DETAILED ACTION
Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Priority
This application is a continuation (CON or bypass application) of PCT/JP2022/026916, filed 7/7/2022, which claims benefit of prior-filed application JP2021-112526, filed 7/7/2021.
Acknowledgment is made of Applicant's claim for foreign priority based on application JP2021-112526, filed in Japan on 7/7/2021. It is noted, however, that Applicant has not filed a certified copy of the JP2021-112526 application as required by 37 CFR 1.55. Correction is required to perfect the priority benefit to the foreign-filed priority document.
Information Disclosure Statement
The information disclosure statements (IDS) submitted on 3/12/2024, 11/6/2025, and 7/24/2026 are acknowledged. The submissions are in compliance with the provisions of 37 CFR 1.97. Accordingly, the information disclosure statements are being considered by the examiner.
The listing of references in the specification is not a proper information disclosure statement. 37 CFR 1.98(b) requires a list of all patents, publications, or other information submitted for consideration by the Office, and MPEP § 609.04(a) states, "the list may not be incorporated into the specification but must be submitted in a separate paper." Therefore, unless the references have been cited by the examiner on form PTO-892, they have not been considered.
Election/Restrictions
Applicant's election, with traverse, of the species of the invention of:
I. p-azido-L-phenylalanine as the noncanonical amino acid;
II. a tRNA(Tyr) having SEQ ID NO: 42 for the tRNA (as in claim 8);
III. A tyrosyl tRNA synthetase comprising the amino acid sequence of SEQ ID NO: 50 for the aminoacyl tRNA synthetase;
IV. a PhoS protein comprising the amino acid sequence of SEQ ID NO: 2;
V. the Sec-dependent signal peptide SlpA (as in claims 23-24);
VI. the method using two expression vectors pPK and pVC (as in claims 31-34); and
VII. wherein the noncanonical amino acid-containing protein is a VHH fragment,
in the reply filed on 7/14/2026 is acknowledged.
The traversal is on the grounds that examining all of the species together would not pose an undue burden on the Examiner because the genus of all of the species are related. This is not found persuasive because the distinct species of the invention set out in the election requirement mailed 5/15/2026 represent different chemical structures and/or distinct sequences to be expressed in the bacterial cells, or the species pertain to specific and distinct embodiments (e.g. the use of two different expression vectors having distinct compositions). Different RNA and amino acid sequences are predicted to have different structures and distinct activities. This will require distinct search strategies and consideration for each alternative species and combination of features, including searches for each and every combination of the various embodiments and sequences recited in alternative.
Further, the Applicant’s argument does not provide any convincing evidence that the species of the invention are all indeed obvious variants of each other.
The requirement is still deemed proper and is therefore made FINAL.
Claims 9, 13-14, 19-22, and 35-37 are withdrawn from further consideration pursuant to 37 CFR 1.142(b), as being drawn to a nonelected species of the invention, there being no allowable generic or linking claim. Applicant timely traversed the election requirement in the reply filed on 7/14/2026. Upon indication of an allowable generic claim, consideration of additional species of the invention will be given.
Claims 1-8, 10-12, 15-18, 23-34, and 38-39 are pending and examined on the merits.
Nucleotide and/or Amino Acid Sequence Disclosures
Summary of Requirements for Patent Applications Filed On Or After July 1, 2022, That Have Sequence Disclosures
37 CFR 1.831(a) requires that patent applications which contain disclosures of nucleotide and/or amino acid sequences that fall within the definitions of 37 CFR 1.831(b) must contain a “Sequence Listing XML”, as a separate part of the disclosure, which presents the nucleotide and/or amino acid sequences and associated information using the symbols and format in accordance with the requirements of 37 CFR 1.831-1.835. This “Sequence Listing XML” part of the disclosure may be submitted:
1. In accordance with 37 CFR 1.831(a) using the symbols and format requirements of 37 CFR 1.832 through 1.834 via the USPTO patent electronic filing system (see Section I.1 of the Legal Framework for Patent Electronic System (https://www.uspto.gov/PatentLegalFramework), hereinafter “Legal Framework”) in XML format, together with an incorporation by reference statement of the material in the XML file in a separate paragraph of the specification (an incorporation by reference paragraph) as required by 37 CFR 1.835(a)(2) or 1.835(b)(2) identifying:
a. the name of the XML file
b. the date of creation; and
c. the size of the XML file in bytes; or
2. In accordance with 37 CFR 1.831(a) using the symbols and format requirements of 37 CFR 1.832 through 1.834 on read-only optical disc(s) as permitted by 37 CFR 1.52(e)(1)(ii), labeled according to 37 CFR 1.52(e)(5), with an incorporation by reference statement of the material in the XML format according to 37 CFR 1.52(e)(8) and 37 CFR 1.835(a)(2) or 1.835(b)(2) in a separate paragraph of the specification identifying:
a. the name of the XML file;
b. the date of creation; and
c. the size of the XML file in bytes.
SPECIFIC DEFICIENCIES AND THE REQUIRED RESPONSE TO THIS NOTICE ARE AS FOLLOWS:
Specific deficiency - Sequences appearing in the drawings are not identified by sequence identifiers in accordance with 37 CFR 1.831(c). Sequence identifiers for sequences (i.e., “SEQ ID NO:X” or the like) must appear either in the drawings or in the Brief Description of the Drawings. Sequences appear in FIGs. 16 and 18 without sequence identifiers in either the drawing itself or in the description in the specification for these figures.
Required response – Applicant must provide:
Amended drawings in accordance with 37 CFR 1.121(d) inserting the required sequence identifiers;
AND/OR
A substitute specification in compliance with 37 CFR 1.52, 1.121(b)(3), and 1.125 inserting the required sequence identifiers (i.e., “SEQ ID NO:X” or the like) into the Brief Description of the Drawings, consisting of:
• A copy of the previously-submitted specification, with deletions shown with strikethrough or brackets and insertions shown with underlining (marked-up version);
• A copy of the amended specification without markings (clean version); and
• A statement that the substitute specification contains no new matter.
Specification
The lengthy specification has not been checked to the extent necessary to determine the presence of all possible minor errors. Applicant’s cooperation is requested in correcting any errors of which applicant may become aware in the specification.
The disclosure is objected to because of the following informalities:
TRADE NAMES, TRADEMARKS, AND OTHER MARKS USED IN COMMERCE:
The use of the following terms which are each a trade name or a mark used in commerce, has been noted in this application: ChemiDoc™ ([000502],[000504]); Amicon® ([000534]); ZipTip® ([000537]); Opti-MEM™ ([000542]); Glo™ ([000542]); and Nivo™ ([000544]).
The terms should be accompanied by the generic terminology; furthermore the terms should be capitalized wherever it appears or, where appropriate, include a proper symbol indicating use in commerce such as ™, SM , or ® following the terms. Although the use of trade names and marks used in commerce (i.e., trademarks, service marks, certification marks, and collective marks) are permissible in patent applications, the proprietary nature of the marks should be respected and every effort made to prevent their use in any manner which might adversely affect their validity as commercial marks (see MPEP 608.01(v) and 608.01(u)).
Appropriate correction is required.
Claim Objections
Claim 17 is objected to because of the following informalities:
In claim 17, the phrase “than aromatic an amino acid” is a typographical error and should instead say “than an aromatic amino acid” (as in claim 16).
Appropriate correction is required.
Claim Rejections - 35 USC § 112(b)
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 5, 12, 16-18, 29, and 30 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.
Claim 5 recites the phrase “a tyrosine derivative or a lysine derivative”. One of ordinary skill would not be appraised of the metes and bounds of this claim element from the claim language, nor does there appear to be a limiting definition of “derivative” in the specification. How closely related or how different can a molecule be from Tyr or Lys and still be considered to be a Tyr derivative or a Lys derivative? Further, it is noted that the elected species of a p-azido-L-phenylalanine is a phenylalanine derivative, but because Tyr and Phe share substantially the same phenyl-based structure, is p-azido-L-phenylalanine considered a tyrosine derivative or not? The claim is indefinite because the claim includes elements not actually disclosed (any and all possible derivatives), thereby rendering the scope of the claim unascertainable.
Claim 12 recites: “wherein the mutation that modifies the substrate specificity is selected from the group consisting of Y32, H70, E107, D158, I159, L162, D286, and combinations thereof”. No reference sequence is recited from which one is to determine for these numbered positions. Is it mean to be SEQ ID NO: 50 or 48, or something else? The resulting claim is indefinite because there is no reference sequence to compare the recited positions to, and thus, one cannot determine the metes and bounds of the claimed mutations.
Claim 16 recites the limitation "the mutation is replacing" in line 1. There is insufficient antecedent basis for this limitation in the claim. Claim 16 depends on claim 15, which recites “The method according to claim 1, wherein the coryneform bacterium has been further modified so as to harbor a phoS gene encoding a mutant PhoS protein.” Therefore “the mutation” as used in claim 16 lacks antecedent basis because there is no prevision recitation of “a mutation” and because there exist other mutations in the coryneform bacterium. The claim is indefinite because one cannot readily determine what mutation is referred to. It is suggested to overcome this rejection, that the claim be amended to recite: “wherein the mutant PhoS protein comprises a mutation of an amino acid residue corresponding to...”, or language similar thereto.
Claims 17 and 18 are dependent on claim 16 and are, therefore, also rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, for the reasons set forth above.
Regarding claim 29, the parenthetical phrase “(FERM BP-734)” renders the claim indefinite because it is unclear whether the Corynebacterium glutamicum AJ12036 strain is required to have the accession number FERM BP-734. The instant specification discloses that the accession number of Corynebacterium glutamicum AJ12036 is FERM BP-734 ([000137]). However, it is unclear whether the accession number FERM BP-734 is optional or required in claim 29, because the accession number is recited within parentheses. To obviate this rejection, the parentheses may be deleted if the FERM BP-734 accession number is required, or “(FERM BP-734)” may be deleted if the accession number is not required.
Claim 30 recites the limitation “the number of molecules of a cell surface layer protein per cell” in line 2. There is insufficient antecedent basis for this limitation in the claim. There is no previous recitation of any number of molecules of a cell surface layer protein per cell in claim 1, from which this claim depends. As such, the claim is indefinite because it is unclear what “number of molecules of a cell surface layer protein per cell” is being referred to. Is this any possible cell surface protein? Or a specific one? Further, the claim states: “reduced as compared with a non-modified coryneform bacterium”. It is noted that this is so broad as to encompass any unmodified coryneform bacterium, not limited to just the species or strain from which the modified coryneform bacterium is derived from. Thus, because there is no way to reasonably determine what the claimed comparison is in reference to, and what non-modified bacterium is used for the comparison, the claim is indefinite.
Claim Interpretation
In light of the indefiniteness rejections discussed above, for the purpose of searching and comparing the claims to the prior art, claims 16 and 17 have been interpreted as requiring a phoS protein comprising any amino acid sequence as long as the tryptophan corresponding to position 302 of SEQ ID NO: 2 is replaced with another amino acid other than an aromatic or histidine residue, or for claim 17, one of the specific residues recited therein. It is noted that claim 18 recites that the protein comprises the elected amino acid sequence of SEQ ID NO: 2.
Claim Rejections - 35 USC § 112(a)
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.
Claim 29 is rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, as failing to comply with the enablement requirement. The claim contains subject matter which was not described in the specification in such a way as to enable one skilled in the art to which it pertains, or with which it is most nearly connected, to make and/or use the invention.
A modified strain of Corynebacterium glutamicum derived from Corynebacterium glutamicum AJ12036 (FERM BP-734) or Corynebacterium glutamicum ATCC 13869 is required to practice the claimed invention. As such the biological material must be known and readily available or obtainable by a repeatable method set forth in the specification, or otherwise known and readily available to the public. If it is not so obtainable or available, the requirements of 35 U.S.C. § 112(a) or pre-AIA 35 U.S.C. § 112, first paragraph, may also be satisfied by a perfected deposit of the modified strains obtained from Corynebacterium glutamicum AJ12036 (FERM BP-734) and/or Corynebacterium glutamicum ATCC 13869.
The process disclosed in the specification does not appear to be repeatable without extensive experimentation, as there is no evidence that the invention will work with other strains of the bacteria, and it is not apparent if the biological materials considered necessary to make and use the invention of claim 29 is both known and readily available to the public.
According to the instant specification, C. glutamicum AJ12036, accession number FERM BP-734, is a streptomycin resistant mutant strain from C. glutamicum ATCC 13869 (see [000137] of the instant specification). The specification suggests that the modified C. glutamicum AJ12036, FERM BP-1539, has a mutation that allows it to have a higher secretory production, as compared to the parent strain ([000137]). Under optimum culture conditions, modified C. glutamicum AJ12036 may accumulate about 2 to 3 times more protein than the parent strain ([000137]). Thus, it is unclear whether the same secreted protein production can be achieved with any other C. glutamicum strains.
With respect to C. glutamicum AJ12036, it is noted that Applicant has deposited the AJ12036 strain but in the instant case there is no indication in the specification or in a signed declaration as to public availability of the required modified strain. The AJ12036 strain was deposited at the Fermentation Research Institute, Agency of Industrial Science and Technology on March 26, 1984, assigned the accession number FERM BP-734. However, the public availability of the modified strains of AJ12036 and ATCC 13869 are unknown, as there is no deposit information of record. Therefore, a perfected deposit at a recognized depository may be made to obviate this rejection.
If the deposit is made under the terms of the Budapest Treaty, then a statement, affidavit or declaration by Applicants, or by an attorney of record over his or her signature and registration number, or by someone in a position to corroborate the facts of the deposit, that the instant invention will be irrevocably and without restriction released to the public upon the issuance of a patent, would satisfy the deposit requirement made herein.
If the deposit is a non-Budapest Treaty deposit, then in order to certify that the deposit meets the requirements set forth in 37 CFR 1.801-1.809 and MPEP 2402-2411.05, a statement, affidavit or declaration by Applicant or by an attorney of record over his or her signature and registration number, or by someone in a position to corroborate the facts of the deposit would satisfy the requirements herein by stating and providing that:
(a) During the pendency of the application, access to the invention will be afforded to the Commissioner upon request;
(b) All restrictions upon availability to the public will be irrevocably removed upon granting of the patent;
(c) The deposit will be maintained in a public depository for a period of 30 years, or 5 years after the last request or for the enforceable life of the patent, whichever is longer; and
(d) Provide evidence of the test of the viability of the biological material at the time of deposit (see 37 CFR 1.807).
In the instant case, it is clear that the C. glutamicum AJ12036 (FERM BP-734) deposit was made under the Budapest Treaty, but it is unclear whether the C. glutamicum ATCC 13869 deposit was made under the Budapest Treaty. An international form filed in the instant application on 1/2/2024 indicates that the C. glutamicum AJ12036 strain was deposited under the Budapest Treaty. The international form references “Brevibacterium lactofermentum” AJ12036, however Brevibacterium lactofermentum is synonymous C. glutamicum. Therefore, to obviate this rejection the record needs to clearly indicate that the modified strains of C. glutamicum AJ12036 (FERM BP-734) or ATCC 13869 of the instant invention will be irrevocably and without restriction released to the public upon the issuance of a patent.
Moreover, an appropriate statement, affidavit or declaration for both deposits is required.
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.
Claims 1-8, 10-12, 15-18, 23-34, and 38-39 are rejected under 35 U.S.C. 103 as being unpatentable over Matsuda et al. (US20180037918A1, on IDS filed 3/12/2024) in view of Robertson et al. (WO2022248061A1, a WIPO/PCT application that names the U.S. and has a filing date of May 28, 2021, before the earliest purported effective filing date of the claimed invention, corresponding to US20240117295A1) and Mureev et al. (US20180171321A1, on IDS filed 3/12/2024).
Matsuda et al., hereafter “Matsuda”, pertains to methods for the secretory production of a heterologous protein by coryneform bacteria modified to have a specific mutation so as to harbor a phoS gene, and when cultured, are able to produce the desired heterologous protein by secretory production (Abstract).
Particularly, Matsuda teaches culturing a coryneform bacterium having a genetic construct that allows for secretory expression of a heterologous protein, collecting the heterologous protein produced by secretory production, wherein the coryneform bacterium has been modified so as to harbor a phoS gene encoding PhoS protein with a mutation, and wherein the genetic construct comprises, in the direction from 5′ to 3′, a promoter sequence that is able to function in the coryneform bacterium, a nucleic acid sequence encoding a signal peptide that is able to function in the coryneform bacterium, and a nucleic acid sequence encoding the heterologous protein, wherein the secreted heterologous protein is expressed as a fusion protein with the signal peptide (Claims 1 and 5 of Matsuda and [0015]).
Regarding claims 15-17, Matsuda teaches that mutation of the tryptophan residue at position 302 of the wild-type PhoS protein leads to a significant improvement of the amount of a heterologous protein that is secreted, when the residue is mutated to be an arbitrary amino acid residue other than an aromatic amino acid or histidine residues, such as a cysteine residue (see e.g. [0015], [0376], Table 9).
Regarding claim 18, Matsuda teaches that the wild-type PhoS protein is “selected from the group consisting of (a) a protein comprising the amino acid sequence of SEQ ID NO: 4, 54, 55, 56, 57, or 58; (b) a protein comprising the amino acid sequence of SEQ ID NO: 4, 54, 55, 56, 57, or 58, but which includes substitution, deletion, insertion, or addition of 1 to 10 amino acid residues; and (c) a protein comprising an amino acid sequence having an identity of 90% or higher to the amino acid sequence of SEQ ID NO: 4, 54, 55, 56, 57, or 58” ([0017]; [0134]). SEQ ID NO: 4 in Matsuda is 100% identical to the elected amino acid sequence of SEQ ID NO: 2 (see Appendix A included at the end of this Action).
Regarding claims 23-24, Matsuda teaches that the signal peptide is a Sec-dependent signal peptide and that the Sec-dependent signal peptide is selected from the group consisting of PS1, PS2, and SlpA ([0022]-[0023]).
Regarding claims 25 and 26, Matsuda teaches that “the genetic construct further comprises a nucleic acid sequence encoding an amino acid sequence comprising Gln-Glu-Thr between the nucleic acid sequence encoding the signal peptide that is able to function in the coryneform bacterium and the nucleic acid sequence encoding the heterologous protein” ([0024]) and that “ the genetic construct further comprises a nucleic acid sequence encoding an amino acid sequence capable of enzymatic digestion between the nucleic acid sequence encoding the amino acid sequence comprising Gln-Glu-Thr and the nucleic acid sequence encoding the heterologous protein” ([0025]).
Regarding claims 27-29, Matsuda teaches that the coryneform bacterium in some embodiments is Corynebacterium glutamicum. and teaches that the coryneform bacterium is a modified Corynebacterium glutamicum AJ12036 (FERM BP-734) strain or a modified Corynebacterium glutamicum ATCC13869 strain ([0026]-[0028]; [0106]-[0107]).
Regarding claims 30, Matsuda teaches that the coryneform bacterium has reduced activity of a cell surface layer protein ([0029]; [0055]-[0056]), which fulfills the B.R.I. of the instant claim limitations as best understood in view of the 112(b) rejection discussed above.
Regarding claims 32-34, Matsuda teaches that when expressing two or more genes in a host cell, the genes may be on two or more expression vectors, or on a single or two or more expression vectors and a chromosome ([0226]). Matsuda teaches specific examples of vectors that are autonomously replicable in coryneform bacteria include pVC vectors, including pVC7 ([0222]) and Matsuda also teaches cloning and expression of secreted proteins using the vectors pPK4 and pPK5 ([0319]-[0321], and [0333]-[0334], see also Figure 5).
Regarding claims 38 and 39, Matsuda teaches that the secreted protein can include a physiologically active proteins and/or antibody-related molecules ([0271], [0276]), including molecules comprising the heavy chain domain VH, i.e. the elected VHH fragment ([0277]).
However, Matsuda does not expressly teach that the secreted heterologous protein produced by the method contains a non-canonical amino acid, wherein the coryneform bacteria have been further modified to express an orthogonal pair of a tRNA corresponding to the noncanonical amino acid and an aminoacyl tRNA synthetase, as required of the instant claims.
Robertson et al. (hereafter “Robertson”) pertains to novel prokaryotic cells for the production of polymers (e.g. polypeptides) containing non-canonical amino acids, and to methods for making said cells, comprising the use of pairs of orthogonal aminoacyl-tRNA synthetases (aaRSs) and orthogonal tRNAs (pg. 1, lines 3-9). Robertson teaches that the art recognizes various platforms for the synthesis of polymers containing non-canonical amino acids using orthogonal aaRS/tRNA pairs to insert the non-canonical amino acid during protein synthesis (see e.g. pg. 2, lines 9-16). Robertson suggests that such a platform for producing peptides with non-canonical amino acids will enable the programmable and encoded cellular synthesis of an expanded set of non-canonical heteropolymers with emergent and potentially useful properties (pg. 134, lines 8-12).
Robertson teaches that the prokaryotic cell selected for the production method is a bacterial cell, of any species suitable for heterologous protein production, in particular the production of polypeptides comprising one or more non-canonical amino acids (pg. 52, lines 1-17). Robertson also teaches that coryneform bacteria, including Corynebacterium glutamicum, are suitable bacteria for the host cell and production process taught therein (see pg. 52, line 12).
Robertson teaches that the non-canonical amino acid can comprise, inter alia, p-azidophenylalanine (“p-Az-Phe”, see pg. 80, lines 5-30), which is the same as p-azido-L-phenylalanine, elected as the noncanonical amino acid, as in the instant claim 6. Robertson also teaches that the non-canonical amino acids can include various tyrosine and lysine derivatives, including, inter alia, 3-nitrotyrosine, sulfotyrosine, acetyllysine, and pyrrolysine (pg. 80).
Robertson teaches that the orthogonal aminoacyl-tRNA synthetase - tRNA pair may comprise MjTyrRS or a variant with altered selectivity to a non-canonical amino acid, and Mj-tRNATyrYYY, thus teaching using a tyrosine derivative (pg. 5, lines 23-25). Robertson teaches that “Another aaRS disclosed herein is M. jannaschii tyrosyl-tRNA synthetase (MjTyrRS), and teaches that MjTyrRS is an aaRS suitable for use in the prokaryotic cell of the present invention” (pg. 64, line 30-pg. 65, line 17). Robertson teaches that MjTyrRS may be comprised of the amino acid sequence of SEQ ID NO: 28, which is about 98% identical to the elected SEQ ID NO: 50 of the instant invention, as recited in the instant claim 11 (see Appendix B below).
Regarding claim 12, Robertson teaches that “the synthetase is a variant of MjTyrRS further comprising mutations necessary to allow the recognition of, or to alter the specificity for, a non-canonical amino acid. In particular embodiments, the aaRS may be according to SEQ ID NO: 28, comprising any one or a combination of substitutions at the following residues: Y32, L65, H70, F108, Q109, D158, I159, or L162. Mutation at the aforementioned residues may be used to generate variants of MjTyrRS with altered selectivity to non-canonical amino acids” (pg. 65, lines 12-17). Robertson also teaches that the synthetase may be MjTyrRS(p-Az-Phe), which is a mutant of MjTyrRS comprising the mutations Y32L, L65V, F108W, Q109M, D158G, and I159A and is suitable for directing the incorporation p-Az-Phe (see also SEQ ID NO: 30 of Robertson, which is about 97% identical to the instantly elected sequence of SEQ ID NO: 50).
Mureev et al. (hereafter “Mureev”) teaches a method whereby tRNA anticodons are “reassigned” from natural amino acids to non-natural moieties, such as non-natural amino acids, to enable the production of recombinant proteins that include one or a plurality of non-natural moieties (Abstract, [0010]-[0011], claim 29). Mureev teaches that one of the most commonly used approach for the generation of orthogonal codons in the art is “nonsense suppression”, which makes use of three stop codons in the genetic code, including the amber (UAG), opal (UGA) or ochre (UAA) codons ([0008]). Mureev also teaches that the noncanonical amino acid may be encoded by a four-residue codon ([0009]).
Mureev teaches that TyrRS from M. jannaschii ([0084]) is a widely used orthogonal tRNA charging enzyme engineered to mediate incorporation of a range of benzyl side-chain analogs, and teaches that p-Azido-L-phenylalanine (AzF, FIG. 22A) as well as the engineered version of MjTyrRS-AzFRS were chosen based on the reports of their successful application for the amber-codon suppression in vivo and in vitro ([0260]).
Mureev teaches that the benefit of incorporating the azide-containing nnAA AzF is that the added functional group in the resulting protein can be used for further modifications involving click chemistry reactions with the azido-group, and successfully demonstrates adding fluorescent dyes to a modified calmodulin protein via combination of AGG and amber codon reassignment-based system taught therein, for the purpose of building a FRET sensor (see [0272], [0051], FIG. 25).
Regarding the selection of the tRNA sequence, Mureev teaches four previously reported MjYtRNA species, including that of MjY1, corresponding to the tRNAs named pAzPhe1, which has a sequence of ([0054], FIG. 28, [0243]). Mureev teaches (see FIG. 28) that the MjY1_tRNA RNA comprises the sequence of SEQ ID NO: 69, which is 100% identical to that of the instantly recited SEQ ID NO: 42 (i.e. the elected sequence for the tRNA(Tyr)).
To one of ordinary skill in the art, prior to the effective filing date of the instant invention, it would have been prima facie obvious to modify the method for producing a secreted protein using the modified coryneform bacteria taught in Matsuda such that the coryneform bacteria is genetically modified to express an orthogonal pair of a tRNA corresponding to the noncanonical amino acid and an aminoacyl tRNA synthetase, according to the teachings of Robertson and Mureev, for the expected benefit of improved production of secreted proteins comprising a non-canonical amino acid.
One would have been motivated to do so in view of the teachings of Robertson that the use of programmable and encoded cellular synthesis of proteins having non-canonical amino acids results in industrially useful proteins with emergent and potentially useful properties. Additionally, Mureev teaches that the incorporation of p-azidophenylalanine facilitates the further modification of the non-canonical amino acid containing proteins by click chemistry methods, and various applications of this are known in the art (e.g. attaching a fluorescent probe). One of ordinary skill in the art would have been knowledgeable in the application of orthogonal pairs of tRNAs and aminoacyl tRNA synthetases, designed to incorporate a non-canonical amino acid encoded by a stop codon such as UAG, as such synthetic protein production systems are taught in both Mureev and Robertson. Robertson further suggests that coryneform bacteria such as Corynebacterium glutamicum are suitable for use in the production methods with the genetic modifications taught therein, while Matsuda pertains to modified Corynebacterium glutamicum that are optimized for increased production of secreted proteins.
Thus, one having ordinary skill in the art would have predicted with a reasonable expectation of success that combining the modified coryneform bacteria of Matsuda having improved secretary production with at least one orthogonal pair of a tRNA and a corresponding aminoacyl tRNA synthetase would result in a high amount of secreted proteins having the desired non-canonical amino acid modification. The selection of the tRNAs and the aminoacyl tRNA synthetase would amount to a matter of judicious selection from the alternatives taught in Mureev and Robertson, and both teach the use of genes from M. jannaschii.
The inclusion of the modified Corynebacterium glutamicum taught in Matsuda would have predictably led to improved protein production and easier collection of the desired protein, secreted into the cell media and thus avoiding the need for cell harvesting and lysing steps. Such benefits of secreted protein production are known in the art, as evidenced by Matsuda.
Regarding claim 2, it would have been prima facie obvious tin view of the express teachings of Matsuda to generate a genetic construct as claimed therein, selected to have a nucleic acid sequence encoding the protein that will contain a noncanonical amino acid, as suggested by at least Robertson.
Regarding claims 3-4, these selections would have been obvious in view of all the cited teachings of Robertson and Mureev.
Regarding claims 5-6, multiple non-canonical amino acids are taught in Robertson and Mureev, and the selection of a suitable one, such as p-azidophenylalanine would have been a matter of judicious selection to one having ordinary skill. Mureev also teaches that the incorporation of p-azidophenylalanine facilitates the further modification of the non-canonical amino acid containing proteins by click chemistry.
Regarding claims 7-8, both Robertson and Mureev teach nucleic acids encoding tRNA(Tyr), and Mureev teaches the sequence and use of MjY1_tRNA, which is identical to that of the instantly claimed SEQ ID NO: 42.
Regarding claims 10-12, the selection of the tyrosyl tRNA synthetase would have been prima facie obvious in view of the teachings of Robertson and Mureev. Further, Robertson teaches that tyrosyl tRNA synthetase comprise a sequence about 98% identical to the elected SEQ ID NO: 50 and that tyrosyl tRNA synthetase may further comprise substitutions at residues Y32, H70, D158, I159, or L162, as recited in claim 12.
The selection of the tRNA sequence and the tyrosyl tRNA synthetase would thus be a matter of judicious selection from among a defined list of known options in the art.
Regarding claims 15-18, all of the claimed features are taught expressly in Matsuda, and the preferred embodiments of the modified Corynebacterium glutamicum taught therein includes a mutant PhoS protein, including one having a sequence that is identical to the instant SEQ ID NO: 2. Because Matsuda teaches improved protein production in such strains, these would have been obvious to selected for the host cell.
For claims 23-24, Matsuda also teaches that, in preferred embodiments, the secreted protein has a signal peptide including SlpA. The selection of the signal peptide as claimed would have been obvious.
Regarding claims 25-26, these modifications are also expressly taught in Matsuda, and would have been obvious features to include for the benefits extolled therein. These appear to be linker sequences and the inclusion of cleavage site allows for the removal of the signal peptide, which may be necessary to ensure the functionality of the protein remains intact. Such modifications are standard in the art, as evidenced in Matsuda.
Regarding claims 27-29, the preferred embodiments of Matsuda include these strains of Corynebacterium glutamicum, which have desired properties for secreted protein production.
Regarding claims 30, Matsuda teaches that the coryneform bacterium has reduced activity of a cell surface layer protein and the selection of such would have naturally flowed from the teachings expressed therein.
Regarding claims 32-34, Matsuda teaches that when expressing two or more genes in a host cell, the genes may be on two or more expression vectors and the reference teaches specific examples of vectors that are autonomously replicable in coryneform bacteria that include pVC7 and the vectors pPK4 and pPK5 (see Figure 5). The selection of such vectors are thus known in the art and one having ordinary skill would have been adequately motivated to include the necessary nucleic acid sequences taught in Robertson and Mureev, e.g. the gene encoding the tRNA and the aminoacyl tRNA synthetase, on vectors used for expression in C. glutamicum. The arrangement of the nucleic acids in any manner suitable to the art would have been a matter of routine optimization. The arrangement of the expression constructs is not critical to the instant invention. Further, there is no convincing evidence of unexpected success when two expression vectors are used as opposed to the presented alterative of a single expression vector.
Regarding claims 38 and 39, Matsuda expressly teaches that the secreted protein can be an antibody-related molecules (as in claim 38) including molecules comprising the heavy chain domain VH, i.e. the elected VHH fragment (as in claim 39). The selection of the protein that contains the noncanonical amino acid does not appear to be critical to the success of the method. Further, the alternatives of claims 38 and 39, such as a physiologically active protein or a fluorescent protein are obvious selections when producing a non-canonical amino acid containing protein, as taught in the cited references. Mureev teaches using non-canonical amino acids to modify the affected proteins by click chemistry methods, including, the generation of fluorescent proteins. Further, the production of fluorescent proteins, particularly GFP, is taught in both Mureev and Robertson and is extremely well known in the art. Thus, the production of at least one of the recited proteins would have likewise been obvious over the teachings of the art.
From the teachings of the cited references, it is apparent that there would have been a reasonable expectation of success in combining the teachings therein to arrive at the claimed invention because both Robertson and Mureev pertain to the successful production of proteins containing non-canonical amino acids (and indeed each teaching highly similar tRNAs and enzymes for the incorporation of p-azido-phenylalanine), Robertson suggests coryneform bacteria as a suitable host for expressing the orthogonal pairs of tRNAs and aminoacyl tRNA synthetases, and Matsuda teaches successfully producing high amounts of secreted heterologous proteins using modified strains of Corynebacterium glutamicum. Combining the teachings of Matsuda, Robertson, and Mureev to produce large amounts of secreted proteins having non-canonical amino acids would have thus been predictable to one of ordinary skill.
Therefore, the invention as a whole would have been prima facie obvious to one of ordinary skill in the art prior to the effective filing date, as evidenced by the cited references, especially in the absence of evidence to the contrary.
Citation of Pertinent Art
The prior art made of record and not relied upon is considered pertinent to applicant's disclosure.
Sakamoto et al. (US20210107997A1, published 4/15/2021) pertains to monoclonal antibodies or antibody fragments, including a human IgG antibody, comprising at least one lysine derivative; transformed cells obtained by introducing a vector into a host cell; and a method for producing the antibody or antibody fragment thereof (Abstract). Sakamoto teaches that the vectors and cells comprise a tRNA linked to an appropriate promoter to recognize a UAG codon (hereinafter, simply represented as “tRNA”) and the nucleotide sequence encoding the amino acid sequence of aminoacyl tRNA synthetase to acylate the tRNA with a non-natural amino acid ([0163]). The antibody fragments of Sakamoto include VH fragments and are taught to include secretion signal sequences ([0067]; [0195]).
Hirasawa et al. (US20190055300A1, on IDS filed 11/6/2025) pertains to methods for manufacturing an azide group-containing Fc protein based on a specific enzymatic reaction and using the manufactured azide group-containing Fc protein as a raw material to produce a fusion protein of a protein of interest and an Fc protein ([0014]). Hirasawa teaches producing Fc proteins that have non-naturally occurring amino acids (e.g. Examples 8 and 9). Hirasawa teaches reacting an azide group-containing phenylalanine derivative with the Fc protein having the above peptide linker at/on the N-terminus by using phenylalanyl tRNA, aminoacyl tRNA synthetase, and leucyl/phenylalanyl tRNA transferase, to yield the azide group-containing Fc protein ([0096]; [0170]-[0172]; [0277], claim 22). Hirasawa also teaches expression vectors including pPK5, which contain signal peptides including those of the Tat-system e.g. tatABC ([0327]-[0330])
“Ningbo Kunpeng Biotech Co” (WO2020187271A1, on IDS filed 11/6/2025) discloses a dual-plasmid system comprising a first plasmid for an expression cassette for expressing a target protein containing a predetermined modified amino acid, a second plasmid for an expression cassette for expressing aminoacyl-tRNA synthetase, and a third expression cassette in the first plasmid or the second plasmid for encoding artificial tRNA (Abstract). This reference teaches the application of lysyl-tRNA synthetases for the incorporation of lysine derivatives (see Examples 2-4).
Conclusion
No claims are allowable.
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/A.T.M./Examiner, Art Unit 1655
/ANAND U DESAI/Supervisory Patent Examiner, Art Unit 1655
APPENDIX A: Alignment of SEQ ID NO: 2 (Qy) with SEQ ID NO: 4 (Db) of Matsuda
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APPENDIX B: Alignment of SEQ ID NO: 50 (Qy) with SEQ ID NO: 28 (Db) of Robertson
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APPENDIX C: Alignment of SEQ ID NO: 42 (Qy) with SEQ ID NO: 69 (Db) of Mureev
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Prosecution Insights