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 .
Priority
This application is a continuation (CON) application of PCT/JP2022/006280, filed 2/17/2022. This application claims benefit to foreign application JAPAN 2021-034870, filed 3/05/2021. Claims 1-21 are pending.
Information Disclosure Statement
The information disclosure statement submitted on 9/5/2023 has been considered by the examiner.
Election/Restrictions
Applicant's election with traverse of Group I, claims 1-6 and Formula 4 as the species of fluorescent dye in the reply filed on 1/23/2026 is acknowledged. The traversal is on the ground that it should be no undue burden on the Examiner to consider all claims in the single application. Applicant’s arguments have been fully considered but are not found to be persuasive because despite the resources available to the examiner, the groups are patentably distinct for reasons set forth in the restriction and require separate considerations and search limitations when searching. The groups are in separate classes. Further, burden consists not only of specific searching of classes and subclasses, but also of searching multiple databases for foreign references and literature searches. Burden also resides in the examination of independent claim sets for clarity, enablement and double patenting issues. In view of these requirements, it is the Examiner's position that searching and examining all of the claims in the same application presents a serious burden on the Examiner for the reasons given above and in the previous restriction requirement; hence, claims 1-6 will be examined on the merits. Claims 7-21 are withdrawn from further consideration pursuant to 37 CFR 1.142(b), as being drawn to a nonelected invention and/or species, there being no allowable generic or linking claim. NOTE: Claims 3-4 are not withdrawn even though Formula 4 from claim 5 was elected as the species because Formula 4 is encompassed by the limitations of claims 3-4.
The requirement is still deemed proper and is therefore made FINAL.
Drawings
The drawings are objected to for the following reasons:
37 CFR 1.84 (u)(1) states "View numbers must be preceded by the abbreviation "FIG."" In the instant application, the view numbers for Figures 1-4 are preceded by the word "Figure" instead of the abbreviation "FIG.". Additionally, the figure labels are in brackets which should be removed. Hence, “[Figure 1]”, “[Figure 2]”, “[Figure 3]” and “[Figure 4]” should be “Fig. 1”, “Fig. 2”, “Fig. 3” and “Fig. 4”, 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.
Claims 1-2 are 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, attached PTO-892; 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 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, i.e., selenocystine is reacted with a reducing agent to generate 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 one or both of fluorescence wavelength and fluorescence intensity, or selenocysteine is reacted with a fluorescent dye under the condition that the fluorescent dye specifically reacts with selenocysteine to change in one or both of fluorescence wavelength and 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, claims 1-2 are prima facie obvious.
Claims 1-2 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, attached PTO-892; herein “Lee”).
The discussion of Webster, Nickel and Zhang regarding claims 1-2 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.
Claims 1-6 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 1-2 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.
Conclusion
No claims are allowed.
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