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 .
Status of Application, Amendments, And/Or Claims
The Applicant’s amendments/remarks received 6/11/2026 are acknowledged. Claims 1, 3 and 6 are amended; claim 2 is canceled; claims 1 and 3-21 are pending; claims 7-21 are withdrawn; claims 1 and 3-6 have been examined on the merits.
Drawings
The replacement drawings received 6/11/2026 have fixed the objection regarding labeling the figures as “Fig. 1”, “Fig. 2”, “Fig. 3” and “Fig. 4”, respectively; however, Applicant has inexplicably removed the sheet numbering; hence, the replacement drawings are objected to for the following reasons:
37 CFR 1.84 (t) states "Numbering of sheets of drawings. The sheets of drawings should be numbered in consecutive Arabic numerals, starting with 1, within the sight as defined in paragraph (g) of this section. These numbers, if present, must be placed in the middle of the top of the sheet, but not in the margin. The numbers can be placed on the right-hand side if the drawing extends too close to the middle of the top edge of the usable surface. The drawing sheet numbering must be clear and larger than the numbers used as reference characters to avoid confusion. The number of each sheet should be shown by two Arabic numerals placed on either side of an oblique line, with the first being the sheet number and the second being the total number of sheets of drawings, with no other marking.” Hence, the replacement sheets should be numbered, as they were previously, as “1/2” and “2/2”, respectively.
Corrected drawing sheets in compliance with 37 CFR 1.121(d) are required in reply to the Office action to avoid abandonment of the application. Any amended replacement drawing sheet should include all of the figures appearing on the immediate prior version of the sheet, even if only one figure is being amended. The figure or figure number of an amended drawing should not be labeled as “amended.” If a drawing figure is to be canceled, the appropriate figure must be removed from the replacement sheet, and where necessary, the remaining figures must be renumbered and appropriate changes made to the brief description of the several views of the drawings for consistency. Additional replacement sheets may be necessary to show the renumbering of the remaining figures. Each drawing sheet submitted after the filing date of an application must be labeled in the top margin as either “Replacement Sheet” or “New Sheet” pursuant to 37 CFR 1.121(d). If the changes are not accepted by the examiner, the applicant will be notified and informed of any required corrective action in the next Office action. The objection to the drawings will not be held in abeyance.
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.
Claim 1 is rejected under 35 U.S.C. 103 as being unpatentable over Webster et al., US 2010/0272641 (U.S. Patent Application Publication cite 1, IDS, 9/5/2023; herein “Webster”) in view of Nickel et al., 2009 (NPL cite 7, IDS, 9/5/2023; herein “Nickel”) and Zhang et al., 2018 (cite U, PTO-892, 3/12/2026; herein “Zhang”).
Webster teaches producing labeled substrates of the cystine/glutamate antiporter of cells, i.e., a cystine analog; incubating cells with the cystine analog, reducing the cystine analog with a reducing agent to generate a cysteine analog, i.e., labeled cysteine, and detecting the cysteine analog transported into the cells (Abst.; [0010], [0026], [0029-30], [0035]). Webster teaches that the cystine content of the cells can be directly assayed by collecting the cells, washing the cells and lysing the cells to quantify the labeled cystine molecules [0056]. A person of ordinary skill in the art at the time of filing would have found it obvious that other methods of liberating the cytoplasm of the cells, such as crushing, could be substituted for lysing the cells in Webster’s method.
Webster teaches that the cystine transporter in mammalian cells comprises xCT, also known as rBAT [0026].
Webster does not state that the cystine analog can be selenocystine; however, a person of ordinary skill in the art at the time of filing would have found it obvious that selenocystine could be the cystine analog in view of the disclosure of Nickel.
Nickel discloses that selenocystine uses the same transporter as cystine and is the same transporter as disclosed by Webster, i.e., rBAT (Abst.; Fig. 1; Table 3; p. 240, “4.1.4. Cystine and selenocystine”). Hence, a person of ordinary skill in the art at the time of filing would have found it obvious that selenocystine could be used as the cystine analog to measure cystine uptake in cells in the method of Webster because selenocystine uses the cystine transporter.
Neither Webster nor Nickel discloses a method for specifically detecting selenocysteine; however, a person of ordinary skill in the art at the time of filing would have found it obvious that the selenocysteine produced by the method of Webster in view of Nickel could be detected by the method of Zhang.
Zhang discloses methods of detecting selenocysteine in cells comprising incubating the cells with selenocystine for a defined time period, collecting the cells, washing the cells, then determining the selenocysteine content of the cells with a fluorophore which covalently reacts with selenocysteine (p. 477, “2.4. Imaging of Sec in living cells”) wherein the fluorescence intensity of the fluorophore linearly increases with the selenocysteine content (Abst.), i.e., quantifying the selenocysteine by the fluorescence intensity.
Zhang discloses that the fluorophore has previously been reported for reacting with biothiols (Abst.), i.e., cysteine, etc., but because selenocysteine has a smaller pKa value and better nucleophilicity (p. 475, “Introduction”, ¶2), the fluorophore reacts better with selenocysteine than with cysteine or other biothiols (about 8-fold better as seen in Fig. 2b) by adjusting the detection conditions (Abst.).
Hence, a person of ordinary skill in the art at the time of filing would have found it obvious to practice the method made obvious by Webster in view of Nickel and Zhang for evaluating the cystine uptake ability of cells wherein the method comprises contacting cells with the cystine analog selenocystine; washing the cells with PBS, i.e., washing away selenocystine not taken up by the cells; crushing the cells, i.e., lysing the cells; and determining the selenocystine contained in cytoplasm by contacting the crushed cells with a reducing agent, reducing the selenocystine to selenocysteine, contacting the reduced cell extract with a selenocysteine-specific fluorophore, i.e., selenocysteine is contacted with a fluorescent dye that specifically reacts with selenocysteine to change in fluorescence intensity; and determining the concentration of selenocysteine (and hence, selenocystine when divided by 2) by the fluorescence intensity of the fluorophore, i.e., the fluorescence intensity is measured to determine selenocystine, because Webster teaches assaying cystine uptake ability of cells by contacting the cells with a cystine analog, reducing the cystine analog to produce cysteine and determining the concentration of the cysteine; Nickel demonstrates that selenocystine uses the cystine transporter; and Zhang teaches contacting cells with selenocystine wherein the selenocystine is endogenously reduced to selenocysteine in the cells and the selenocysteine is quantified by reacting with a selenocysteine-specific fluorophore wherein the fluorescence intensity of the fluorophore linearly increases with the selenocysteine content; therefore, claim 1 is prima facie obvious.
Response to Arguments
Applicant's arguments filed 6/11/2026 have been fully considered but they are not persuasive. Notably, regarding the rejection of claims 1-2 under 35 U.S.C. 103 over Webster in view of Nickel and Zhang, Applicant states that the rejection is over Webster in view of Nickel (Remarks, p. 13) and regarding the rejection of claims 1-2 and 6 under 35 U.S.C. 103 over Webster in view of Nickel, Zhang and Lee, Applicant states that the rejection is over Webster in view of Nickel and Lee (Remarks, p. 13).
Regarding the rejection of claims 1-2 under 35 U.S.C. 103 over Webster in view of Nickel and Zhang, Applicant argues against the references individually.
In response to applicant's arguments against the references individually, one cannot show nonobviousness by attacking references individually where the rejections are based on combinations of references. See In re Keller, 642 F.2d 413, 208 USPQ 871 (CCPA 1981); In re Merck & Co., 800 F.2d 1091, 231 USPQ 375 (Fed. Cir. 1986).
Regarding the Webster reference, Applicant argues (p. 13) that Webster requires a labeled cystine for their method and that selenocystine does not qualify as a labeled cystine. Applicant argues that the instant invention is fundamentally different in that “the claimed invention uses selenocystine that is not provided as a labeled imaging substrate and performs detection through post-treatment processing after cellular uptake. In particular, after selenocystine not taken up by the cells is washed away and the cells are crushed, selenocystine contained in the cytoplasm is reduced to selenocysteine, and the generated selenocysteine is detected by fluorescence measurement” (p. 13).
The rejection is not over Webster in isolation but over Webster in view of Nickel and Zhang. As set forth in the previous Office action and in the rejection above, Zhang clearly discloses methods of detecting selenocysteine in cells comprising incubating the cells with selenocystine for a defined time period, collecting the cells, washing the cells, then determining the selenocysteine content of the cells with a fluorophore which covalently reacts with selenocysteine (p. 477, “2.4. Imaging of Sec in living cells”) wherein the fluorescence intensity of the fluorophore linearly increases with the selenocysteine content (Abst.), i.e., quantifying the selenocysteine by the fluorescence intensity. Zhang only differs from the claimed method of claim 1 in that in Zhang the selenocystine is reduced to selenocysteine endogenously in the cell rather than being reduced with a reducing agent exogenously.
Hence, Applicant’s argument that a person of ordinary skill in the art at the time of filing would have found it novel and non-obvious to perform a method of cystine determination comprising using selenocystine that is not provided as a labeled imaging substrate and performing detection through post-treatment processing after cellular uptake; in particular, after selenocystine not taken up by the cells is washed away and the cells are crushed, selenocystine contained in the cytoplasm is reduced to selenocysteine, and the generated selenocysteine is detected by fluorescence measurement is completely unpersuasive because Zhang teaches the method but with endogenous reduction of selenocystine to selenocysteine and Webster teaches cystine uptake assays wherein the cystine analog is incubated with cells, the cells are washed, lysed, the cystine analog reduced with an exogenous reducing agent, and the amount of cysteine quantified; hence, the claimed method is clearly obvious over Webster in view of Nickel and Zhang.
Applicant states (p. 15) “Zhang concerns fluorescence-based detection chemistry for free selenocysteine, rather than transporter uptake quantification using unlabeled selenocystine.” And (p. 15) “Even when Zhang and Wang are considered, those references at most provide separate teachings relating to fluorescence detection chemistry.” This is incorrect. Zhang discloses methods of detecting selenocysteine in cells comprising incubating the cells with selenocystine for a defined time period, collecting the cells, washing the cells, then determining the selenocysteine content of the cells with a fluorophore which covalently reacts with selenocysteine (p. 477, “2.4. Imaging of Sec in living cells”) wherein the fluorescence intensity of the fluorophore linearly increases with the selenocysteine content (Abst.), i.e., quantifying the selenocysteine by the fluorescence intensity. Applicant’s arguments are unpersuasive and the rejections are maintained with modification to address claim amendments and/or for clarity.
Claims 1 and 6 are rejected under 35 U.S.C. 103 as being unpatentable over Webster in view of Nickel, Zhang and Lee et al., US 2023/0272221 (cite A, PTO-892, 3/12/2026; herein “Lee”).
The discussion of Webster, Nickel and Zhang regarding claim 1 set forth in the rejection above is incorporated herein.
Webster does not specifically teach that the reducing agent is tris(carboxyethyl)phosphine (TCEP); however, a person of ordinary skill in the art at the time of filing would have found it obvious for the reducing agent to be TCEP in view of the disclosure of Lee.
Lee is drawn to labeling reagents and linkers (Abst.) and discloses that TCEP is an effective reducing agent for cleaving cleavable groups such as disulfides, azido groups, etc. ([0012], [0033], [0045], [0150]). Hence, a person of ordinary skill in the art at the time of filing would have found it obvious that TCEP is a sufficiently strong reducing agent to reduce the selenium-selenium bond in selenocystine; therefore, claim 6 is prima facie obvious.
Response to Arguments
Applicant does not specifically address the rejection of claims 1-2 and 6 under 35 U.S.C. 103 over Webster in view of Nickel, Zhang and Lee; hence, the rejection is maintained with modification to address claim amendments and/or for clarity.
Claims 1 and 3-6 are rejected under 35 U.S.C. 103 as being unpatentable over Webster in view of Nickel, Zhang, Lee and Wang et al., 2012 (NPL cite 5, IDS, 9/5/2023).
The discussion of Webster, Nickel, Zhang and Lee regarding claims 1 and 6 set forth in the rejection above is incorporated herein.
Zhang teaches that the selenocysteine-specific probe can be a fluorescent probe which has previously been known for detecting biothiols, i.e., reacting with biothiols (Abst.). Zhang demonstrates that the fluorescent biothiol probe reacts more strongly with selenocysteine than with other biothiols because of the lower pKa and better nucleophilicity of selenocysteine as compared to cysteine. Selenocysteine has the exact molecular structure of cysteine wherein the sulfur atom is replaced with a selenium atom. Because of the greater nucleophilicity of selenocysteine as compared to cysteine and the lower pKa of selenocysteine as compared to cysteine, selenocysteine will more strongly and more quickly react with a cysteine-specific fluorophore than cysteine itself will. Hence, a person of ordinary skill in the art at the time of filing would have found it obvious that the method made obvious by Webster in view of Nickel, Zhang and Lee can be practiced with a reasonable expectation of success wherein the fluorescent probe of Zhang is substituted with other cysteine-specific fluorescent probes wherein the detection of selenocysteine is optimized over the detection of cysteine by optimizing the reaction time, pH, ionic strength, etc., i.e., normal experimental optimization of the method to increase selectivity for selenocysteine over cysteine which is in the purview of one of ordinary skill in the art.
None of Webster, Nickel, Zhang or Lee teach that the fluorescent probe comprises the structure of Formula 4 in claim 5; however, a person of ordinary skill in the art at the time of filing would have found it obvious that the cysteine-specific probe to be optimized for the detection of the more nucleophilic selenocysteine can be the cysteine-specific probe taught by Wang, which is identical to Formula 4.
Wang teaches that Formula 4 (chemodosimeter 1 in Wang; Abst.; Scheme 1) reacts with the nucleophiles cysteine and homocysteine by a conjugate addition/cyclization sequence (Scheme 2) wherein the intermolecular cyclization of the nucleophile is 25-fold faster for cysteine than for homocysteine (p. 276, “3.2. Effects of reaction time on sensing Cys and Hcy”). Wang does not examine the reactivity of Formula 4 toward selenocysteine; however, because of the greater nucleophilicity of selenocysteine over cysteine, the lower pKa of selenocysteine over cysteine and the same chain length and molecular structure of selenocysteine and cysteine; a person of ordinary skill in the art at the time of filing would have found it obvious that Formula 4 would have greater and faster reactivity with selenocysteine than with cysteine.
Hence, a person of ordinary skill in the art at the time of filing would have found it obvious that the cysteine-selective fluorophore of Wang, i.e., Formula 4, can be used in the method made obvious by Webster in view of Nickel, Zhang and Lee as a selenocysteine-specific probe with minor optimization of the reaction conditions, i.e., reaction time, pH, ionic strength, etc., with a reasonable expectation of success; therefore, claims 3-5 are prima facie obvious.
Response to Arguments
Regarding the rejection of claims 1-6 under 35 U.S.C. 103 over Webster in view of Nickel, Zhang, Lee and Wang, Applicant argues (p. 13) “Wang concerns cysteine detection chemistry and does not suggest applying such chemistry to intracellular uptake quantification of unlabeled selenocystine. Even when Zhang and Wang are considered, those references at most provide separate teachings relating to fluorescence detection chemistry”.
These allegations, regarding claims 1-2 and 6 and the Zhang reference, are unpersuasive and are addressed on pp. 7-9 above. Regarding Wang, Wang is not relied on to provide a method of intracellular uptake quantification, rather, Wang is relied on as providing other selenocysteine detection agents which could substitute for Zhang’s coumarin-based fluorescent selenocysteine probe. Additionally, the rejection does not rely solely on Wang, but also on Zhang’s disclosure of the greater nucleophilicity of selenocysteine over cysteine, the lower pKa of selenocysteine over cysteine and the same chain length and molecular structure of selenocysteine and cysteine; hence, a person of ordinary skill in the art at the time of filing would have found it obvious that Formula 4 of Wang would have greater and faster reactivity with selenocysteine than with cysteine and could be used in the method made obvious by Webster in view of Nickel, Zhang and Lee as a selenocysteine-specific probe with minor optimization of the reaction conditions, i.e., reaction time, pH, ionic strength, etc., with a reasonable expectation of success; therefore, claims 3-5 are prima facie obvious.
Applicant’s arguments are unpersuasive; thus, the rejection is maintained with modification to address claim amendments and/or for clarity.
Conclusion
No claims are allowed.
THIS ACTION IS MADE FINAL. 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.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to Trent R Clarke whose telephone number is (571)272-2904. The examiner can normally be reached M-F 10-7 MST.
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/TRENT R CLARKE/ Examiner, Art Unit 1651
/DAVID W BERKE-SCHLESSEL/ Primary Examiner, Art Unit 1651
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