomassiliicoccales, a Thermoplasmatales-related seventh order of methanogenic archaea that encodes pyrrolysine. BMC Genomics. 2014 Aug 13;15:679 - form PTO-892, page 19 “Data access” “CP004049 and see the sequence alignment below).
Regarding claim 21, Fladischer discloses a composition comprising MaPylRS (bottom of page 195).
Fladischer does not disclose a MaPylRS variant having M129L + V168C amino acid substitutions.
Regarding claims 1, 3-4, 6-10, and 12-13, Fladischer discloses that AzideK (NƐ-((2-azidoethoxy)carbonyl)-L-lysine) is barely accepted as a substrate by MaPylRS but MmPylRS accepts Azide K very well (bottom of page 196 through top of page 197). Fladischer discloses that the ability of PylRS to accommodate different Lysine derivatives is correlated with the size and plasticity of the active site (middle of page 200). Fladischer discloses that in MmPylRS, residues lining the active site, N346, Y306, Y384, W417, C348, V401 and L309, were shown to be important for substrate recognition (middle of page 200). Fladischer discloses that in MaPylRS, M129, A223, and V168 occupies in the active sites correlating to L309, V401, and C348, respectively, of MmPylRS (middle of page 200). One having ordinary skill in the art would have recognized to mutate M129, A223, and V168 in the substrate recognition site of MaPylRS to M129L, A223V, and V168C in order to mimic the residues in the substrate recognitions site of MmPylRS since MmPylRS accepts Azide K very well. The resulting MaPylRS variant would have at least 80% sequence identity to SEQ ID NO:1 or 2 of the instant application.
Therefore, it would have been obvious to one having ordinary skill in the art before the time the claimed invention was effectively filed to make a MaPylRS variant having M129L + V168C +A233V amino acid substitutions. One having ordinary skill in the art would have been motivated to do so in order to endow MaPylRS to accept Azide K as a substrate by changing the substrate recognition site to have the same residues as MmPylRS which accepts Azide K very well. One having ordinary skill in the art would have has a reasonable expectation of success since Fladischer discloses MaPylRS having pyrrolysyl-tRNA synthetase activity, identifies that M129, A223, and V168 of MaPylRS occupies in the active sites, which correlates to L309, V401, and C348, respectively, of MmPylRS, which has accepted Azide K very well.
Therefore, the above references render claims 1, 3-4, 6-13, and 21 prima facie obvious.
Conclusion
Claims 1-18, 20-21, and 23-24 are pending.
Claims 2, 14-18, 20, and 23-24 are withdrawn.
Claims 1, 3-13, and 21 are rejected.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to YONG D PAK whose telephone number is (571)272-0935. The examiner can normally be reached M-Th: 5:30 am - 3:30 pm.
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/YONG D PAK/Primary Examiner, Art Unit 1652
Sequence alignment between the MaPylRS of SEQ ID NO:1 of the instant application (“Qy”) and the MaPylRS of Tugel/ Fladischer (“Db”)
M9SC49_METAX
ID M9SC49_METAX Unreviewed; 275 AA.
AC M9SC49;
DT 26-JUN-2013, integrated into UniProtKB/TrEMBL.
DT 26-JUN-2013, sequence version 1.
DT 05-FEB-2025, entry version 50.
DE SubName: Full=Pyrrolysyl-tRNA synthetase {ECO:0000313|EMBL:AGI85861.1};
DE EC=6.1.1.26 {ECO:0000313|EMBL:AGI85861.1};
GN ORFNames=MMALV_11280 {ECO:0000313|EMBL:AGI85861.1};
OS Methanomethylophilus alvi (strain Mx1201).
OC Archaea; Methanobacteriati; Thermoplasmatota; Thermoplasmata;
OC Methanomassiliicoccales; Methanomethylophilaceae; Methanomethylophilus.
OX NCBI_TaxID=1236689 {ECO:0000313|EMBL:AGI85861.1, ECO:0000313|Proteomes:UP000012672};
RN [1] {ECO:0000313|EMBL:AGI85861.1, ECO:0000313|Proteomes:UP000012672}
RP NUCLEOTIDE SEQUENCE [LARGE SCALE GENOMIC DNA].
RC STRAIN=Mx1201 {ECO:0000313|EMBL:AGI85861.1,
RC ECO:0000313|Proteomes:UP000012672};
RX PubMed=23209209; DOI=10.1128/JB.01867-12;
RA Borrel G., Harris H.M., Tottey W., Mihajlovski A., Parisot N.,
RA Peyretaillade E., Peyret P., Gribaldo S., O'Toole P.W., Brugere J.F.;
RT "Genome sequence of 'Candidatus Methanomethylophilus alvus' Mx1201, a
RT methanogenic archaeon from the human gut belonging to a seventh order of
RT methanogens.";
RL J. Bacteriol. 194:6944-6945(2012).
RN [2] {ECO:0007829|PDB:6EZD}
RP X-RAY CRYSTALLOGRAPHY (2.40 ANGSTROMS).
RA Fladischer P., Blamauer J., Pavkov-Keller T., Schweiger K., Darnhofer B.,
RA Birner-Gruenberger R., Gruber K., Wiltschi B.;
RT "A new archaeal pyrrolysyl-tRNA synthetase/amber suppressor tRNA pair for
RT orthogonal protein translation.";
RL Submitted (NOV-2017) to the PDB data bank.
RN [3] {ECO:0007829|PDB:6JP2}
RP X-RAY CRYSTALLOGRAPHY (2.27 ANGSTROMS).
RX PubMed=32182048; DOI=10.1021/acssynbio.9b00288;
RA Seki E., Yanagisawa T., Kuratani M., Sakamoto K., Yokoyama S.;
RT "Fully Productive Cell-Free Genetic Code Expansion by Structure-Based
RT Engineering of <i>Methanomethylophilus alvus</i> Pyrrolysyl-tRNA
RT Synthetase.";
RL ACS Synth. Biol. 9:718-732(2020).
RN [4] {ECO:0007829|PDB:7U0R}
RP X-RAY CRYSTALLOGRAPHY (1.80 ANGSTROMS) IN COMPLEX WITH MG(2+).
RX PubMed=37264106; DOI=10.1038/s41557-023-01224-y;
RA Fricke R., Swenson C.V., Roe L.T., Hamlish N.X., Shah B., Zhang Z.,
RA Ficaretta E., Ad O., Smaga S., Gee C.L., Chatterjee A., Schepartz A.;
RT "Expanding the substrate scope of pyrrolysyl-transfer RNA synthetase
RT enzymes to include non-alpha-amino acids in vitro and in vivo.";
RL Nat. Chem. 15:960-971(2023).
RN [5] {ECO:0007829|PDB:8C49}
RP X-RAY CRYSTALLOGRAPHY (1.82 ANGSTROMS) IN COMPLEX WITH MG(2+).
RX PubMed=37051694; DOI=10.1002/pro.4640;
RA Taylor C.J., Hardy F.J., Burke A.J., Bednar