DETAILED ACTION
Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Claim Status
Claims 2-3, 21-22 were cancelled.
Claims 1, 4-10, 12, 14-18, and 20 were amended.
Claims 1, 4-20 are examined on the merits.
Priority
The applicant’s application is a U.S. National Stage application of PCT International Patent Application Serial No. PCT/IB2021/061624, filed December 13, 2021, which itself claims the benefit of Indian Patent Application Serial No. IN202041054332, filed December 14, 2020 is acknowledged.
Receipt is acknowledged of certified copies of papers required by 37 CFR 1.55.
Nucleotide and/or Amino Acid Sequence Disclosures
REQUIREMENTS FOR PATENT APPLICATIONS CONTAINING NUCLEOTIDE AND/OR AMINO ACID SEQUENCE DISCLOSURES
Items 1) and 2) provide general guidance related to requirements for sequence disclosures.
37 CFR 1.821(c) requires that patent applications which contain disclosures of nucleotide and/or amino acid sequences that fall within the definitions of 37 CFR 1.821(a) must contain a "Sequence Listing," 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 1.821 - 1.825. This "Sequence Listing" part of the disclosure may be submitted:
In accordance with 37 CFR 1.821(c)(1) 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") as an ASCII text file, together with an incorporation-by-reference of the material in the ASCII text file in a separate paragraph of the specification as required by 37 CFR 1.823(b)(1) identifying:
the name of the ASCII text file;
ii) the date of creation; and
iii) the size of the ASCII text file in bytes;
In accordance with 37 CFR 1.821(c)(1) 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 of the material in the ASCII text file according to 37 CFR 1.52(e)(8) and 37 CFR 1.823(b)(1) in a separate paragraph of the specification identifying:
the name of the ASCII text file;
the date of creation; and
the size of the ASCII text file in bytes;
In accordance with 37 CFR 1.821(c)(2) via the USPTO patent electronic filing system as a PDF file (not recommended); or
In accordance with 37 CFR 1.821(c)(3) on physical sheets of paper (not recommended).
When a “Sequence Listing” has been submitted as a PDF file as in 1(c) above (37 CFR 1.821(c)(2)) or on physical sheets of paper as in 1(d) above (37 CFR 1.821(c)(3)), 37 CFR 1.821(e)(1) requires a computer readable form (CRF) of the “Sequence Listing” in accordance with the requirements of 37 CFR 1.824.
If the "Sequence Listing" required by 37 CFR 1.821(c) is filed via the USPTO patent electronic filing system as a PDF, then 37 CFR 1.821(e)(1)(ii) or 1.821(e)(2)(ii) requires submission of a statement that the "Sequence Listing" content of the PDF copy and the CRF copy (the ASCII text file copy) are identical.
If the "Sequence Listing" required by 37 CFR 1.821(c) is filed on paper or read-only optical disc, then 37 CFR 1.821(e)(1)(ii) or 1.821(e)(2)(ii) requires submission of a statement that the "Sequence Listing" content of the paper or read-only optical disc copy and the CRF are identical.
Specific deficiencies and the required response to this Office Action are as follows:
Specific deficiency – Nucleotide and/or amino acid sequences appearing in the specification are not identified by sequence identifiers in accordance with 37 CFR 1.821(d). See nucleotide sequences at page 28.
Required response – Applicant must provide:
A substitute specification in compliance with 37 CFR 1.52, 1.121(b)(3) and 1.125 inserting the required sequence identifiers, 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.
Claim Rejections - 35 USC § 101
35 U.S.C. 101 reads as follows:
Whoever invents or discovers any new and useful process, machine, manufacture, or composition of matter, or any new and useful improvement thereof, may obtain a patent therefor, subject to the conditions and requirements of this title.
Claims 1, 4-5 are rejected under 35 U.S.C. 101 because the claimed inventions are directed to judicial exception(s) (i.e., a law of nature, a natural product, and/or an abstract idea) without significantly more. The rationale for this determination is explained below:
Claims 1, 4-5 recites a composition comprising SEQ ID NO 1 or SEQ ID NO 2, which corresponds to GDH2 promoter of Pichia pastoris and PEPC promoter of Pichia pastoris, respectively. The claimed SEQ ID NO 1 is not markedly different than the naturally occurring counterpart from Komagataella phaffii GS115 (Pichia Pastoris) (see Fig. below).
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The claimed SEQ ID NO 2 is not markedly different than the naturally occurring counterpart from Pichia pastoris GS115 (see Fig. below).
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This judicial exception is not integrated into a practical application because no elements in addition of the judicial exception are recited in the claims.
The claims do not include additional elements that are sufficient to amount to significantly more than the judicial exception because no elements in addition to the judicial exception are recited in the claims.
Claim Rejections - 35 USC § 112
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, 6-20 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 applications subject to pre-AIA 35 U.S.C. 112, the inventor(s), at the time the application was filed, had possession of the claimed invention.
Claim 1, is drawn to a genus of nucleotide sequences from Pichia pastoris. The rejected claim comprise a genus consisted of a Nucleotide Sequence selected from a sequence having at least 90% identity to sequence set forth as SEQ ID No.1; or a sequence having at least 90% identity to sequence set forth as SEQ ID No. 2.
To provide adequate written description and evidence of possession of a claimed genus, the specification must provide sufficient distinguishing identifying characteristics of the genus. The factors to be considered include disclosure of a complete or partial structure, physical and/or chemical properties, functional characteristics, structure/function correlation, and any combination thereof.
The specification envisions nucleotide sequences selected from a group comprising sequence set forth as SEQ ID No. 1 and SEQ ID No. 2, wherein the said nucleotide sequence possess promoter activity. The nucleotide sequence enhances the production of protein of interest/heterologous protein/recombinant protein in yeast expression system, particularly Pichia pastoris, wherein the said nucleotide sequence is an inducible promoter (e.g., lanes 7-14, page 2). The specification envisions Pichia pastoris Glutamate dehydrogenase 2 (GDH2) promoter (SEQ ID No. 1) up to 995 bp upstream of the ATG start codon (e.g., lane 20, page 3; Fig. 1). The specification envisions nucleotide sequence of the Pichia pastoris phosphoenolpyruvate carboxykinase (PEPCK) promoter (SEQ ID No. 2) up to 1000 bp upstream of the ATG start codon (e.g., lane 23; Fig. 2). The specification envisions the nucleic acid sequence having at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to nucleic acid sequence set forth as SEQ ID No. 1, possess promoter activity capable of influencing effective production of protein of interest or recombinant protein (e.g., lane 17, page 9).
The specification envisions the nucleic acid sequence having at least
90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to nucleic acid sequence set forth as SEQ ID No. 2, possess promoter activity capable of influencing effective production of protein of interest or recombinant protein (e.g. lane 23, page 9). The specification envisions the gene encoding protein of interest is
cloned downstream of the SEQ ID No. 1 and/or SEQ ID No. 2 in the expression vector. In some embodiments of the present disclosure, the expression vector comprising the nucleic acid sequence set forth as SEQ ID No. 1 includes but is not limited to pTRl-IX and pTRl-SX (e.g., lane 6, page 12). The specification envisions the expression vector comprising the
nucleic acid sequence set forth as SEQ ID No. 2 includes but is not limited to pTR2-IX and pTR2-SX (e.g., lane 12, page 12).
The examples described in the specification does not meet the limitation of the rejected claim (at least 90% identity), the examples are only representative of SED ID NO 1 or SEQ ID NO 2. “At least 90 % identity” encompasses a very large number of different nucleotide sequences (i.e., every sequence at least 90% identical to SEQ ID NO: 1 or SEQ ID NO 2), but there is insufficient guidance provided indicating any of the elements that are critical to the functioning of the promoter, thus it cannot be determined which nucleotides can be changed without disrupting the function of the promoter; thus further experimentation would be required to determine which variants of SEQ ID NO: 1 and SEQ ID NO 2 are functional and which are not.
As such, the claims encompasses significantly more than what is disclosed in the specification and does not satisfy the written description requirement under 35 U.S.C 112(a).
Therefore, the skilled artisan would have reasonably concluded applicants were not in possession of the claimed invention for claims 1, 6-20.
The claims listed in the statement of rejection but not otherwise discussed are rejected because they are similarly not limited to particular polynucleotides that are considered to be adequately described by the specification.
Claim Rejections - 35 USC § 102
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
The following is a quotation of the appropriate paragraphs of 35 U.S.C. 102 that form the basis for the rejections under this section made in this Office action:
A person shall be entitled to a patent unless –
(a)(1) the claimed invention was patented, described in a printed publication, or in public use, on sale, or otherwise available to the public before the effective filing date of the claimed invention.
Claims 1, 4 is rejected under 35 U.S.C. 102(a)(1) as being anticipated by Achmuller et al. (“Achmuller”, US 2018/0079788 A1).
Regarding claim 1, 4, Achmuller teaches modified eukaryotic cell, or expression system, respectively, comprising a modified gene, a polynucleotide sequence comprising said modified gene, an expression vector comprising said polynucleotide sequence, a host cell comprising said expression vector (e.g., paragraph, 0001). Achmuller teaches The modified eukaryotic cell, wherein the ssn6-like gene and/or the ssn6-like related gene is/are modified in a regulatory sequence, such as promoter(s), enhancer(s), terminator(s), silencer(s), IRES-sequence(s ), ribosome-binding site(s ), and sequence (s) stabilizing or destabilizing the mRNA by secondary structure( s ), and/ or in a coding sequence (e.g., paragraph 0020). Achmuller teaches the modified eukaryotic cell according to any of the preceding items, wherein said cell is a fungal cell, preferably a yeast cell, more preferably selected from the group consisting of Saccharomyces species ( e.g., Saccharomyces cerevisiae), Kluyveromyces species (e.g., Kluyveromyces lactis), Torulaspora species, Yarrowia species (e.g., Yarrowia lipolitica ), Schizosaccharomyces species ( e.g., Schizosaccharomyces pombe), Pichia species (e.g., Pichia pastoris or Pichia methanolica), (e.g., paragraph 0028). Achmuller teaches SEQ ID NO 96 with 100% homology to SEQ ID NO 1 of the instant claim (see alignment below).
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Claims 1, 5 are rejected under 35 U.S.C. 102(a)(1) as being anticipated by Callewaert et al. (“Callewaert”, US 8,440,456 B2).
Callewaert teaches novel nucleic acids, proteins, and related expression vectors useful for genetic engineering of methylotrophic yeast strains, as well as engineered methylotrophic yeast strains particularly Pichia pastoris, and use thereof for recombinant production of heterologous proteins including glycoproteins suitable for use in mammals including humans (e.g., abstract). Callewaert teaches SEQ ID NO 252 with 99.9% similarity with SEQ ID NO 2 of the instant claims (see alignment below).
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Claim Rejections - 35 USC § 103
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. Considering objective evidence present in the application indicating obviousness or nonobviousness.
Claims 6-11, 13-15, 20 are rejected under 35 U.S.C. 103 as being unpatentable over Callewaert et al. (“Callewaert”, US 8,440,456 B2) as applied to claims 1, 5 above, and further in view of Mattanovich et al. (“Mattanovich, US 2019/0337998 A1).
Regarding claim 6-8, 20, Callewaert teaches a set of vectors useful for identification of the most effective choice of signal peptide for any given heterologous protein. Each vector contains a promoter and the coding sequence of one of the signal peptides an and a linker sequence or cloning site for inserting or receiving the coding sequence of the heterologous protein. In a specific embodiment, the linker sequence or cloning site simply includes a restriction endonuclease site. The heterologous coding sequence, with the same restriction site at its 5' end, can be joined to the signal peptide coding sequence via restriction enzyme digestion and subsequent ligation (e.g., lane 14, column 2). Callewaert teaches specific promoters of the present invention include those identified for genes involved in glycolysis pathway, genes showing high expression levels, homologs of S. cerevisiae genes whose promoters are frequently used for recombinant expression, methanol metabolism genes, and genes involved in xylose, arabinose or trehalose metabolism. The promoters of these genes are located within the 1000 bp of the 5' region provided herein, and generally are located within the 500 bp immediately before the start codon of the gene, in some embodiments within 250 bp, 200 bp, 150 bp, 125 bp, 100 bp, 75 bp, 50 bp, 40 bp, or 55 even 25 bp immediately before the start codon of the gene (e.g., lane 43, column 2).
Regarding claim 9, Callewaert teaches promoters for expression
of a heterologous gene, including the related expression vectors and cells transformed with any such expression vectors. (e.g., lane 58, column 2). Callewaert teaches a methylotrophic yeast strain such as Pichia can be transformed with a heterologous nucleic acid, i.e., a nucleic acid which the non-engineered Pichia strain does not have. The resulting engineered strain will express the protein encoded by the heterologous nucleic acid, i.e., a heterologous protein (e.g., lane 6, column 8).
Regarding claim 11, 13-14, Callewaert teaches expression vector capable of directing the expression and secretion of a heterologous protein in Pichia pastoris or another methylotrophic yeast. The expression vector contains, from 5' to 3', a promoter functional in the recipient strain, operably linked to a coding sequence for the fusion of a signal peptide and the heterologous protein. Host cells transformed with such an expression vector (e.g., lane 12, column 10).
Regarding claim 15, Callewaert teaches that the promoters can be placed in an operable linkage to a heterologous gene for methanol-inducible recombinant expression in Pichia pastoris. Therefore, expression vectors, host cells and methods of recombinant expression by utilizing any of the promoters (e.g., lane 43, column 18).
Callewaert does not teach the expression vector where the gene encodes a protein selected from an antibody, hormone, enzyme, growth factor, vaccine, as required by instant claim 10. However, this is cured by Mattanovich.
Regarding claim 10, Mattanovich teaches the recombinant host cell which comprises the nucleic acid sequence of the expression construct of the invention, preferably a eukaryotic cell, more preferably a yeast or filamentous fungal cell, more preferably a yeast cell of the Saccharomyces or Pichia genus (e.g., paragraph 0039). Mattanovich teaches the protein of interest is a recombinant or heterologous protein, preferably selected from therapeutic proteins, including antibodies or fragments thereof, enzymes and peptides, protein antibiotics, toxin fusion proteins, carbohydrate-protein conjugates, structural proteins, regulatory proteins, vaccines and vaccine like proteins or particles, process enzymes, growth factors, hormones and cytokines (e.g., paragraph 0156).
Based on these teachings, it would have been prima facie obvious to one of ordinary skill in the art, before the effective filing date of the claimed invention, to combine the teachings of Callewaert - expression vectors useful for genetic engineering of methylotrophic yeast strains, as well as engineered methylotrophic yeast strains particularly Pichia pastoris, and use for recombinant production of heterologous proteins by using specific promoters including those identified for genes involved in glycolysis pathway, methanol metabolism, and genes involved in xylose, arabinose or trehalose metabolism, like SEQ ID NO 252, with the teachings of Mattanovich -recombinant host cell preferable the Saccharomyces or Pichia genus, which comprises the nucleic acid sequence of the expression construct carrying the protein of interest, preferably selected from therapeutic proteins, including antibodies or fragments thereof, enzymes and peptides, protein antibiotics, toxin fusion proteins, carbohydrate-protein conjugates, structural proteins, regulatory proteins, vaccines and vaccine like proteins or particles, process enzymes, growth factors, hormones and cytokines; for someone skilled in the art would have been obvious to use these teachings to achieve the predictable result of developing an expression vector comprising the promoter SEQ ID NO 252 and a gene for the heterologous protein for expression in a yeast like Saccharomyces or Pichia pastoris.
One of ordinary skill in the art before the effective filing date of the invention would have been motivated to develop an expression vector for expression in yeast Saccharomyces or Pichia genus of therapeutic proteins, including antibodies or fragments thereof, enzymes and peptides, protein antibiotics, toxin fusion proteins, carbohydrate-protein conjugates, structural proteins, regulatory proteins, vaccines and vaccine like proteins or particles, process enzymes, growth factors, hormones and cytokines.
Claims 16-19 are rejected under 35 U.S.C. 103 as being unpatentable over Callewaert et al. (“Callewaert”, US 8,440,456 B2) and Mattanovich et al. (“Mattanovich, US 2019/0337998 A1) as applied to claims 1, 5-11, 13-20 above, and further in view of Sahu et al. (“Sahu”, The J. OF Biol. Chem., Mar. 2016, cited as reference CD on IDS filed 06/09/2023).
Regarding claim 19, Mattanovich teaches that the protein of interest is secreted from the cells, it can be isolated and purified from the culture medium using state of the art techniques. Secretion of the recombinant expression products from the host cells is generally advantageous for reasons that include facilitating the purification process, since the products are recovered from the culture supernatant rather than from the complex mixture of proteins that results when yeast cells are disrupted to release intracellular proteins (e.g., paragraph 0207). Mattanovich teaches The cultured transformant cells may also be ruptured sonically or mechanically, enzymatically or chemically to obtain a cell extract containing the desired POI, from which the POI is isolated and purified (e.g., paragraph 0208).
Callewaert and Mattanovich do not teach replacing the carbon source with a component selected from glutamate, monosodium glutamate as required by the instant claims 16-18. However, this is cured by Sahu.
Regarding claim 16, Sahu teaches that unlike Saccharomyces cerevisiae, the methylotrophic yeast Pichia pastoris can assimilate amino acids as the sole source of carbon and nitrogen. It can grow in media containing yeast extract and peptone (YP), yeast nitrogen base (YNB) + glutamate (YNB+Glu), or YNB + aspartate (YNB+Asp). Sahu teaches that methanol expression regulator 1 (Mxr1p), a zinc finger transcription factor, is essential for growth in these media. Mxr1p regulates the expression of several genes involved in the utilization of amino acids as the sole source of carbon and nitrogen. These include the following: (i) GDH2 encoding NAD-dependent glutamate dehydrogenase; (ii) AAT1 and AAT2 encoding mitochondrial and cytosolic aspartate aminotransferases, respectively; (iii) MDH1 and MDH2 encoding mitochondrial and cytosolic malate dehydrogenases (e.g., abstract). Sahu teaches that in addition to GDH2, enzymes such as aspartate aminotransferase (AAT),2 malate dehydrogenase (MDH), and glutamine synthetase (GLN1) also play key roles in the metabolism of amino acids. Sahu teaches P. pastoris, a methylotrophic yeast, is extensively used for the production of recombinant proteins. Being a respiratory yeast, P. pastoris completely oxidizes sugars, avoiding formation of ethanol, and this results in efficient utilization of carbon sources yielding high biomass (e.g., paragraph 2nd, column right, page 20588). Sahu teaches that the ability of P. pastoris to utilize amino acids as the sole source of carbon and nitrogen has not been investigated. (e.g., paragraph 2nd, column left, page 20589). Sahu teaches that P. pastoris (GS115, his) was cultured in either minimal medium containing 0.17% yeast nitrogen base (YNB) without amino acids, and 0.5% ammonium sulfate supplemented with 2.0% glucose (YNBD) (It reads as required by instant claim 16 [a]), 1.0% glutamate (YNB+Glu) (It reads as required by instant claim 16 [b]), or 1% aspartate (YNB+Asp) or nutrient rich YP medium (1.0% yeast extract and 2.0% peptone) alone or YP medium containing 2.0% glucose (YPD) or 2% methanol (YPM) (e.g., paragraph 2nd, column right, page 20597). Sahu teaches that knockout mxr1 Pichia pastoris strain fails to grow on media where amino acid are the only available energy source (e.g., Fig. 1).
Fig. 1A-B:
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Regarding claims 17-18, Sahu teaches the generation of P. pastoris strain expressing GDH2His from its own promoter, the gene encoding GDH2 along with 545 bp of its promoter was cloned into pIB3 vector as a histidine-tagged protein (GDH2His) (e.g., paragraph 3rd, column right, page 20597). Sahu teaches that P. pastoris GS115 and knockout mxr1 were transformed with pIB3-GDH2His, and expression of Gdh2pHis was examined in cells cultured in different carbon sources by Western blotting. GDH2His expression levels were higher in cells cultured in YP, YNB+Glu, and YNB+Asp than those cultured in YPD or YNBD (e.g., paragraph 1st, column right, page 20589; Fig. 1. F and G). (It reads that there is no expression of GDH2His in YPD or YNBD media [claim 17]).
Fig. 1F-G:
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Sahu teaches that the assimilation of glutamate requires the activity of NAD-dependent glutamate dehydrogenase 2 (GDH2), and a knockout gdh2 strain cannot utilize glutamate or aspartate as either a carbon or a nitrogen source (e.g., paragraph 2nd, column right, page 20588; Fig. 2).
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Sahu teaches that Mxr1p as a regulator of synthesis of key enzymes involved in the utilization of amino acids. Biosynthesis of AAT1, AAT2,
MDH1, MDH2, and GLN1 is regulated by Mxr1p at the level of transcription as evident from the significant reduction in mRNA levels of genes encoding these enzymes in knockout mxr1 strain. Promoters of AAT2, MDH2, and GLN1 contain MXREs to which recombinant Mxr1p binds specifically in vitro. Using AAT2 and GLN1 as examples, demonstrating the
importance of promoter occupancy by Mxr1p in vivo for transcriptional
activation of these genes. Key enzymes identified in this study, whose synthesis is regulated by Mxr1p, are depicted below. Mxr1p functions as a global regulator of multiple metabolic pathways in P. pastoris (e.g., paragraph 2nd, column right, page 20595; Fig. 8 [see below]). Sahu teaches that amino acids enhance growth rate as well as recombinant
protein production in several yeast species when added to media containing conventional carbon sources, primarily by serving as precursors for the synthesis of proteins as well as participating in anaplerotic reactions. In the case of P. pastoris, mixed feeds of methanol and a multicarbon source instead of methanol as the sole carbon source have been shown to improve recombinant protein production (e.g., paragraph 3rd, column right, page 20595).
Fig. 8:
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Based on these teachings, it would have been prima facie obvious to one of ordinary skill in the art, before the effective filing date of the claimed invention, to combine the teachings of Callewaert -expression vectors useful for genetic engineering of methylotrophic yeast strains, as well as engineered methylotrophic yeast strains particularly Pichia pastoris, and use for recombinant production of heterologous proteins by using specific promoters including those identified for genes involved in glycolysis pathway, methanol metabolism, and genes involved in xylose, arabinose or trehalose metabolism, like SEQ ID NO 252, with the teachings of Mattanovich -recombinant host cell preferable the Saccharomyces or Pichia genus, which comprises the nucleic acid sequence of the expression construct carrying the protein of interest, preferably selected from therapeutic proteins, including antibodies or fragments thereof, enzymes and peptides, protein antibiotics, toxin fusion proteins, carbohydrate-protein conjugates, structural proteins, regulatory proteins, vaccines and vaccine like proteins or particles, process enzymes, growth factors, hormones and cytokines and the teachings of Sahu -respiratory yeasts such as P. pastoris and P. stipitis have evolved pathways for efficient utilization of amino acids both as a source of carbon and nitrogen. The generation of TCA cycle intermediates such as alpha-ketoglutarate and oxaloacetate from glutamate and aspartate is the first step in the utilization of amino acids as the sole source of carbon. P. pastoris GS115 but not knockout mxr1 strain can grow in media containing amino acids as the sole source of carbon. enzymes such as GDH2, cAAT, mAAT, cMDH, and mMDH, which play a crucial role in the inter-conversion of amino acids and keto acids in the cytosolic and mitochondrial compartments as well as glutamine synthetase involved in glutamine synthesis, are important for the utilization of amino acids as the sole source of carbon and nitrogen in P. pastoris; for someone skilled in the art would have been obvious to use these teachings to achieve the predictable result of developing an expression vector comprising the promoter SEQ ID NO 252 and a gene for the heterologous protein for expression in a yeast like Pichia pastoris and growth in media containing amino acids like glutamate as a source of carbon to enhance the growth rate as well as recombinant protein production.
One of ordinary skill in the art before the effective filing date of the invention would have been motivated to develop method to enhance growth rate as well as recombinant protein production in different yeast species, like Pichia pastoris by using amino acids like glutamate as a source of carbon.
Conclusion
Any inquiry concerning this communication or earlier communications from the examiner should be directed to JULIO GOMEZ RODRIGUEZ whose telephone number is (571)270-0991. The examiner can normally be reached Monday - Friday 8:00 am - 5:00 pm.
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/JULIO WASHINGTON GOMEZ RODRIGUEZ/Examiner, Art Unit 1637
/J. E. ANGELL/Primary Examiner, Art Unit 1637