DETAILED ACTION
Status of claim rejections
The rejections of record under 35 USC 103 are maintained in view of Applicant’s arguments in the response filed 07/07/2026.
This Action is FINAL.
Claim Interpretation
Claim 32 recites, inter alia, “a detector luciferase configured to catalyze oxidation of a substrate to emit light at a first wavelength, wherein the detector luciferase is responsive to the analyte of interest such that the presence or amount of the analyte modulates the bioluminescent emission of the detector luciferase without serving as the substrate for the luciferase reaction.” The examiner has interpreted the “configured to catalyze oxidation of a substrate” limitation to encompass the detector luciferase being capable of catalyzing oxidation of a substrate to emit light, including but not limited to a NanoLuc. This interpretation is supported by pg. 3 of the instant specification. The examiner has also interpreted “such that the presence or amount of the analyte modulates the bioluminescent emission of the detector luciferase without serving as the substrate for the luciferase reaction” under broadest reasonable interpretation as inherent property or inherent result of the detector luciferase being responsive to the analyte of interest. Where the claimed and prior art products are identical or substantially identical in structure or composition, or are produced by identical or substantially identical processes, a prima facie case of either anticipation or obviousness has been established. In re Best, 562 F.2d 1252, 1255, 195 USPQ 430, 433 (CCPA 1977) (MPEP 2112.01). In short, any detector luciferase that has the recited structures claimed in claim 32 would inherently have the recited functional characteristic.
Claim 32 also recites “wherein the ratio of light intensities emitted at the first and second wavelengths provides a time- and substrate-concentration-independent quantification of the analyte of interest”. The specification on pgs. 3-5 recites “the emission ratio of measured bioluminescence intensity of the detector luciferase and measured bioluminescence intensity of the calibrator luciferase remains stable over time” (see pg. 3) and “any type of bioluminescent assay may be used in order to provide the time-independent and concentration-independent quantification assay of the present invention. As long as a calibrator luciferase is selected having the same luciferase domain as the detector luciferase, detection and quantification of the analyte of interest of a sample to be analysed can be performed in a reliable manner” (see pg. 5). The examiner has interpreted this limitation to encompass the intensity of the light emitted by the luciferases remain stable over time, and that the time and concentration-independence depends on the use of the same luciferase domains in the detector and calibrator luciferase.
Maintained 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.
This application currently names joint inventors. In considering patentability of the claims the examiner 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.
First rejection
Claims 16-17, 20-23, and 32-36 are rejected under 35 U.S.C. 103 as being unpatentable over Stacer et al (NanoLuc Reporter for Dual Luciferase Imaging in Living Animals. Molecular Imaging. 2013;12(7). doi:10.2310/7290.2013.00062; hereinafter “Stacer”; prior art of record) in view of Arts (Luminescent sensor proteins for point-of-care diagnostics, published 23 October 2017 [Phd Thesis 1 (Research TU/e /Graduation TU/e), Biomedical Engineering]. Technische Universiteit Eindhoven)) and Leu et al (US 20150191769 A1; prior art of record).
Stacer teaches the NanoLuc reporter for luciferase imaging in animal cells, and teaches that luciferase from the deep-sea shrimp Oplophorus gracilirostris engineered for enhanced protein stability (NanoLuc [NL]) and its optimally designed substrate, furimazine (pg. 458, col 1, paragraph 2). Stacer teaches the use of Nanoluc in bioluminescence imaging of tumors and internal organs of living mice and in Western blot analysis of antibodies (a bioluminescence assay to quantify analyte of interest as in claim 16 and 32; detector luciferase as in claim 16 and 32) compared to Gaussia luciferase bioluminescence (see pg. 458, col 1, paragraph 3; pg. 459, col 1-2; pg. 460, Fig. 1). Stacer also teaches the NL emits a blue light in response to furimazine within the mice lung metastases (detector luciferase is reactive to a substrate to emit light at a first wavelength and is responsive to the analyte of interest as in claim 16) (see Fig. 4; see pg. 464, col 1, paragraph 1).
The difference between Stacer and the instant claims is that Stacer does not teach a calibrator luciferase, wherein the calibrator luciferase is reactive to the same substrate to emit light at a second wavelength which second wavelength is different from the first wavelength emitted by the detector luciferase.
However, Arts teaches luminescent sensor proteins for point-of-care diagnostics (title). Arts teaches a sensor platform called LUCID and LUMABS based on bioluminescence resonance energy transfer that allows detection of antibodies in solution (pg. 46, abstract). Arts teaches the luciferase NanoLuc that is reactive to the substrate furimazine, connected via a semi-flexible linker to the green fluorescent protein mNeonGreen, which results in change in color upon addition of antibodies in the sample from green to blue (a calibrator luciferase reactive to the same substrate to emit light at a second wavelength where the second wavelength is different from the first wavelength emitted by the detector luciferase as in claim 16 and 32) (abstract; pg. 48, paragraph 2; see Fig. 2.1). Arts specifically teaches that the use of semisynthetic LUMABS sensors significantly expands the range of antibody targets and enables the application of LUMABS sensors for the ratiometric bioluminescent detection of small molecules using a competitive immunoassay format. (pg. 54, paragraph 1 and pg.102, paragraph 1). Please note the NanoLuc-mNeonGreen conjugate luciferase of Arts is a variant that contains the same luciferase domain (NanoLuc) as the luciferase of Stacer.
Therefore, it would have been prima facie obvious to one of ordinary skill in the art at the time of filing to modify the NanoLuc bioluminescent assay of Stacer by including the NanoLuc-mNeonGreen LUMAB luciferase as taught by Arts to arrive at the claimed invention. As Stacer teaches the bioluminescent detection of mice tumor using NanoLuc luciferase and Arts teaches a luciferase conjugated to a fluorescent protein capable of detecting small molecules in immunoassays, one of ordinary skill would have been motivated to modify the assay of Stacer with the luciferase conjugate of Arts with a reasonable expectation of success. One of ordinary skill would have been motivated to make the modification because Arts teaches that the NanoLuc-mNeonGreen luciferase reporter advantageously change in color upon addition of antibodies in the sample from green to blue and expands the range of antibody targets and enables the application of LUMABS sensors for the ratiometric bioluminescent detection of small molecules.
Neither Stacer nor Arts explicitly teaches that the calibrator luciferase is non-responsive to the analyte of interest.
However, Leu teaches dual and triple luciferase assays to activate fluorescence from a fluorescent protein in close proximity to the luciferase in a sample (paragraph 0024) comprising a first luciferase and a second luciferase wherein the first and second luciferases may utilize the same or different substrate (paragraph 0032), and the luminescent signals generated by the luciferases may be produced at different wavelengths (paragraph 0033 and 0172; paragraph 0181). Leu also teaches the addition of a reducing agent to the sample that inactivates one of the luciferases but not the other (paragraph 0066, paragraph 0073). Leu teaches that the inactivation of one of the luciferases by inhibiting or abolishing the catalytic activity of the luciferase and/or converting the luciferase into an inactive conformation (see paragraph 0064) allows for the use of the luciferases in a multi-luciferase assay, as well as the desirable removal of unwanted signal so the sample can be applied to other assays that use luminescence or fluorescence as a read out or to reduce light leakage (paragraph 0137).
Therefore, it would have been prima facie obvious to one of ordinary skill at the time of filing to modify the bioluminescent assay of Stacer and Arts by adding a reducing agent that inactivates one of the luciferases as taught by Leu to arrive at the claimed invention. As Stacer and Leu teach the use of luciferases for biometric detection using a luciferase and a luciferase conjugate, and Leu teaches a dual luciferase system where one luciferase is inactivated in response to a reducing agent in a sample, one of ordinary skill would have been motivated to modify the assay of Stacer and Arts with the reducing agent of Leu with a reasonable expectation of success. One of ordinary skill would have been motivated to make the modification because Leu teaches that inactivation of one of the luciferases allows for the use of the luciferases in a multi-luciferase assay, as well as the desirable removal of unwanted signal so the sample can be applied to other assays that use luminescence or fluorescence as a read out or to reduce light leakage.
Further regarding claim 32, “the ratio of light intensities emitted at the first and second wavelengths provides a time- and substrate-concentration-independent quantification of the analyte of interest” was interpreted to encompass the intensity of the light emitted by the luciferases remain stable over time, and that the time and concentration-independence depends on the use of the same luciferase domains in the detector and calibrator luciferase (see above and Applicant’s specification). Arts teaches that the NanoLuc luciferase was developed to have improved brightness, thermodynamic stability and substrate preference over Renilla and firefly luciferases, in combination with the substrate furimazine and was shown to be 100-fold brighter than Renilla and firefly luciferases (figure 1.13). NanoLuc produces a glow-type luminescence that is relatively stable over time (see pg. 24). Further, as the combination of Stacer, Arts, and Leu teaches the use of luciferases that contain the same luciferase domain that emit light at different wavelengths, absent evidence to the contrary, the luciferases would be capable of providing a time- and substrate-concentration-independent quantification of the analyte of interest.
Regarding claim 17, 32, and 35, Arts teaches that the luciferase is connected to mNeonGreen, a fluorescent protein (i.e., functionalized with a fluorescent acceptor) (see above).
Regarding claim 20 and 33, Arts teaches using the luciferase for competitive assay or sandwich immunoassay (see above, and for example, Fig. 5.1).
Regarding claim 21 and 34, Arts and Stacer both teach using the NanoLuc luciferase domain (see above).
Regarding claim 22, Arts teaches using mNeonGreen as fluorescent acceptor conjugated to (i.e., functionalized with) NanoLuc (see above).
Regarding claim 23 and 36, Arts teaches using the luciferase for antibody detection (i.e., the analyte of interest is an antibody) (see above).
Accordingly, the claimed invention was prima facie obvious to one of ordinary skill at the time of filing, especially in the absence of evidence to the contrary.
Second rejection
Claim 31 is rejected under 35 U.S.C. 103 as being unpatentable over Stacer, Arts, and Leu as applied to claims 16-17, 20-23, and 32-36 above, and further in view of Dixon et al (NanoLuc Complementation Reporter Optimized for Accurate Measurement of Protein Interactions in Cells. ACS Chem Biol. 2016 Feb 19;11(2):400-8; cited in 12/27/2021 IDS; hereinafter “Dixon”).
As discussed above, claims 16-17, 20-23, and 32-36 were rendered prima facie obvious by the combined teachings of Stacer, Arts, and Leu.
The difference between the references and the instant claims is that none of the references explicitly teach the luciferase domain of the detector luciferase is a split luciferase domain.
However, Dixon teaches optimized NanoLuc reporters for accurate measurement of protein interactions in cells (title, abstract). Dixon teaches the use of the NanoLuc reporter to create a new complementation reporter called NanoBiT, which is a 13 kDa peptide and 18 kDa peptide split luciferase that has bright and sustained luminescence, where the subunits weakly associate such that their assembly into a luminescent complex is dictated by interaction characteristics of the target proteins they bind to (see abstract; pg. 400, col 2; pg. 401, Fig. 1; Fig. 2). Dixon further teaches that assays based on split luciferases are often preferred due to their sensitivity and simplicity (pg. 400, col 2, paragraph 2).
Therefore, it would have been prima facie obvious to one of ordinary skill at the time of filing to modify the bioluminescent assay of Stacer and Arts and use a split luciferase reporter as taught by Dixon to arrive at the claimed invention. As Dixon teaches optimized Nanoluc reporter split luciferase system with sustained and bright luminescence, one of ordinary skill would have been motivated to perform a simple substitution of one known element (the luciferase of Stacer) with another (the split luciferase of Dixon) with a reasonable expectation of success. One of ordinary skill would have been motivated to make the modification because Dixon teaches that split luciferase systems advantageously has bright and sustained luminescence, where the subunits weakly associate such that their assembly into a luminescent complex is dictated by interaction characteristics of the target proteins they bind to, and are preferred due to their sensitivity and simplicity.
Accordingly, the claimed invention was prima facie obvious to one of ordinary skill at the time of filing, especially in the absence of evidence to the contrary.
Response to Arguments
Applicant's arguments filed 7/7/2026 have been fully considered but they are not persuasive.
On pg. 8-9 of the remarks, Applicant argues that the combination of Stacer, Leu and Arts is improper and based on impermissible hindsight. Applicant argues the examiner reconstructs Applicant’s invention by taking a detector from Stacer, incorporating a wavelength-shifting concept from Arts, and relying on Leu to render one luciferase “non-responsive” and that while each reference addresses luminescent detection in a different context, none of the references contemplates/teaches/suggests Applicant’s architecture nor would a PHOSITA have motivation to combine the references. Applicant argues Arts achieves ratiometric detection within a single molecular construct to generate both optical signals necessary for ratiometric analysis and that a PHOSITA would simply employ the disclosed construct of Arts. Applicant argues that nothing in Arts suggests separating the sensing and reference functions into 2 distinct proteins or suggest abandoning the integrated single-protein architecture in favor of a more complicated multi-component system because one would have to dismantle the integrated LUMABS sensor, extract the selected BRET concepts and reconstruct those with a different assay architecture based on Stacer which runs contrary to the teachings of Arts and represents a step backward in complexity, reliability, and assay design.
In response, the examiner disagrees. First, in response to applicant's argument that the examiner's conclusion of obviousness is based upon improper hindsight reasoning, it must be recognized that any judgment on obviousness is in a sense necessarily a reconstruction based upon hindsight reasoning. But so long as it takes into account only knowledge which was within the level of ordinary skill at the time the claimed invention was made, and does not include knowledge gleaned only from the applicant's disclosure, such a reconstruction is proper. See In re McLaughlin, 443 F.2d 1392, 170 USPQ 209 (CCPA 1971). Here, the combination of Stacer, Arts, and Leu explicitly teach a PHOSITA to combine two luciferases for ratiometric detection of analytes in a sample. Stacer teaches the use of NanoLuc in bioluminescence imaging of tumors and internal organs of living mice and in Western blot analysis of antibodies (i.e., a bioluminescence assay to quantify analyte of interest as in claim 16 and 32; a detector luciferase as in claim 16 and 32) (see pg. 458, col 1, paragraph 3; pg. 459, col 1-2; pg. 460, Fig. 1). Arts explicitly discloses that use of NanoLuc provides improved brightness, thermodynamic stability and substrate preference over Renilla and firefly luciferases, expands the range of antibody targets and enables the application of sensors for the ratiometric bioluminescent detection of small molecules. As shown above, Arts provides a PHOSITA the teaching, suggestion, and motivation to use a second luciferase as instantly claimed. Indeed, an obviousness rejection under 35 U.S.C 103 requires one of ordinary skill to balance each individual teaching of the prior art references with the totality of the references in combination. Applicant’s arguments do not take into account the combination of teachings of the prior art references, and do not appreciate all of the evidence as a whole.
On pg. 9-11 of the remarks, Applicant argues the characterization of Leu overlooks the actual purpose of Leu’s disclosure. Specifically, Applicant argues that Leu teaches temporary signal termination through chemical treatment so that a second, different luciferase can be measured without interference. Applicant argues Leu is not concerned with creating a stable internal calibrator that coexists with the detector throughout the assay because Leu’s inactivation step intentionally eliminates one reporter signal rather than preserving it. Applicant argues that the calibrator luciferase must remain continuously active throughout the assay to provide analyte-independent reference signals and remain operational during measurement. Applicant argues a PHOSITA would not look to Leu’s destructive inactivation methodology because applying the reagent of Leu would introduce substantial uncertainty into a system in which the detector and calibrator share the same luciferase domain and substrate so it would be entirely unpredictable how chemical inactivation intended for one luciferase would affect the activity, kinetics, or stability of the other and would undermine the objective of achieving a robust, simultaneously operating calibration system. Applicant then urges that the combination of references relies on impermissible hindsight. Applicant further argues that the claimed functional result recited in claim 32 is not an inherent property but flows from Applicant’s specific architecture because the luciferases comprise identical domains and catalyze oxidation of the same substrate and substrate concentration, reaction kinetics, etc. affect both luciferases in the same manner, and that because only the detector luciferase remains responsive to the analyte, the experimental variations cancel in the measured ratio to produce the quantification. Applicant argues the examiner has not provided any technical explanation or evidentiary support demonstrating the proposed combination would necessarily achieve this result because the claimed quantification is a direct consequence of the designed calibrator/detector architecture. Applicant argues much of the same regarding the rejection of the dependent claims and claim 31.
In response, the examiner disagrees for much of the same reasons as set forth above. First, Leu teaches dual and triple luciferase assays to activate fluorescence from a fluorescent protein in close proximity to the luciferase in a sample (paragraph 0024) comprising a first luciferase and a second luciferase wherein the first and second luciferases may utilize the same or different substrate (paragraph 0032), and the luminescent signals generated by the luciferases may be produced at different wavelengths (paragraph 0033 and 0172; paragraph 0181). Leu also teaches the addition of a reducing agent to the sample that inactivates one of the luciferases but not the other (paragraph 0066, paragraph 0073). Leu teaches that the inactivation of one of the luciferases by inhibiting or abolishing the catalytic activity of the luciferase and/or converting the luciferase into an inactive conformation (see paragraph 0064) allows for the use of the luciferases in a multi-luciferase assay, as well as the desirable removal of unwanted signal so the sample can be applied to other assays that use luminescence or fluorescence as a read out or to reduce light leakage (paragraph 0137).
Second, it is important to note that Applicant’s argument regarding “the experimental variability” flows from the teachings of the prior art, namely Leu. As discussed above, Leu provides a teaching, suggestion, and motivation to use a luciferase that is reactive to the same substrate as another luciferase, and inhibiting or abolishing the catalytic activity of the luciferase and/or converting the luciferase into an inactive conformation that allows for the use of the luciferases in a multi-luciferase assay. Furthermore, the stability and time-independence as instantly claimed was interpreted to encompass the intensity of the light emitted by the luciferases remaining stable over time, and that the time and concentration-independence depends on the use of the same luciferase domains in the detector and calibrator luciferase (see interpretation above). Thus, absent evidence to the contrary, the luciferases would be capable of providing a time- and substrate-concentration-independent quantification of the analyte of interest. Leu provides a teaching, suggestion, and motivation to use a luciferase that is reactive to the same substrate as another luciferase, and inhibiting or abolishing the catalytic activity of the luciferase and/or converting the luciferase into an inactive conformation that allows for the use of the luciferases in a multi-luciferase assay.
Finally, regarding Applicant’s argument regarding impermissible hindsight, the examiner notes that the prior art references, before Applicant’s filing date, provides one of ordinary skill the necessary teachings, suggestion, and motivation to combine the teachings of the references to arrive at the claimed invention. Solely to rebut Applicant’s argument, the examiner points Applicant to Thorne et al (Illuminating insights into firefly luciferase and other bioluminescent reporters used in chemical biology. Chem Biol. 2010 June 25; 17(6): 646–657) which teaches dual luciferase assays (with single color or two color) that utilizes different or the same luciferase domains (see abstract; Fig. 2 reproduced below). Fig. 2B demonstrates the use of 2 CBLuciferase variants (i.e., the same luciferase domains) that emit a red or green light and are reactive to the same substrate, requires a single substrate addition with activity from the two variant luciferases differentiated based on their shifted bioluminescent emission wavelengths (se pg. 3; see also Figure 2B). Thus, the rejection is maintained as set forth above.
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Conclusion
NO CLAIMS 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 GEORGIANA C REGLAS whose telephone number is (571)270-0995. The examiner can normally be reached M-Th: 8:00am-2:00pm.
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/G.C.R./Examiner, Art Unit 1651
/THOMAS J. VISONE/Supervisory Patent Examiner, Art Unit 1672
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