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 the Claims
Claims 1-11 were pending. Claims 1, 3, 7, and 11 are amended.
Claims 1-11 are examined herein.
Claim Objections
Claims 1-3, 7, and 11 are objected to because of the following informalities:
Claims 1, 3, 7, and 11 recite lysosomal system flux function, while claim 2 recites lysosomal system function. Terminology should be consistent.
Appropriate correction is required.
Withdrawn Rejections
The rejection of claims 1-11 under 35 U.S.C. §112(b) is withdrawn in view of claims 1, 3, 7, and 11 amendments.
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.
The factual inquiries set forth in Graham v. John Deere Co., 383 U.S. 1, 148 USPQ 459 (1966), that are applied for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
Determining the scope and contents of the prior art.
Ascertaining the differences between the prior art and the claims at issue.
Resolving the level of ordinary skill in the pertinent art.
Considering objective evidence present in the application indicating obviousness or nonobviousness.
This application currently names joint inventors. In considering patentability of the claims the examiner presumes that the subject matter of the various claims presumes that the subject matter of the various claims was commonly owned as of the effective filing date of the claimed invention(s) absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and effective filing dates of each claim that was not commonly owned as of the effective filing date of the later invention in order for the examiner to consider the applicability of 35 U.S.C. 102(b)(2)(C) for any potential 35 U.S.C. 102(a)(2) prior art against the later invention.
Claims 1-11 are rejected under 35 U.S.C. 103 as being unpatentable over Chittaranjan et al. (Cold Spring Harb Protoc. 2015 Aug 3;2015(8):743-50) in view of Groeneveld et al. (IDS; Research and Practice in Thrombosis and Haemostasis, Vol. 1, Supp. 1, pp. 1238-9, Abstract Number PB 1280, 26th International Society on Thrombosis and Haemostasis Congress (Berlin Germany, July 8-13, 2017); a new copy is provided due to poor quality of the submitted copy), for reasons of record which are reiterated herein below.
Regarding claim 1, Chittaranjan teaches a method for “Monitoring Autophagic Flux by Using Lysosomal Inhibitors and Western Blotting of Endogenous MAP1LC3B” (Title). The reference teaches monitoring of autophagic flux using LC3B-II as a lysosomal system marker, and LC3B-II levels were determined by western blotting (Abstract). The term autophagic flux refers to the same process as the lysosomal system flux of instant disclosure. Lysosomal inhibitors are inhibitors of lysosomal system function of instant disclosure.
Chittaranjan does not specifically teach determining the level of the lysosomal system marker in a sample of whole blood from a subject.
Regarding claim 1, Groeneveld teaches disruption of the autophagy machinery by treating plasma or whole blood from healthy volunteers with different concentrations of chloroquine or bafilomycin A1 (pg. 1239, col. 1, par. 3).
It would have been obvious to one having ordinary skill in the art before the effective filing date of the claimed invention to modify the method of Chittaranjan by employing treatment of whole blood with lysosomal inhibitors as taught by Groeneveld, in order to provide a method for measuring lysosomal system flux because treatment of whole blood samples with lysosomal inhibitors does not require preliminary cell isolation. One having ordinary skill in the art would have been motivated to treat whole blood samples because it simplifies experiments. This combination would have been desirable to those of ordinary skill in the art for the reasons mentioned above.
One having ordinary skill in the art would have had a reasonable expectation of success in combining the prior art references because Groeneveld demonstrates that treatment of whole blood samples does not negatively affect the cells.
Regarding claim 2, Chittaranjan teaches chloroquine or bafilomycin A1 inhibitors (pg. 743, last par.) meeting the limitation of claim 2 reciting chloroquine, bafilomycin A 1, E-64d, leupeptin, and pepstatin A.
Regarding claim 3, Chittaranjan in view of Groeneveld teaches using platelet-rich plasma or whole blood from healthy volunteers (Groeneveld, pg. 1239, col. 1, par. 3), meeting the limitation of claim 3 reciting other cellular populations derived from the whole blood. Platelet-rich plasma is a cellular population enriched with platelets and derived from whole blood.
Regarding claims 4-6, Chittaranjan teaches LC3B-II as a lysosomal system marker (Abstract), meeting the limitation of claim 6 reciting the lysosomal system marker comprises LC3B-II protein. LC3B-II is the lipidated form of LC3B (id.), meeting the limitation of claim 5 reciting the lysosomal system marker comprises LC3B protein. LC3B one of the variants of a protein marker known in the art as LC3 protein - an LC3 protein with light chain B, thus meeting the limitation of claim 4 reciting the lysosomal system marker comprises an LC3 protein.
Regarding claim 7, Chittaranjan teaches quantitation of the levels of LC3B-II using different concentrations of the lysosomal inhibitor bafilomycin A1, including a sample with no inhibitor treatment (Fig. 2A and B), which corresponds to the limitation of the comparison of the level of the lysosomal system marker with a level of the lysosomal system marker in a sample of whole blood from the subject which has not been treated with the inhibitor of lysosomal system flux function.
Regarding claims 8 and 10, Chittaranjan teaches determining the level of the lysosomal system marker using immunological detection. Specifically, the reference teaches using western blotting with primary antibody for LC3B-II (Title and pg. 744, par. 1).
Regarding claim 11, Chittaranjan in view of Groeneveld teaches treating a sample of whole blood with the inhibitor of lysosomal system function (Groeneveld, pg. 1239, col. 1, par. 3) and determining the level of the lysosomal system marker in the sample of whole blood so treated as compared to the level of the lysosomal system marker in sample of whole blood without treatment – quantitation of the levels of LC3B-II using different concentrations of the lysosomal inhibitor bafilomycin A1, including a sample with no inhibitor treatment (Chittaranjan, Fig. 2A and B).
Claim 9 is rejected under 35 U.S.C. 103 as being unpatentable over Chittaranjan in view of Groeneveld, as applied to claim 8 above, and further in view of Oh et al. (Anal Biochem. 2017 Aug 1; 530:57-67), for reasons of record which are reiterated herein below.
The teachings of Chittaranjan and Groeneveld have been set forth above.
Chittaranjan and Groeneveld fail to teach the immunological detection comprises ELISA or immunocytochemical staining.
Regarding claim 9, Oh teaches “Quantification of autophagy flux using LC3 ELISA” (Title). Specifically, Oh teaches that LC3 marker was analyzed by a sandwich ELISA using two LC3 antibodies, LC3 capture and HRP-conjugated LC3 detection antibodies (Abstract).
It would have been obvious to one having ordinary skill in the art before the effective filing date of the claimed invention to modify the method of Chittaranjan and Groeneveld by employing ELISA as taught by Oh, as an obvious matter of simple substitution of one known element (ELISA method) for another (western blotting) to obtain predictable results.
One having ordinary skill in the art would have had a reasonable expectation of success in combining the prior art references because both ELISA and western blotting detection methods rely on similar antibodies specific to a lysosomal flux marker, and Oh successfully demonstrates application of LC3 ELISA for quantification of autophagy flux.
Response to Arguments
Applicant’s arguments filed on February 2, 2026 have been fully considered.
Applicant argues that “Without the benefit of hindsight knowledge of the present invention, a person of ordinary skill would not have contemplated the combining of the teachings of Chittaranjan and Groeneveld. A person of ordinary skill would have understood that Groeneveld has a different focus and is directed to an entirely different mechanism than Chittaranjan” (pg. 6, par. 1). The argument of the “different focus” of Groeneveld is not persuasive because Groeneveld is cited of its teaching of the lysosome inhibitors chloroquine and bafilomycin being able to successfully disrupt the autophagic process when added to whole blood samples; the other aspects of the Groeneveld’s teachings including the “different focus” of the entire study are not used in the rejection. Please see explanation 2(b) below for details.
Applicant argues that “Groeneveld did not add lysosome inhibitors to whole blood to measure autophagic flux. Groeneveld used lysosome inhibitors chloroquine and bafilomycin to disrupt the autophagic process. This distinction is fundamental, and it would have dissuaded a person of ordinary skill from the addition of lysosomal inhibitors to whole blood to measure autophagic flux. The distinction is more evident when one examines the materials and methods of Groeneveld” (pg. 6, par. 2).
The arguments are not persuasive because: (a) the autophagic process is the same as the autophagic flux, as evidenced by Zhang et al. (Acta Pharmacol Sin. 2013 May;34(5):595-9) provides the following definition: “The term “autophagic flux” is used to represent the dynamic process of autophagy. In detail, autophagic flux refers to the whole process of autophagy, including autophagosome formation, maturation, fusion with lysosomes, subsequent break down and the release of macromolecules back into the cytosol” (pg. 595, col. 2, par. 2); and (b) Groeneveld is cited of its teaching of the lysosome inhibitors chloroquine and bafilomycin being able to successfully disrupt the autophagic process when added to whole blood samples, instead of the culture media as taught by Chittaranjan. The measurement of the autophagic flux is taught by Chittaranjan, using detection of LC3B-II biomarker by western blotting, which requires harvesting and washing cells prior to proteins separation (Abstract, and par. 3-4 at the bottom of pg. 745). The washing step removes all components of whole blood, plasma, or culture media, so they cannot affect the results of Western blot analysis. As such, it is unclear why a person of ordinary skill would have been dissuaded from the addition of lysosomal inhibitors to whole blood. Centrifugation and washing of cells is a routine procedure with a predictable outcome.
Applicant argues that “The 3-methyladenine referred to in Groeneveld inhibits the initiation of autophagy, not autophagic flux function” (pg. 6, par. 2). The arguments are not persuasive because the non-final OA (mailed October 1, 2025) did not use the teaching of 3-methyladenine. Groeneveld was cited of its teaching of the lysosome inhibitors chloroquine and bafilomycin being able to successfully disrupt the autophagic process when added to whole blood.
Paragraphs 4-5 on pg. 6 provide an explanation of chloroquine and bafilomycin activity without any argument.
Applicant argues again that “Groeneveld did not add lysosome inhibitors chloroquine and bafilomycin to whole blood in order to measure autophagic flux. These inhibitors were used in platelet-rich plasma or whole blood to disrupt the autophagic process” (pg. 6, last par. – pg. 7, par. 1). The arguments are not persuasive, because Groeneveld is cited of its teaching of the lysosome inhibitors chloroquine and bafilomycin being able to successfully disrupt the autophagic process when added to whole blood samples. Please, see 2(b) above for detailed explanation.
Applicant argues that “No reasonable expectation of success” (pg. 7, par. 2). Specifically, “If a person of ordinary skill had chanced upon the Groeneveld abstract, they would have noticed a critical detail described in this study: while chloroquine (CQ) did disrupt platelet function, bafilomycin (BAF) did not …” (id.) and “this difference in effect of the two compounds suggests that the platelet-based functions” (pg. 7, par. 4-7). The arguments are not persuasive, because as has already been discussed in 2(b) Groeneveld is cited of its teaching of the lysosome inhibitors chloroquine and bafilomycin being able to successfully disrupt the autophagic process when added to whole blood samples, while the measurement of the autophagic flux is taught by Chittaranjan. The effects of chloroquine and bafilomycin on platelet functions taught by Groeneveld were not relied upon for rejection of claims 1-11.
Applicant argues that Groeneveld would have directed a scientist in the field addressee away from adding either compound to whole blood in order to measure autophagy” (pg. 7, par. 3). The arguments are not persuasive, because Applicant fails to provide specific arguments of teaching away in Groeneveld’s reference. Additionally, Groeneveld is cited of its teaching of the lysosome inhibitors chloroquine and bafilomycin being able to successfully disrupt the autophagic process when added to whole blood samples, but not the measurement of autophagy. The measurement of the autophagic flux is taught by Chittaranjan. There is nothing in the teachings of Groeneveld and Chittaranjan to direct a scientist from applying the inhibitors to whole blood samples.
Applicant argues that “at the time of the invention there were no methods that directly measured autophagic flux in human samples. The citation of Chittaranjan and Groeneveld does not negate this understanding” (pg. 8, par. 2). The argument is not persuasive, because Chittaranjan does teach measurement of the autophagic flux using detection of LC3B-II biomarker by western blotting as evidenced by the title “Monitoring Autophagic Flux by Using Lysosomal Inhibitors and Western Blotting of Endogenous MAP1LC3B”.
Applicant argues that “it would not have been obvious for a person of ordinary skill to try to combine the teachings of Chittaranjan and Groeneveld. But if the combination were made, then there would have been no expectation of success arriving at the present invention, in view of the data provided by Groeneveld pertaining to the effects of the molecules that they tested, and in view of the lack of precedent for successful measurement of autophagic flux in human samples” (pg. 8, par. 3). The arguments are not persuasive, because the combination of Chittaranjan and Groeneveld has already been discussed above in 2(b) and there is a motivation for using whole blood (Groeneveld) instead of the culture media (Chittaranjan) because the whole blood does not require preliminary cell purification before applying the inhibitors (OA, pg. 5, par. 2). The fact that chloroquine and bafilomycin are able to successfully disrupt the autophagic process is the basis for a reasonable expectation of success.
Applicant argues that “The Office added the Oh reference to its rejection of claim 9, alleging that Oh teaches the use of ELISA. Oh does not remedy the deficiencies of Chittaranjan and Groeneveld discussed above in relation to the base claims. Thus, the rejection based on Chittaranjan and Groeneveld and Oh should also be withdrawn” (pg. 8, par. 4). The arguments are not persuasive, because as has already been discussed above, the teachings of Chittaranjan and Groeneveld do not have deficiencies regarding claims 1-8 and 10-11. Chittaranjan and Groeneveld fail to teach the immunological detection comprises ELISA. Oh teaches “Quantification of autophagy flux using LC3 ELISA” (Title). Specifically, Oh teaches that LC3 marker was analyzed by a sandwich ELISA using two LC3 antibodies, LC3 capture and HRP-conjugated LC3 detection antibodies (Abstract).
Conclusion
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 date of this final action.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to Alexander Volkov whose telephone number is (571) 272-1899. The examiner can normally be reached M-F 9:00AM-5:00PM (EST).
If attempts to reach the examiner by telephone are unsuccessful, the examiner’s supervisor, Bao-Thuy Nguyen can be reached on (571) 272-0824. The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300.
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/ALEXANDER ALEXANDROVIC VOLKOV/
Examiner, Art Unit 1677
/BAO-THUY L NGUYEN/Supervisory Patent Examiner, Art Unit 1677 May 4, 2026
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