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 CON of 15/538,479 (06/21/2017 ABN)
15/538,479 is a 371 of PCT/EP2015/080858 (12/21/2015)
and claims foreign priority to EP 14199729.6 (12/22/2014).
New Claim Rejections - 35 USC § 103
Claims 11-30 are rejected under 35 U.S.C. 103 as being unpatentable over Sarkar et al. (PNAS, September 30, 2008, 105 (39), p. 14808-14813) and Dodevski et al. (J. Mol. Biol. (2011) 408, 599–615) in view of Junge et al. (Cell. Mol. Life Sci. 65 (2008), p. 1729 – 1755), OMalley et al. (Protein Science 2009, vol. 18: p. 2356-2370) and Thompson (US5879891).
Regarding claim 11, Dodevski and Sarkar teach:
A method for increasing surface expression of functional G protein- coupled receptors (GPCRs) in yeast cells, wherein said functional GPCRs are capable of ligand binding (Dodevski Title: “Evolution of Three Human GPCRs for Higher Expression and Stability”; p. 600: “By applying this evolutionary selection method to the model GPCR … we had identified mutations that strongly improve functional surface expression”; p. 610: “we have developed a directed evolution method that allows the rapid identification of mutations in a receptor sequence that lead to higher functional expression”, “Yet other techniques aim at identifying amino acids that
are critical for receptor function and signaling by employing screening assays in yeast.”) (Sarkar, Title: “Directed evolution of a G protein-coupled receptor for expression”; Abstract: “For a mammalian G protein-coupled receptor, we arrived at a sequence with an order-of-magnitude increase in functional expression that still retains the biochemical properties of wild type.”; Fig. 1), and the method comprises:
a) providing a plurality of yeast cells, wherein each yeast cell comprises a nucleic acid sequence member of a randomized mutant library, the nucleic acid sequence member being expressed as a GPCR in a plasma membrane of the yeast cell; (Dodevski p. 613: “To create genetic diversity, we amplified the DNA encoding the GPCR (excluding the fusion adducts) by error-prone PCR using the GeneMorph II Random Mutagenesis Kit (Stratagene).”) (Sarkar p. 14808: “The general approach is given in Fig. 1. The expression vector containing the GPCR library of interest (e.g., from an error-prone PCR of the receptor gene) with two constant fusion partners (N-terminal maltose binding protein and C-terminal thioredoxin) is used to express the corresponding proteins in functional form in the inner membrane of E. coli DH5a (see Methods).”),
b) permeabilizing the cell wall of said plurality of yeast cells by a non-enzymatic chemical treatment; (Dodevski p. 601: “binding buffer must therefore be formulated such that the outer membrane becomes permeable”, p. 613) (Sarkar p. 14808: “After expression, cells are incubated at 4°C in an optimized buffer that renders the outer membrane permeable to small molecules to allow binding of fluorescent ligand to the receptors, and at the same time maximizes cell viability (see Methods).”),
c) labeling the plurality of permeabilized yeast cells with a fluorescent dye capable of passing through the permeabilized cell wall and binding to functional GPCRs in the plasma membranes of the yeast cells; (Dodevski p. 613: “libraries were labeled with 200 nM prazosin-BODIPY-FL”, “labeled with 300 nM Substance P Oregon Green”) (Sarkar p. 14808: “the buffer was specifically optimized to allow for saturable, specific binding of fluorescently labeled agonist [BODIPY-NT(8–13)] to NTR1, while maximizing cell viability after fluorescence-activated cell sorting (FACS)”),
d) washing the plurality of labeled yeast cells; (Dodevski p. 613: “cells were washed by centrifugation”),
e) measuring functional GPCR expression levels of the plurality of labeled yeast cells with a binding assay and selecting a subset of the plurality of labeled yeast cells that exhibits the greatest fluorescence; (Dodevski p. 613: “Sorting was done on a FACS Aria II (BD Biosciences) at 20,000–30,000 events/s in yield mode (for sorting naïve libraries) or purity mode (for subsequent sorting rounds). The most fluorescent 0.5–1.5% of the cells in the population (107 to 108 cells) were sorted directly into 2xYT (1% glucose and 100 μg/ml Carb) for regrowth and further selection. The most fluorescent cells were enriched by sorting the libraries for three to six rounds by FACS.”)(Sarkar p. 14809: “After incubation with saturating concentrations of BODIPYNT(8–13), bacteria expressing the largest number of functional receptors correspondingly exhibit the greatest fluorescence, and these cells were collected directly in growth medium and then expanded for a subsequent round. A single selection round, which consisted of library expansion, induced receptor expression, incubation with fluorescent ligand, and FACS to recover the most fluorescent bacterial cells”), and
f) expanding said subset in an expansion step, to yield a plurality of expanded yeast cells (Dodevski p. 613: “The most fluorescent 0.5–1.5% of the cells in the population (107 to 108 cells) were sorted directly into 2xYT (1% glucose and 100 μg/ml Carb) for regrowth and further selection. The most fluorescent cells were enriched
by sorting the libraries for three to six rounds by FACS.”); and
g) repeating steps b) to f) one or more times with the expanded yeast cells yielded by the preceding step f) to yield a subset of viable, labeled yeast cells; (Dodevski p. 601: “During the process of receptor evolution, the functional expression level of the evolving receptor pools increased for each of the receptors with each round of mutagenesis and selection, as evidenced by an increase in the specific mean fluorescence intensity (MFI). Figure 1 shows the increase in MFI after two rounds of evolution (ep2 pool) and four rounds of evolution (ep4 pool) for the three receptor targets.”; p. 608-609: “We sequenced single clones after two and four rounds of evolution (ep2 pool and ep4 pool, respectively).”, “To analyze which mutations led to an increase in expression and stability in the evolution of α1aAR, we sequenced single clones of the ep2 pool”)(Sarkar p. 14809: “From the enriched pool, 96 single clones were sequenced and analyzed for receptor expression level (see Methods and Figs. S6 and S7).”)
wherein the subset of viable, labeled yeast cells of step g) have an ability for expression of at least 40,000 total functional GPCRs per cell (Dodevski p. 607-608: “Based on these measurements, clone NK1-E11 is expressed at 3000 receptors/cell (10-fold better than wt NK1), clone A1a-05 is expressed at 1900 receptors/cell (5-fold better than wt α1aAR), and clone A1b-C10 is expressed at 3400 receptors/cell (1.3 times better than wt α1bAR). These numbers correspond to about 1 mg of functional and thermostable GPCR”) (Sarkar p. 14809: “optimization of the binding buffer and FACS gating conditions resulted in a specific signal in the gating window that was ~900-fold above background”; Table 1:
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Sarkar p. 14809: “From the enriched pool, 96 single clones were sequenced and analyzed for receptor expression level (see Methods and Figs. S6 and S7).”; Sarkar p. 14810: “The functional expression level for D03, as assayed by specific radioligand binding to membrane preparations, was ~ 12-fold higher than that for WT NTR1 (Table 1)”); and
wherein the subset of viable, labeled yeast cells of step g) have increased surface expression of functional GPCRs as compared to the plurality of yeast cells of step a) (Dodevski p. 601: “During the process of receptor evolution, the functional expression level of the evolving receptor pools increased for each of the receptors with each round of mutagenesis and selection, as evidenced by an increase in the specific mean fluorescence intensity (MFI). Figure 1 shows the increase in MFI after two rounds of evolution (ep2 pool) and four rounds of evolution (ep4 pool) for the three receptor targets.”.
wherein said detectable label is a fluorescent dye and said selection step is accomplished by fluorescent cell sorting (Dodevski p. 613: “Sorting was done on a FACS Aria II (BD Biosciences) … The most fluorescent cells were enriched by sorting the libraries for three to six rounds by FACS.”; p. 613: “libraries were labeled with 200 nM prazosin-BODIPY-FL”, “labeled with 300 nM Substance P Oregon Green”) (Sarkar p. 14809: “After incubation with saturating concentrations of BODIPYNT(8–13)”, “A single selection round, which consisted of library expansion, induced receptor expression, incubation with fluorescent ligand, and FACS”).
Although Sarkar does teach successful GPCR expression of the screened sequence in yeast (Pichia pastoris), Dodevski and Sarkar teach the GPCR expression/selection or directed evolution in E. Coli, but the process in a yeast cells as in claim 1. However, Junge (Title: “Large-scale production of functional membrane proteins”) which reviews and compares the membrane protein (MP) expression systems including E. Coli and Yeast, teaches “Yeasts are a preferred host for the production of GPCRs” (p. 1743) and “A systematic optimization screen was used to increase the production levels of 20 selected GPCRs in protease deficient P. pastoris mutants” (p. 1743) which one of ordinary skill in the art would consider in improving GPCR expression and reasonably consider using a yeast host cell in place of Dodevski’s and Sarkar’s E. Coli and arrive at the claimed invention.
Regarding the language of “have an ability for expression of at least 40,000 total functional GPCRs per cell” Dodevski teaches after applying the technique for two rounds provided significant increases in expression levels in E. Coli (p. 602: “After two additional rounds of evolution, expression increased further, and the best clones expressed up to 6500 receptors/cell, which is about 18-fold better than wt”) and noted that E. coli might be limited (Dodevski p. 610: “a similar final level is reached for all three receptors, as well as for the evolved NTR1 (∼6000 receptors/cell), independent of the starting number.”, with discussion of a possible “intrinsic limit in the E. coli biosynthesis of such proteins”) which Sarkar demonstrated was feasible to implement in yeast (Sarkar p. 14810: “we also expressed WT NTR1, D03, and D03-L167R in the methylotrophic yeast P. pastoris and compared functional and total expression yields. The functional expression level for D03, as assayed by specific radioligand binding to membrane preparations, was ~12-fold higher than that for WT NTR1 (Table 1)”), including Sarkar’s teaching of 58,800 per cell in Table 1 (D03) which one of ordinary skill in the art would have considered in view of Jung which teaches optimization of parameters for GPCR production in yeast (Jung p. 1743: successful optimization to more that 20 pmol/mg, “In a comprehensive expression study of 25 human ABC transporters in P. pastoris, amounts between 3 and 5 mg/100 g of cells have been produced.”). The combined teaching of the art would have suggested that continuing with rounds of evolution expression would have increased further, particularly when considering the success of OMalley (OMalley p. 2358, i.e. Table 1: yeast expression of GPCRs at levels of > 100,000 to 465,000 molecules per cell).
Jung points out the benefits of utilizing yeast, including post translational modifications (PTM) (Jung Table 1, p. 1742-43: “yeasts have become the second most popular MP expression system after E. coli, with more cost effective than expression in mammalian or insect cell lines and often higher yields.”, “Some eukaryotic-specific PTMs such as proteolytic processing and lipidation can be performed in yeasts”, “Since beginning of the 1990 s, the methylotrophic yeast P. pastoris has become one of the standard tools in molecular biology for the generation of recombinant proteins (Fig. 1).”) which would be expected to motivate one of ordinary skill in the art to utilize yeast to improve quality and functionality of the membrane protein (“MP”) (Jung p. 1740: “Expression in E. coli is frequently the first and only choice for prokaryotic MPs, while eukaryotic MPs are often attempted to be produced in prokaryotic as well as in eukaryotic backgrounds. Reasons to switch to the latter more expensive and time-consuming systems are almost exclusively the intention to improve functionality or the overall quality of the MP samples.”). Thus, one of ordinary skill in the art would have considered yeast in an effort to improve function and quality and arrive at the claimed invention.
Jung teaches that yeast have been used to produce a number of functional GPCRs, including optimization of the expression of the functional form on p. 1743:
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Thus, one of ordinary skill in the art would have reasonably considered yeast for optimizing functional production yields as was taught by Jung and arrive at the claimed invention. Jung clearly teaches optimization of production of functional GPCRs on p. 1743:
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where [31] cited Andre et al. (Protein Sci. 2006 May; 15(5): 1115–1126) and is an optimization for functional GPCRs as assessed by specific binding constants. Thus, the cited art teaches optimization of expression to produce functional GPCRs in yeast and one of ordinary skill in the art would have had an expectation that combining optimization using the techniques of Dodevski and Sarkar (Dodevski p. 610: “we have developed a directed evolution method that allows the rapid identification of mutations in a receptor sequence that lead to higher functional expression”; Sarkar Abstract: “For a mammalian G protein-coupled receptor, we arrived at a sequence with an order-of-magnitude increase in functional expression that still retains the biochemical properties of wild type.”) would be successful.
Dodevski teaches after applying the technique for two rounds provided significant increases in expression levels in E. Coli (p. 602: “After two additional rounds of evolution, expression increased further, and the best clones expressed up to 6500 receptors/cell, which is about 18-fold better than wt”) and noted that E. coli might be limited (Dodevski p. 610: “a similar final level is reached for all three receptors, as well as for the evolved NTR1 (∼6000 receptors/cell), independent of the starting number.”, with discussion of a possible “intrinsic limit in the E. coli biosynthesis of such proteins”) which Sarkar demonstrated was feasible to implement in yeast (Sarkar p. 14810: “we also expressed WT NTR1, D03, and D03-L167R in the methylotrophic yeast P. pastoris and compared functional and total expression yields. The functional expression level for D03, as assayed by specific radioligand binding to membrane preparations, was ~12-fold higher than that for WT NTR1 (Table 1)”) which one of ordinary skill in the art would have considered in view of Jung which teaches optimization of parameters for GPCR production in yeast (Jung p. 1743: successful optimization to more that 20 pmol/mg, “In a comprehensive expression study of 25 human ABC transporters in P. pastoris, amounts between 3 and 5 mg/100 g of cells have been produced.”, “Yeasts are a preferred host for the production of GPCRs [183]. In a concerted approach to express 100 different GPCRs, more than 90% of the targets were successfully produced in P. pastoris” “Oxygen supply and defined pH values were further found to be valuable for the expression of functional GPCRs”). The combined teaching of the art would have suggested that continuing with rounds of evolution expression would have increased further, particularly when considering the success of OMalley (OMalley p. 2358, i.e. Table 1: yeast expression of GPCRs at levels of > 100,000 to 465,000 molecules per cell, p. 2264: “the goal of achieving high functional yields for these proteins”).
Regarding the new claim language in step b’s permeabilizing step of wherein the non-enzymatic chemical treatment comprises exposing said plurality of yeast cells to a buffer of alkaline pH comprising lithium ions, a reducing agent and/or a chelating agent, the non-enzymatic lithium chemical treatment method for yeast permeabilization was well-known in the art as taught by Thompson, including the use of using a buffer comprising alkaline pH, lithium ions (LiAc), reducing agent (DTT), and a chelating agent (EDTA) (Thompson Example 1, cols 3-4). One of ordinary skill in the art would consider routine the use of well-known permeabilization technique as taught by Thompson and readily apply it to studies on yeast cells.
Therefore, one of ordinary skill in the art following the teaching in the art would have had a reasonable expectation of continuing rounds of evolution would have resulted in GPCR expression level as in the instant claims, thereby rendering the claim obvious.
Regarding claim 12 Sarkar and Dodevski teach isolating, amplifying, transferring, and repeating as claimed (Sarkar p. 14809: “Whenever additional diversity was desired after any FACS round, the sorted pool of cells was grown and harvested, the enriched plasmid collection was purified, the GPCR sequences (excluding the fusion partners) were further randomized, and fresh bacteria were transformed for the next selection.”)(Dodevski p. 613: “To create genetic diversity, we amplified the DNA encoding the GPCR (excluding the fusion adducts) by error-prone PCR using the GeneMorph II Random Mutagenesis Kit”).
Regarding claim 13, Dodevski and Sarkar teaches increasing the functional expression which corresponds to a decreased subpopulation of non-expressing GPCR cells compared to the starting population (Dodevski Fig. 1; Sarkar Table 1).
Regarding claim 14, Sarkar teaches intracellular Ca2+ pools via phospholipase decreased relative to WT or the starting population (p. 14809-10).
Regarding claim 15, Dodevski teaches isolating an expressed nucleic acid sequence of the cells (p. 609: “we sequenced single clones of the ep2 pool”).
Regarding claim 16, Sarkar teaches high levels of expression per cell as in the claim (Sarkar p. 14809: “optimization of the binding buffer and FACS gating conditions resulted in a specific signal in the gating window that was ~900-fold above background”; Table 1).
Regarding claim 17-18, Dodevski teaches the most florescent are within the claim range (p. 613: “The most fluorescent 0.5–1.5% of the cells in the population (107 to 108 cells) were sorted”).
Regarding claim 19 wherein the plurality of yeast cells comprises Saccharomyces cerevisiae, Jung teaches that Saccharomyces cerevisiae is widely used producing functional MPs with successful expression of a GPCR including optimization of expression (p. 1743: “Widely used yeasts for the production of functional MPs are P. pastoris, S. cerevisiae and S. pombe”, “Activity of the human b2-adrenergic receptor could be observed after co-expression with a mammalian G-protein subunit in S. cerevisiae by partial activation of the yeast pheromone-response pathway [182]. Expression could be improved by (I) replacing the 5’ untranslated region, (II) use of the strong GAL1 promotor and co-expression of its transcriptional activator and (III) induction in the presence of its ligand up to final amounts of 115 pmol of b2-adrenergic receptor per milligram of total MP[182].”). Thus, one of ordinary skill in the art would have had a reasonable expectation of success in the selection of Saccharomyces cerevisiae as the yeast and arrive at the claimed invention.
Regarding claim 20, Dodevski teaches radioligand binding assays (p. 613) and Sarkar teaches flow cytometry (p. S1).
Regarding claim 21, the selection process for increased functional GPCRs in the prior art is the same (Sarkar Fig. 1; Dodevski Fig. 1).
Regarding claim 22, Dodevski teaches four rounds (Dodevski p. 601: “During the process of receptor evolution, the functional expression level of the evolving receptor pools increased for each of the receptors with each round of mutagenesis and selection, as evidenced by an increase in the specific mean fluorescence intensity (MFI). Figure 1 shows the increase in MFI after two rounds of evolution (ep2 pool) and four rounds of evolution (ep4 pool) for the three receptor targets.”; p. 608-609: “We sequenced single clones after two and four rounds of evolution (ep2 pool and ep4 pool, respectively). Regarding claim 23 specifying five rounds, in view of the teaching of the prior art one of ordinary skill in the art would have had an expectation that repeating the process further would lead to improved selection with a reasonable expectation of success and perform five rounds and arrive at the claimed invention.
Regarding claims 24-25 wherein the cells are adapted yeast cells, the prior art, Dodevski and Sarkar, tech cell selection for adaption which are variants (Dodevski p. 608-609: “We sequenced single clones after two and four rounds of evolution (ep2 pool and ep4 pool, respectively).”, “To analyze which mutations led to an increase in expression and stability in the evolution of α1aAR, we sequenced single clones of the ep2 pool”)(Sarkar p. 14809: “From the enriched pool, 96 single clones were sequenced and analyzed for receptor expression level (see Methods and Figs. S6 and S7).”) (Junge p 1743: “A systematic optimization screen was used to increase the production levels of 20 selected GPCRs in protease deficient P. pastoris mutants”).
Regarding claim 26 specifying non-enzymatic chemical treatment, Sarkar and Omalley teach DTT treatment of yeast (Omalley p. 2360; Sarkar p. 14813).
Regarding claim 27 reading on a process parallel to claim 11 for expressing the same receptor after applying another round of evolution, as with claim 11 one of ordinary skill in the art would have reasonably considered repeating the technique that created the initial plurality of yeast cells in the course of evolving desirable properties as taught by Dodevski and Sarkar (Dodevski p. 608-609: “We sequenced single clones after two and four rounds of evolution (ep2 pool and ep4 pool, respectively).”, “To analyze which mutations led to an increase in expression and stability in the evolution of α1aAR, we sequenced single clones of the ep2 pool”) (Sarkar p. 14809: “From the enriched pool, 96 single clones were sequenced and analyzed for receptor expression level (see Methods and Figs. S6 and S7).”).
Regarding claim 28, as with claim 13, Dodevski and Sarkar teaches increasing the functional expression which corresponds to a decreased subpopulation of non-expressing GPCR cells compared to the starting population (Dodevski Fig. 1; Sarkar Table 1).
Regarding claim 29, as with claim 14, Sarkar teaches intracellular Ca2+ pools via phospholipase decreased relative to WT or the starting population (p. 14809-10).
Regarding claim 30, the selection process for increased functional GPCRs in the prior art is the same (Sarkar Fig. 1; Dodevski Fig. 1).
The Supreme Court stated in KSR "if a technique has been used to improve one device, and a person of ordinary skill in the art would recognize that it would improve similar devices in the same way, using the technique is obvious unless its actual application is beyond that person's skill." KSR Intern. Co. v. Teleflex Inc., 127 S.Ct. 1727, 1731 (2007).
In the instant case, utilizing yeast in place of E Coli as suggested by Junge to optimize expression of GPCR using Dodevski and Sarkar’s technique improves similar processes in a similar way and the application of the technique is within the skill of one of ordinary skill in the art. Furthermore, one of ordinary skill in the art would consider using yeast as suggested by Junge in Sarkar and Dodevski’s GPCR screening library technique because they both relate to screening membrane protein for improved expression. In addition, one of ordinary skill in the art routinely adapts or combines techniques to make improvements through substitutions of one known element for another which Junge describes as MP expression systems including E. Coli and yeast. One of ordinary skill in the art could have considered a yeast library screening technique in a directed evolution technique similar to the success described by Sarkar and Dodevski with GPCRs, and the results of the substitution would have been predictable because both Sarkar (p. 14810: confirmed expression in yeast P. pastoris) and Dodevski (p. 612: “screening assays in yeast”) discuss expression of their GPCRs in yeast. Thus, one of ordinary skill in the art had a reasonable expectation of success.
With each of the claims, the level of skill in the art is very high such that one of ordinary skill in the art would consider routine the combination of elements from the teaching of the art. One of ordinary skill in the art would have recognized that the results of the combination would be predictable due to the well-known nature and optimizations routinely performed in the art. Thus, one of ordinary skill in the art would have arrived at the invention as claimed before the effective filing date with a reasonable expectation of success.
Response to Remarks - 35 USC § 103
Applicant argues that none of the cited art teaches the amended steps. This is not persuasive because the amended rejection above including Thompson does teach the claim as amended.
Applicant argues that the claimed invention is to the surprising discovery that the permeabilization step is key to enable directed evolution. This argument is not persuasive because one of ordinary skill in the art would have known about and utilized the lithium permeabilization step in yeast as was well-known and routine in the art.
Conclusion
No claims allowed.
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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/ROBERT H HAVLIN/Primary Patent Examiner, Art Unit 1626