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 .
The Response of 7 July 2026 has been entered.
Claims 1-3 and 11-27 are currently pending.
Election/Restrictions
Applicant's election with traverse of the invention of Group I, claims 1-3, 11-15 and 19-26, in the reply filed on 7 July 2026 is acknowledged. The traversal is on the ground(s) that there would be no undue burden to examiner the restricted groups together. This is not found persuasive because the restriction was made on the basis of unity of invention, and thus search burden is not at issue.
The requirement is still deemed proper and is therefore made FINAL.
Claims 16-18 and 27 are withdrawn from further consideration pursuant to 37 CFR 1.142(b), as being drawn to a nonelected invention, there being no allowable generic or linking claim. Applicant timely traversed the restriction (election) requirement in the reply filed on 7 July 2026.
Claims 1-3, 11-15 and 19-26 are considered here.
Claim Rejections - 35 USC § 112(b) (indefiniteness)
The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
Claim 11 is rejected 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 (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
Claim 11 recites "a medium suitable for the normal growth of the reporter cell". The term "normal" is a relative and/or subjective term that renders the claim indefinite (see MPEP 2173.05(b)). The specification does not provide a basis for determining what type of growth would be considered "normal", and thus one of ordinary skill in the art would not be able to reasonably ascertain what media would fall within the scope of the claim. The rejection can be overcome by amending to delete the term "normal".
Claim Rejections - 35 USC § 103
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
Claims 1-3, 11-13, 15 and 19-26 are rejected under 35 U.S.C. 103 as being unpatentable over the combination of US20090075314 to Barak et al. (previously cited in RR of 7 May 2026) in view of US20140199704 to Horton (cited in IDS of 22 Oct. 2024) and Sumit et al., Integrative Biology 7.11 (2015): 1378-1386 (previously cited in RR of 7 May 2026), as evidenced by Xu et al., Inflammation Research 63.6 (2014): 463-473.
Regarding claim 1, Barak teaches a method of screening for an acetylcholinesterase inhibitor (e.g., an organophosphate pesticide), comprising 1) mixing a sample to be screened with an acetylcholinesterase and an acetylcholine; 2) contacting a reporter cell expressing a muscarinic acetylcholine receptor and a GFP-labeled reporter protein (arrestin) with the mixture; and 3) detecting the degree of activation of the reporter cell ([0009]-[0013]; [0015]; [0021]; [0027]-[0028]; [0050]; [0140]; [0159]-[0162]; Example 9). Upon activation of the muscarinic receptor, the arrestin-GFP translocates from the cytosol to the plasma membrane where it binds to the activated muscarinic receptor, and the degree of receptor activation can thus be detected by measuring the distribution of arrestin-GFP within the cell (e.g., at the plasma membrane vs. the cytosol) ([0016]; [0150]; Examples 1, 4, 7-9; claims 8 and 14-16). If the sample to be screened contains an acetylcholinesterase inhibitor, the acetylcholinesterase will not break down the acetylcholine and the presence of the inhibitor will be detected as receptor activation (Example 9). Barak teaches that the muscarinic receptor can be a type 3 (i.e. M3) receptor ([0052]).
Regarding the recitation in claims 1 and 11 of mixing the sample with acetylcholinesterase, and then mixing the sample/acetylcholinesterase with acetylcholine, Barak teaches mixing the sample "with a buffer containing acetylcholinesterase protein and an agonist to the muscarinic receptor such as acetylcholine chloride" (Example 9), and it would have been prima facie obvious to mix the ingredients in any order (e.g., either mixing the sample with acetylcholinesterase and acetylcholine as in Barak, or mixing the sample first with acetylcholinesterase and then with acetylcholine) (see MPEP 2144.04, IV., C. - changes in the sequence of adding ingredients are prima facie obvious in the absence of new or unexpected results).
Regarding claim 3, Barak teaches the use of acetylcholine chloride as the acetylcholine, and it would have been obvious to use any similar acetylcholine salt including acetylcholine iodide. Barak further teaches the DNA and amino acid sequences of human acetylcholinesterase and that the expression of such sequences is well known in the art (Fig. 3E; [0162]-[0172]). It would have thus been obvious to use a recombinant version of acetylcholinesterase in the method of Barak.
Regarding claim 12, Barak teaches incubating the mixture for "a period of time between zero and a few hours, between 5 and 60 minutes is most practical" ([0221]). The time period of Barak overlaps with and renders obvious the total incubation times recited in claim 12 (see claims 1 and 11 above re the order of steps; it would have been obvious to add and incubate the ingredients stepwise or together) (see MPEP 2144.05). Barak further teaches carrying out the cell culture at 37 °C ([0030]), and it would have been obvious to carry out the method as a whole at said temperature (i.e. at the native temperature of the cells and enzymes). Moreover, it would have been obvious to discover the optimal and/or workable ranges for the incubations times and temperatures in the method (see MPEP 2144.05).
Regarding claim 13, Barak teaches that the reporter cell can be contacted with the test substance for 15-30 minutes or 1 hour before measuring the distribution of the GFP-reporter protein ([0030]).
Claims 1-3, 11-13, 15 and 19-26 differ from Barak in that: the reporter cell is M3:NFATc1-EGFP U2OS (which differs from Barak in that the labeled reporter protein is NFATc1, and the host cell is a U2OS cell) (claim 1); the degree of activation of the reporter cell is expressed as the degree of translocation of NFATc1-EGFP from cytoplasm to nucleus, quantified according to the number of intracellular NFATc1-EGFP translocated from cytoplasm to nucleus (claim 2); a high-content cell imaging and analysis system is used to detect the degree of activation of the reporter cell (claim 15); the medium is a DMEM high-glucose medium (claim 19); and the concentrations of acetylcholine and acetylcholinesterase are as recited in claims 20-26.
Horton teaches a high-throughput method of screening for test agents that activate a receptor of interest by contacting the test agents with a NFATc1-EGFP U2OS reporter cell, comprising a U2OS host cell expressing an EGFP-labeled NFATc1 transcription factor ([0021]; [0027]-[0028]; [0043]; [0099]-[0170]). NFATc1 resides in the cytosol in its inactive form and is activated by G-protein coupled receptors (GPCRs) that activate PLC and induce IP3-mediated transient release of calcium, resulting in the translocation of NFATc1 from the cytosol to the nucleus ([0099]-[0104]). The degree of translocation is measured via cell imaging ([0026]; [0105]-[0106]).
Sumit teaches a similar NFATc1-GFP reporter cell as in Horton for measuring activation of a recombinant human muscarinic M3 acetylcholine receptor (under RESULTS). Sumit further teaches that the M3 receptor is a GPCR that is activated via the common G-protein-PLC-IP3 pathway that triggers calcium release and induces NFAT nuclear translocation (under INTRODUCTION).
It would have been obvious to one of ordinary skill in the art at the time the invention was made to use the method of Barak to screen for acetylcholinesterase inhibitors by contacting a sample with a reporter cell that measures acetylcholine receptor activation by detecting changes in the cellular distribution of a GFP-labeled protein that is translocated upon receptor activation wherein the reporter cell is an NFATc1-EGFP U2OS reporter cell as taught by Horton (which measures GPCR activation via detection of translocation of NFATc1 from cytosol to nucleus) expressing an M3 acetylcholine receptor as taught by Sumit because it would have been obvious to combine prior art elements according to known methods to yield predictable results. One of ordinary skill would have been motivated to use M3-expressing NFATc1-EGFP U2OS reporter cell in the method of Barak because Horton and Sumit teach that the NFATc1-GFP reporter system provides an established high-throughput method for measuring activation of GPCRs such as the M3 receptor. Using an M3-expressing NFATc1-EGFP U2OS reporter cell as taught by Horton and Sumit in the method of Barak would have led to predictable results with a reasonable expectation of success because Horton teaches that NFATc1-EGFP can be used to measure activation of GPCRs such as M3 receptors that act via the IP3/PLC pathway, and Sumit teaches use of NFATc1-GFP to measure human M3 receptor activation. Moreover, Xu evidences that U2OS cells can stably express a recombinant human M3 receptor (Xu, Abstract). Furthermore, one of ordinary skill in the art would have recognized that the screening methodology taught by Barak (wherein acetylcholinesterase inhibitors are detected via repression of acetylcholinesterase inhibition of receptor activation) is distinct from the reporter cell mechanism by which receptor activation is detected, and thus one could substitute another known reporter cell capable of measuring acetylcholine receptor activation for the arrestin-based reporter taught by Barak with a reasonable expectation of success.
Regarding the recitation in claim 2 that receptor activation is quantified according to the number of intracellular NFATc1-EGFP translocated from cytoplasm to nucleus, Horton teaches measuring the degree of translocation from cytosol to nucleus by measuring an EGFP signal in the cell ([0105]-[0106]). The EGFP signal would correlate with the number of NFATc1-EGFP translocated, and thus it would be obvious to quantify the activity of the acetylcholinesterase inhibitor "according to the number of intracellular NFATc1-EGFP translocated from cytoplasm to nucleus" as in claim 2.
Regarding the recitation in claim 11 that the solvent is a medium suitable for growth of the reporter cell, both Barak and Horton teach mixing test substances with cell culture media (MEM, DMEM) before contacting them with the reporter cell (Barak, [0192]; Horton, [0166]). Moreover, since the assay of the cited combination requires measuring intracellular processes of a live reporter cell, it would have been obvious to mix the sample, acetylcholinesterase and acetylcholine in a medium suitable for growth of the reporter cell.
Regarding claim 15, Horton teaches use of "high-content" imaging and analysis ([0001]; [0020]-[0021]; [0026]; [0106]).
Regarding claim 19, Horton teaches use of DMEM with Glutamax-1 media (i.e. high glucose DMEM) ([0120]).
Regarding claims 20-26, the cited combination teaches using the same reporter cell for the same screening purpose as in the claimed method and it would have thus been obvious to discover the optimal and/or workable ranges for the concentrations of ingredients, including acetylcholine and acetylcholinesterase (see MPEP 2144.05).
Claim 14 is rejected under 35 U.S.C. 103 as being unpatentable over the combination of Barak in view of Horton and Sumit as evidenced by Xu, as applied to claims 1-3, 11-13, 15 and 19-26, further in view of Shen et al., Histochemistry and cell biology 134.4 (2010): 387-402.
The teachings of Barak in view of Horton and Sumit are set forth above. Regarding claim 14, Horton teaches a specific imaging system for analyzing NFATc1-EGFP translocation but also teaches that "the assay can be imaged on other systems" as well ([0106]).
Claim 14 differs from the combination of Barak in view of Horton and Sumit as evidenced by Xu, as applied to claims 1-3, 11-13, 15 and 19-26, in that: before step 4), the method further comprises: fixing the reporter cell with a formaldehyde solution diluted in a phosphate buffer; and staining the nucleus and/or cytoskeleton of the reporter cell.
Shen teaches a method of measuring NFATc1-GFP distribution in cells, comprising stimulating cells to induce NFAT translocation, staining the nucleus of the reporter cell (e.g., with the nuclear stain TO-PRO-3) and fixing the reporter cell with formaldehyde (under Materials and methods; Fig. 1c; Fig. 2).
It would have been obvious to one of ordinary skill in the art at the time the invention was made to use the method of Barak in view of Horton and Sumit to screen for acetylcholinesterase inhibitors using an NFATc1-EGFP reporter cell wherein the NFAT translocation is measured via fixing and staining the cells as taught by Shen because it would have been obvious to combine prior art elements according to known methods to yield predictable results. One of ordinary skill would have been motivated to use the fixation and staining method of Shen to measure NFAT translocation because doing so would provide a more stable measurement system wherein the distributional changes are fixed. Using the fixation and staining method of Shen to measure NFAT translocation would have led to predictable results with a reasonable expectation of success because Horton teaches that systems other than the one exemplified by Horton can be used to measure translocation, and Shen teaches a fixing/staining procedure for measuring translocation of a similar NFAT-GFP reporter protein as used in the method of Barak in view of Horton and Sumit
Conclusion
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/ROBERT J YAMASAKI/Primary Examiner, Art Unit 1657
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