DETAILED CORRESPONDENCE
Status of the Application
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Claims 1-2, 4-5, 8-19, 22, and 26 are pending in this application.
Applicant’s amendment to the claims filed 02/18/2026 is acknowledged. This listing of the claims replaces all prior versions and listings of the claims.
Applicant’s remarks filed on 02/18/2026 in response to the non-final rejection mailed on 09/18/2025 are acknowledged and have been fully considered.
The text of those sections of Title 35, U.S. Code not included in this action can be found in a prior Office action.
Election
The elected subject matter is
Group I, corresponding to claims 1-2, 4-5, 8-13, 22 and 26, drawn to the technical feature of a method of cell culture comprising (i) providing cells in a cell culture medium to start a cell culture process, wherein the cells are modified to reduce the level of synthesis of growth or productivity inhibitors by the cell, wherein the inhibitors are formate or glycerol,
elected without traverse in the reply filed 07/23/2024.
Claims 14-19 are withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected invention, there being no allowable generic or linking claim.
Claims 1-2, 4-5, 8-13, 22 and 26 are being examined on the merits.
Information Disclosure Statement
The Information Disclosure Statement (IDS) submitted on 02/18/2026 is in compliance with the provisions of 37 CFR 1.97. Accordingly, the IDS has been considered by the examiner.
Claim Objections
Claim 2 is objected to for the recitation of the phrase “serine hydroxymethyltransferase (SHMT2)”, for SHMT2 is referred to in the specification as mitochondrial serine hydroxymethyltransferase [p 2]. In the interest of improving claim form, Applicant should consider an amendment to recite “mitochondrial serine hydroxymethyltransferase (SHMT2)”.
Claim 2 is objected to for the phrase “The method of claim 1, wherein expression of serine hydroxymethyltransferase (SHMT2) is decreased in the cells to reduce a level of synthesis of formate as compared to a level of synthesis of formate by corresponding cells without decreased expression of SHMT2”. In the interest of improving claim form, Applicant should consider an amendment to recite “The method of claim 1, further comprising decreasing the expression of mitochondrial serine hydroxymethyltransferase (SHMT2) in the cells to reduce a level of synthesis of formate as compared to a level of synthesis of formate by corresponding cells without decreased expression of SHMT2”.
Claim 4 is objected to for the phrase “wherein the PGP and SHMT2 genes are modified to decrease gene expression”. In the interest of improving claim form, Applicant should consider an amendment to recite “wherein the expression of PGP and SHMT2 genes is decreased due to modification of the PGP and SHMT2 genes of the CHO cells.”
Claim Rejections - 35 USC § 112(b)
The rejection of claims 2, 4-5, 8-9, 12-13 and 26 under 35 U.S.C. 112(b) as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor regards as the invention are withdrawn in view of
the amendment to claim 2 to recite “wherein expression of serine hydroxymethyltransferase (SHMT2) is decreased in the cells to reduce a level of synthesis of formate as compared to a level of synthesis of formate by corresponding cells without decreased expression of SHMT2”, and
the amendments to claims 5, 8-9 and 12 to ultimately depend from claim 1.
Claim Rejections - 35 USC § 112(d)
The rejection of claims 2, 4-5, 8-9, 12-13 and 26 under 35 U.S.C. 112(d) as being of improper dependent form for failing to further limit the subject matter of the claim upon which it depends, and for failing to include all the limitations of the claim upon which it depends is withdrawn.
Claim Rejections - 35 USC § 103
The rejection of claims 1, 10-11 and 22 under 35 U.S.C. 103 as being unpatentable over Pfizer et al. (WO 2017/051347 A2; cited on the IDS submitted 06/02/2021; herein referred to as Pfizer) in view of Possik et al. (Biochimie, 2017, 143:18; cited on the Form PTO-892 mailed 03/21/2025; herein referred to as Possik), and
the rejection of claims 2, 4-5, 8-9, 12-13 and 26 rejected under 35 U.S.C. 103 as being unpatentable over Pfizer in view of Possik, and further in view of Perry et al. (Arch Biochem Biophys, 2007, 462:21; cited on the IDS submitted 06/02/2021; herein referred to as Perry)
are withdrawn in view of the amendment to claim 1 to recite “wherein expression of a phosphoglycolate phosphatase (PGP) gene is decreased in the cells to reduce a level of synthesis of glycerol and lactate”.
Claims 1, 5, 8-12 and 22 are newly rejected under 35 U.S.C. 103 as being unpatentable over Pfizer in view of Possik and Wilson et al. (J Appl Physiol, 2012, 113:1838; cited on the attached Form PTO-892; herein referred to as Wilson).
The instant rejection is newly stated and necessitated by claim amendment.
Claim 1 is drawn to a method of cell culture comprising (i) providing CHO cells in a cell culture medium to start a cell culture process,
wherein expression of a phosphoglycolate phosphatase (PGP) gene is decreased in the cells to reduce a level of synthesis glycerol and lactate by the cells as compared to a level of synthesis glycerol and lactate by corresponding cells without decreased expression of PGP.
Pfizer discusses cells and methods of cell culture [title].
Regarding claim 1, Pfizer discloses a method of cell culture where cells are modified to reduce the level of synthesis of growth and/or productivity of inhibitors by the cell [abstract], wherein the expression of one or more genes is modified to reduce the level of synthesis of cell growth and/or productivity inhibitors [claim 2] in CHO cells [claim 63].
Pfizer additionally discloses a scenario where proteins are increasingly important diagnostic and therapeutic agents, commonly produced commercially in cell culture wherein the cells have been engineered to produce unusually high levels of a protein of interest, and therefore discloses the importance of optimizing culture conditions to address inefficient mammalian cell metabolism that normally results in the consumption of nutrients for the production of byproducts that accumulate over the course of the culture in order to facilitate the successful production of target proteins [p 1, ln 15-24]. Pfizer further discloses lactate and ammonia to be known inhibitors of cells in cell cultures as two major byproducts of cellular metabolism [p 1, beginning on ln 24]. Therefore Pfizer indicates the importance of reducing byproduct formation of inhibitors such as lactate that can inhibit or contend with target protein production in mammalian cell culture.
Pfizer does not teach the limitation of decreasing expression of a PGP gene to reduce a level of synthesis of glycerol and lactate.
Possik relates to glycerol-3-phosphate phosphatase and PGP and their roles in intermediary metabolism [title], and discloses a previously unknown mammalian enzyme phosphoglycolate phosphatase (PGP) can function as a glycerol-3-phosphate (Gro3P) phosphatase (G3PP) to regulate metabolite levels [abstract].
Regarding claim 1, Possik discloses that Gro3P can be formed from DHAP and taken away from glycolysis through the “Gro3P shuttle”, where it is further dephosphorylated by G3PP activity to form glycerol [Figure 1]. As Possik discloses that PGP also acts as a G3PP enzyme, PGP is indicated to influence the production of glycerol that is inversely tied to glycolysis, since the originating DHAP molecule is shunted to the glycerol pathway at the expense of its progression through glycolysis. Possik further discloses that removal of G3PP activity in rat cells resulted in increased O2 consumption and ATP production, but the overexpression of proteins with G3PP activity had the reverse effect [p 21, col 1, para 3], indicating that lower G3PP activity reduces glycerol production while increasing activity through the glycolytic pathway based on the pathway displayed in [Figure 1].
Wilson relates to oxygen, pH, and mitochondrial oxidative phosphorylation [title].
Regarding claim 1, Wilson discloses that the activity of the citric acid cycle is a measure of transition in ATP synthesis from oxidative phosphorylation to glycolytic lactate production, and of the decrease in oxygen consumption [p 1843, col 1, para 3], therefore defining (a) the production of lactate and (b) the production of ATP via oxidative phosphorylation and consumption of oxygen as metabolic alternatives that can be observed based on the relative activity of the citric acid cycle. In view of the teachings of Wilson, one of ordinary skill in the art would have recognized the removal of G3PP activity attributed to PGP that results in increased O2 consumption and ATP production disclosed by Possik corresponds to an increase in oxidative phosphorylation, and therefore would have been expected to result in a decrease in lactate formation as well as the decrease in glycerol formation disclosed by Possik.
In view of Pfizer, Possik and Wilson, one of ordinary skill in the art would have recognized that lowering the expression of PGP would thereby lower the production of glycerol and instead shift metabolism towards O2 consumption and ATP production, which additionally would lower the production of lactate, which is interpreted to correspond to increased growth and activity through the removal of cellular inhibitors. One of ordinary skill in the art would have been motivated to do so as Pfizer discloses the importance of reducing the consumption of nutrients used for production of byproducts in order to facilitate the successful production of target molecules.
It would have been prima facie obvious for one of ordinary skill in the art before the effective filing date of the claimed invention to combine Pfizer, Possik and Wilson to modify the method of Pfizer by lowering expression of PGP to reduce the accumulation of glycerol, as taught by Possik, and reduce the accumulation of lactate, as taught by Wilson to arrive at the claimed invention. One of ordinary skill in the art would have been motivated to modify the method of Pfizer by lowering PGP expression because Pfizer teaches the importance of reducing the consumption of nutrients used for production of byproducts in order to facilitate the successful production of target molecules, Possik teaches lowering the expression of PGP lowers the production of glycerol and shifts metabolism towards O2 consumption and ATP production, and Wilson teaches that the production of ATP via oxidative phosphorylation and the production of glycolytic lactate are inversely related. One of ordinary skill in the art would have had a reasonable expectation of success because Pfizer relates to methods of optimizing metabolism in cells through regulating metabolite levels, Possik relates to the enzyme PGP and its ability to regulate metabolite levels, and Wilson discusses the relationship between metabolic pathways that branch from glycolysis and their effects on ATP production.
Regarding claim 5, Pfizer discloses maintaining formate at a specific concentration below a reference concentration [claim 13], which is below 2 mM [claim 14], wherein formate is identified as a metabolite that inhibits cell growth which can be limited by maintaining concentration in cell culture medium below levels where they inhibit cell growth [p 8, lns 17-26]. As Pfizer additionally identifies lactate as an inhibitor of cell growth [p 1, beginning on ln 24], one of skill in the art would be motivate to similarly maintain lactate concentrations below a reference concentration as taught by Pfizer above.
Regarding claim 8, Pfizer discloses measuring the concentration of formate and when that measured concentration is above a predefined value, that concentration is decreased by reducing the amount of precursor provided to the cells [claim 15], wherein the predefined value of formate is disclosed as 2 mM [claim 14]. As Pfizer identifies formate as a metabolite that inhibits cell growth which can be limited by maintaining concentration in cell culture medium below levels where they inhibit cell growth [p 8, lns 17-26], and additionally identifies lactate as an inhibitor of cell growth [p 1, beginning on ln 24], one of skill in the art would be motivate to similarly measure and maintain lactate concentrations as taught by Pfizer above.
Regarding claim 9, Pfizer discloses measuring the concentration by NMR, HPLC or UPLC [claim 16].
Regarding claim 10, Pfizer discloses the use of a pH sensor to monitor pH of the cell culture, and in response to a rise above a predetermined pH value, glucose is fed to the cell culture [claim 27].
Regarding claim 11, Pfizer discloses the cell culture is a fed batch culture [claim 28].
Regarding claim 12, Pfizer discloses the method comprises a growth phase and a production phase and step (ii) is applied during the growth phase, wherein step (ii) comprises a step of measuring a concentration of formate [claim 29].
Regarding claim 22, Pfizer discloses the method wherein cell growth and/or productivity are increased as compared to a control culture, said control culture being identical except comprises unmodified cells [claim 66].
Therefore, the invention of claims 1, 5, 8-12 and 22 would have been obvious to one of ordinary skill in the art before the effective filing date.
Claims 2, 4, 13 and 26 are newly rejected under 35 U.S.C. 103 as being unpatentable over Pfizer in view of Possik and Wilson as applied to claims 1, 5, 8-12 and 22 above, and further in view of Perry.
The instant rejection is newly stated and necessitated by claim amendment.
Claim 2 is drawn to the method of claim 1, wherein expression of serine hydroxymethyltransferase (SHMT2) is decreased in the cells to reduce a level of synthesis of formate as compared to a level of synthesis of formate by corresponding cells without decreased expression of SHMT2.
The teachings of Pfizer and Possik as applied to claims 1, 5, 8-12 and 22 are discussed above. Additionally, Pfizer discloses a method of cell culture where cells are modified to reduce the level of synthesis of growth and/or productivity of inhibitors by the cell [abstract], wherein the expression of one or more genes is modified to reduce the level of synthesis of cell growth and/or productivity inhibitors [claim 2] in CHO cells [claim 63], wherein the modified gene encodes an enzyme that catalyzes the synthesis of formate [claim 4] which inhibits the growth of cells [p 8, ln 17-23].
These references do not teach expression of serine hydroxymethyltransferase (SHMT2) is decreased in the cells to reduce a level of synthesis of formate as compared to a level of synthesis of formate by corresponding cells without decreased expression of SHMT2.
Perry discusses the effect of vitamin B6 availability on serine hydroxymethyltransferase (SHMT) in MCF-7 cells [title], and discloses that formate is a major source of one-carbon units for cytoplasmic one-carbon metabolism that is generated in the mitochondria from serine [p 21, col 2, para 1].
Regarding claim 2, Perry teaches the modification of MCF-7 cells to generate PLP-deficient cells to reduce the expression and activity of mitochondrial serine hydroxymethyltransferase [p 24, col 1, para 3; p 22, col 1, final paragraph; Figure 2b and Figure 2b caption], and that mitochondrial serine hydroxymethyltransferase is responsible for the conversion of serine to formate [Figure 1 and Figure 1 caption]. As the instant specification refers to SHMT2 as mitochondrial serine hydroxymethyltransferase [p 2], the SHMT of Perry is considered to correspond to the SHMT2 recited in the claims. As such, one of skill in the art would have reasoned that the modification of a cell to reduce the expression of SHMT would result in reduced production of formate as well.
In view of Perry, it would have been prima facie obvious for one of ordinary skill in the art before the effective filing date of the claimed invention to modify the combined method of Pfizer, Possik and Wilson by reducing the expression of SHMT, as taught by Perry, to arrive at the claimed invention. One of ordinary skill in the art would have been motivated to modify the combined method of Pfizer, Possik and Wilson because Pfizer teaches a method of reducing the levels of growth and/or productivity inhibitors by a cell that includes modifying the expression of genes that produce the growth inhibitor formate, and Perry indicates that reduction of SHMT expression will reduce the production of formate from serine. One of ordinary skill in the art would have had a reasonable expectation of success because both Pfizer and Perry discuss methods that regulate production of the metabolite formate.
Regarding claim 4, Possik teaches the reduction of PGP activity via RNAi knockdown [p 21, col 1, para 3], which is not a method of modifying the PGP gene as RNAi knockdown affects translation of mRNA. Perry teaches the modification of MCF-7 cells to generate PLP-deficient cells to reduce the expression and activity of serine hydroxymethyltransferase [p 24, col 1, para 3, p 22, col 1, final paragraph, and Figure 2b], which is not a method of modifying the SHMT2 gene. Pfizer however teaches the modification of genes to decrease expression to reduce the level of synthesis of growth and/or productivity inhibitors by the cell [claim 58] for use in a method to reduce the level of inhibitory metabolites that include formate and lactate [abstract; p 8, ln 17-23; p 1, beginning on ln 24]. In view of Pfizer, Possik and Perry, one of ordinary skill in the art would be motivated to modify the genes encoding PGP and SHMT to reduce the production of lactate and formate, respectively, as Possik and Perry teach methods for reducing the activity of polypeptides encoded by the PGP and SHMT genes, and Pfizer teaches a method of modifying genes to reduce the activity of the encoded polypeptides.
Regarding claim 13, Pfizer discloses modifying the expression of one or more genes comprises gene deletion, disruption, substitution, point mutation, multiple point mutation, insertion mutation or frameshift mutation of the gene, or introduction of one or more nucleic acids comprising the one or more genes into the cell, optionally as an expressible construct or expressible vector construct [claim 40].
Regarding claim 26, Pfizer discloses the method wherein cell growth and/or productivity are increased as compared to a control culture, said control culture being identical except comprises unmodified cells [claim 66].
Therefore, the invention of claims 2, 4, 13 and 26 would have been obvious to one of ordinary skill in the art before the effective filing date.
Response to remarks: beginning page 7 of Applicant’s response to rejections under 35 USC 103; Applicant in summary contends Pfizer and Possik do not teach or suggest each element of the amended claims, especially considering the newly recited limitation in claim 1 of decreasing PGP expression to reduce levels of both glycerol and lactate; Applicant further contends that Possik teaches away from such PGP reductions by stating the G3PP expression is inversely related to glycolytic flux [p 21, col 1, para 3]; Applicant further contends that Mugabo et al. (PNAS, 2016, 113:E430; cited on the IDS filed 09/27/2022) discloses knockdown of G3PP in hepatocytes enhanced lactate production and release; and Applicant further contends one of ordinary skill in the art would not have been motivated to modify PGP to decrease expression to reduce the level of synthesis of growth inhibitors in view of the cited teachings of Possik and Mugabo.
Applicants remarks are considered and found not convincing.
The teachings of Pfizer, Possik and Wilson are discussed in detail in the rejection above. Briefly, Pfizer teaches a method of reducing inhibitors of cell growth such as formate, and additionally names lactate as a well-known inhibitor, but Pfizer does not teach that glycerol is such an inhibitor. Pfizer instead generally discloses the importance of optimizing culture conditions to address inefficient mammalian cell metabolism that normally results in the consumption of nutrients for the production of byproducts that accumulate over the course of the culture in order to facilitate the successful production of target proteins. Possik discloses that Gro3P can be formed from DHAP and taken away from glycolysis through the “Gro3P shuttle”, where it is further dephosphorylated by G3PP activity to form glycerol, which corresponds to the production of glycerol that is inversely tied to glycolysis. This pathway would thereby be concluded by one of skill in the art to be encompassed by “inefficient mammalian cell metabolism that normally results in the consumption of nutrients for the production of byproducts that accumulate over the course of the culture” as disclosed by Pfizer. As Possik shows the decrease in PGP activity corresponds to a decrease in glycerol, and an increase in ATP and O2 consumption (and therefore oxidative phosphorylation), Possik discloses the decrease in PGP activity is related to higher glycolytic activity carried through to oxidative phosphorylation. Wilson discusses the relationship between oxidative phosphorylation and oxygen, and explicitly states that the level of lactate production is inversely related to the amount of ATP produced via oxidative phosphorylation and thus O2 produced, and such a relationship can be monitored through observing the activity of the citric acid cycle. Therefore, as Possik discloses the reduction of PGP activity results in the reduction of glycerol levels which meets the goal of Pfizer in addressing the consumption of nutrients for byproduct production that would otherwise decrease target molecule production, and Wilson discloses that the increase in ATP production through phosphorylation is inversely proportional to glycolytic lactate production, wherein such ATP production and O2 -consumption is reported by Possik to result from reduced PGP activity, one of skill in the art would have been motivated to reduce the activity of PGP production in order to reduce byproduct formation of glycerol as taught by Possik, and in doing so would have arrived at the claimed invention to also have reduced the formation of lactate as taught by Wilson.
Regarding the assertion that Possik teaches away from such PGP reductions by stating the G3PP (note: PGP and G3PP are referred to synonymously in the cited art) expression is inversely related to glycolytic flux: Possik does not criticize, discredit, or otherwise discourage carrying out of the reduction of PGP activity as recited in the claims, and therefore does not teach away from the claimed invention (see MPEP 2143.01.I). Applicants citation of Possik that “G3PP expression was inversely related to glycolytic flux, measured as lactate production” on p 21, col 1, para 3 is in reference to experiments carried out on hepatocytes in the Mugabo reference also cited by Applicant. The data shown by Possik, however, is derived from experiments with islet cells, which are a different cell type, and these islet cells responded in a way that one of skill in the art would interpret to correspond to decreased lactate production in view of Wilson. Therefore, Possik does not teach away from the limitations of the claims, but rather reports the findings of the carried out experiments and emphasizes the complications of mammalian metabolism regarding this enzyme and the levels of lactate, indicating variability among cell types such as hepatocytes and islet cells.
Regarding Applicant’s citation of Possik that lactate is produced through the hydrolysis of 2-P-Lactate [see Figure 2 of Possik] that would not happen under normal metabolic conditions: Possik states that the accumulation of 2-P-Lactate through the activity of pyruvate kinase only occurs through the deletion of PGP [p 23, col 1, para 1], which is considered to correspond to the lowering of lactate concentrations as a result of disruption of PGP activity.
Regarding the assertion that Mugabo et al. (PNAS, 2016, 113:E430; cited on the IDS filed 09/27/2022) discloses knockdown of G3PP in hepatocytes enhanced lactate production and release, which is referenced by the citation of Possik by Applicant above and ties to Applicant’s supporting argument that Possik teaches away: the data in Mugabo, particularly in Figures 4D and 4E, indeed indicate that knockdown of G3PP results in increased lactate content and release at higher concentrations of glucose as stated by Applicant. However, the same figures show that in lower amounts of glucose, the measured lactate concentrations are not significantly different when compared to controls both without any knockdown and with knockdown of a negative control target. Therefore, Mugabo shows G3PP knockdown relates to increases in lactate as well as a lack of increase in lactate production both intracellularly [Figure 4D] and extracellularly [Figure 4E], and that lactate production is dependent on other variables beyond the presence or absence of G3PP alone (e.g., glucose content). Therefore, in view of these data and the teachings of the prior art set forth in the rejection above, one of ordinary skill in the art would be motivated to reduce the activity of PGP production in order to reduce byproduct formation of glycerol as taught by Possik, and in doing so would have arrived at the claimed invention to also have reduced the formation of lactate as taught by Wilson, and in considering the teachings of Mugabo would be aware of the role that glucose concentrations would play in the production of lactate.
Double Patenting
The rejection of claims 2, 4-5, 8-9, 12-13 and 26 on the ground of nonstatutory double patenting as being unpatentable over claims 1 and 5 of U.S. Patent No. 10,544,395 (cited on the Form PTO-892 mailed 10/28/2025) in view of Pfizer and Perry is withdrawn in view of the amendment to claim 1 to recite “wherein expression of a phosphoglycolate phosphatase (PGP) gene is decreased in the cells to reduce a level of synthesis glycerol and lactate by the cells as compared to a level of synthesis glycerol and lactate by corresponding cells without decreased expression of PGP”.
Claims 1, 5, 8-12 and 22 are newly rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1 and 5 of U.S. Patent No. 10,544,395 (herein “patent”) in view of Pfizer, Possik and Wilson.
The instant rejection is newly stated and necessitated by claim amendment.
Regarding instant claim 1, claim 1 of the patent recites a method of cell culture comprising maintaining at least one metabolite below a specific concentration in cell medium, and claim 5 of the patent recites the metabolite is formate.
The claims of the patent do not recite wherein expression of a phosphoglycolate phosphatase (PGP) gene is decreased in the cells to reduce a level of synthesis glycerol and lactate by the cells as compared to a level of synthesis glycerol and lactate by corresponding cells without decreased expression of PGP.
Pfizer discusses cells and methods of cell culture [title].
Regarding instant claim 1, Pfizer discloses a method of cell culture where cells are modified to reduce the level of synthesis of growth and/or productivity of inhibitors by the cell [abstract], wherein the expression of one or more genes is modified to reduce the level of synthesis of cell growth and/or productivity inhibitors [claim 2] in CHO cells [claim 63], wherein the modified gene encodes an enzyme that catalyzes the synthesis of formate [claim 4] which inhibits the growth of cells [p 8, ln 17-23].
Pfizer additionally discloses a scenario where proteins are increasingly important diagnostic and therapeutic agents, commonly produced commercially in cell culture wherein the cells have been engineered to produce unusually high levels of a protein of interest, and therefore discloses the importance of optimizing culture conditions to address inefficient mammalian cell metabolism that normally results in the consumption of nutrients for the production of byproducts that accumulate over the course of the culture in order to facilitate the successful production of target proteins [p 1, ln 15-24]. Pfizer further discloses lactate and ammonia to be known inhibitors of cells in cell cultures as two major byproducts of cellular metabolism [p 1, beginning on ln 24]. Therefore Pfizer indicates the importance of reducing byproduct formation of inhibitors such as lactate that can inhibit or contend with target protein production in mammalian cell culture.
Possik relates to glycerol-3-phosphate phosphatase and PGP and their roles in intermediary metabolism [title], and discloses a previously unknown mammalian enzyme phosphoglycolate phosphatase (PGP) can function as a glycerol-3-phosphate (Gro3P) phosphatase (G3PP) to regulate metabolite levels [abstract].
Regarding instant claim 1, Possik discloses that Gro3P can be formed from DHAP and taken away from glycolysis through the “Gro3P shuttle”, where it is further dephosphorylated by G3PP activity to form glycerol [Figure 1]. As Possik discloses that PGP also acts as a G3PP enzyme, PGP is indicated to influence the production of glycerol that is inversely tied to glycolysis, since the originating DHAP molecule is shunted to the glycerol pathway at the expense of its progression through glycolysis. Possik further discloses that removal of G3PP activity in rat cells resulted in increased O2 consumption and ATP production, but the overexpression of proteins with G3PP activity had the reverse effect [p 21, col 1, para 3], indicating that lower G3PP activity reduces glycerol production while increasing activity through the glycolytic pathway based on the pathway displayed in [Figure 1].
Wilson relates to oxygen, pH, and mitochondrial oxidative phosphorylation [title].
Regarding instant claim 1, Wilson discloses that the activity of the citric acid cycle is a measure of transition in ATP synthesis from oxidative phosphorylation to glycolytic lactate production, and of the decrease in oxygen consumption [p 1843, col 1, para 3], therefore defining (a) the production of lactate and (b) the production of ATP via oxidative phosphorylation and consumption of oxygen as metabolic alternatives that can be observed based on the relative activity of the citric acid cycle. In view of the disclosure of Wilson, one of ordinary skill in the art would recognize the removal of G3PP activity attributed to PGP that results in increased O2 consumption and ATP production disclosed by Possik corresponds to an increase in oxidative phosphorylation, and therefore would be expected to result in a decrease in lactate formation as well as the decrease in glycerol formation disclosed by Possik.
In view of Pfizer, Possik and Wilson, one of ordinary skill in the art would have recognized that lowering the expression of PGP would thereby lower the production of glycerol and instead shift metabolism towards O2 consumption and ATP production, which additionally would lower the production of lactate, which is interpreted to correspond to increased growth and activity through the removal of cellular inhibitors. One of ordinary skill in the art would have been motivated to do so as Pfizer discloses the importance of reducing the consumption of nutrients used for production of byproducts in order to facilitate the successful production of target molecules.
In view of Pfizer, Possik and Wilson, it would have been obvious for one of ordinary skill in the art before the effective filing date of the claimed invention to modify the claims of the patent by lowering expression of PGP to reduce the accumulation of glycerol, as disclosed by Possik, and reduce the accumulation of lactate, as disclosed by Wilson to arrive at the claimed invention. One of ordinary skill in the art would have been motivated to modify the method of claims of the patent because Pfizer discloses a method to reduce the production of cell growth inhibitors such as formate and lactate, and to reduce the production of byproducts which consume nutrients required for target molecule production, Possik discloses lowering the expression of PGP lowers the production of the metabolic byproduct glycerol and shifts metabolism towards O2 consumption and ATP production, and Wilson discloses that the production of ATP via oxidative phosphorylation and the production of glycolytic lactate are inversely related. One of ordinary skill in the art would have had a reasonable expectation of success because the patent relates to method of maintaining low levels of metabolites in cell culture such as formate, Pfizer relates to methods of optimizing metabolism in cells through regulating metabolite levels such as lactate and formate, Possik relates to the enzyme PGP and its ability to regulate byproduct metabolite levels, and Wilson discusses the relationship between metabolic pathways that branch from glycolysis and their effects on ATP production.
Regarding instant claim 5, Pfizer discloses maintaining formate at a specific concentration below a reference concentration [claim 13], which is below 2 mM [claim 14], wherein formate is identified as a metabolite that inhibits cell growth which can be limited by maintaining concentration in cell culture medium below levels where they inhibit cell growth [p 8, lns 17-26]. As Pfizer additionally identifies lactate as an inhibitor of cell growth [p 1, beginning on ln 24], one of skill in the art would be motivate to similarly maintain lactate concentrations below a reference concentration as disclosed by Pfizer above.
Regarding instant claim 8, Pfizer discloses measuring the concentration of formate and when that measured concentration is above a predefined value, that concentration is decreased by reducing the amount of precursor provided to the cells [claim 15], wherein the predefined value of formate is disclosed as 2 mM [claim 14]. As Pfizer identifies formate as a metabolite that inhibits cell growth which can be limited by maintaining concentration in cell culture medium below levels where they inhibit cell growth [p 8, lns 17-26], and additionally identifies lactate as an inhibitor of cell growth [p 1, beginning on ln 24], one of skill in the art would be motivate to similarly measure and maintain lactate concentrations as disclosed by Pfizer above.
Regarding instant claim 9, Pfizer discloses measuring the concentration by NMR, HPLC or UPLC [claim 16].
Regarding instant claim 10, Pfizer discloses the use of a pH sensor to monitor pH of the cell culture, and in response to a rise above a predetermined pH value, glucose is fed to the cell culture [claim 27].
Regarding instant claim 11, Pfizer discloses the cell culture is a fed batch culture [claim 28].
Regarding instant claim 12, Pfizer discloses the method comprises a growth phase and a production phase and step (ii) is applied during the growth phase, wherein step (ii) comprises a step of measuring a concentration of formate [claim 29].
Regarding instant claim 22, Pfizer discloses the method wherein cell growth and/or productivity are increased as compared to a control culture, said control culture being identical except comprises unmodified cells [claim 66].
Claims 2, 4, 13 and 26 are newly rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1 and 5 of U.S. Patent No. 10,544,395 in view of Pfizer, Possik and Wilson as applied to claims 1, 5, 8-12 and 22 above, and further in view of Perry.
The instant rejection is newly stated and necessitated by claim amendment.
The claims of the patent and relevant disclosures of Pfizer, Possik and Wilson as applied to claims 1, 5, 8-12 and 22 are discussed above. The claims of the patent do not recite expression of serine hydroxymethyltransferase (SHMT2) is decreased in the cells to reduce a level of synthesis of formate as compared to a level of synthesis of formate by corresponding cells without decreased expression of SHMT2.
Perry discusses the effect of vitamin B6 availability on serine hydroxymethyltransferase (SHMT) in MCF-7 cells [title], and discloses that formate is a major source of one-carbon units for cytoplasmic one-carbon metabolism that is generated in the mitochondria from serine [p 21, col 2, para 1].
Regarding instant claim 2, Perry discloses the modification of MCF-7 cells to generate PLP-deficient cells to reduce the expression and activity of mitochondrial serine hydroxymethyltransferase [p 24, col 1, para 3; p 22, col 1, final paragraph; Figure 2b and Figure 2b caption], and that mitochondrial serine hydroxymethyltransferase is responsible for the conversion of serine to formate [Figure 1 and Figure 1 caption]. As the instant specification refers to SHMT2 as mitochondrial serine hydroxymethyltransferase [p 2], the SHMT of Perry is considered to correspond to the SHMT2 recited in the claims. As such, one of skill in the art would have reasoned that the modification of a cell to reduce the expression of SHMT would result in reduced production of formate as well.
In view of Perry, it would have been obvious for one of ordinary skill in the art before the effective filing date of the claimed invention to modify the combined method of the claims of the patent, Pfizer, Possik and Wilson by reducing the expression of SHMT, as disclosed by Perry, to arrive at the claimed invention. One of ordinary skill in the art would have been motivated to modify the combined method of the claims of the patent, Pfizer, Possik and Wilson because Pfizer discloses a method of reducing the levels of growth and/or productivity inhibitors by a cell that includes modifying the expression of genes that produce the growth inhibitor formate, and Perry indicates that reduction of SHMT expression will reduce the production of formate from serine. One of ordinary skill in the art would have had a reasonable expectation of success because the patent, Pfizer and Perry discuss methods that regulate the levels of the metabolite formate.
Regarding instant claim 4, Possik discloses the reduction of PGP activity via RNAi knockdown [p 21, col 1, para 3], which is not a method of modifying the PGP gene as RNAi knockdown affects translation of mRNA. Perry discloses the modification of MCF-7 cells to generate PLP-deficient cells to reduce the expression and activity of serine hydroxymethyltransferase [p 24, col 1, para 3, p 22, col 1, final paragraph, and Figure 2b], which is not a method of modifying the SHMT2 gene. Pfizer however discloses the modification of genes to decrease expression to reduce the level of synthesis of growth and/or productivity inhibitors by the cell [claim 58] for use in a method to reduce the level of inhibitory metabolites that include formate and lactate [abstract; p 8, ln 17-23; p 1, beginning on ln 24]. In view of Pfizer, Possik and Perry, one of ordinary skill in the art would be motivated to modify the genes encoding PGP and SHMT to reduce the production of lactate and formate, respectively, as Possik and Perry disclose methods for reducing the activity of polypeptides encoded by the PGP and SHMT genes, and Pfizer discloses a method of modifying genes to reduce the activity of the encoded polypeptides.
Regarding instant claim 13, Pfizer discloses modifying the expression of one or more genes comprises gene deletion, disruption, substitution, point mutation, multiple point mutation, insertion mutation or frameshift mutation of the gene, or introduction of one or more nucleic acids comprising the one or more genes into the cell, optionally as an expressible construct or expressible vector construct [claim 40].
Regarding instant claim 26, Pfizer discloses the method wherein cell growth and/or productivity are increased as compared to a control culture, said control culture being identical except comprises unmodified cells [claim 66].
Response to remarks: beginning on page 10 of applicants response to double patenting rejections; Applicant in summary contends the amendments to the claims renders the double patenting rejections no longer applicable.
Applicant’s response is considered and found not convincing. For the reasons stated above, claims 1-2, 4-5, 8-13, 22 and 26 are newly rejected in view of the amendments to the claims.
Conclusion
Status of the Application:
Claims 1-2, 4-5, 8-19, 22, and 26 are pending.
Claims 14-19 are withdrawn.
Claims 1-2, 4-5, 8-13, 22 and 26 are rejected.
No claim is in condition for allowance.
Applicant's amendment necessitated the new ground(s) of rejection presented in this Office action. Accordingly, THIS ACTION IS MADE FINAL. See MPEP § 706.07(a). Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action. In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any nonprovisional extension fee (37 CFR 1.17(a)) pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action. In no event, however, will the statutory period for reply expire later than SIX MONTHS from the mailing date of this final action.
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/JOSEPH R SPANGLER/
Examiner
Art Unit 1656
/David Steadman/Primary Examiner, Art Unit 1656
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