Notice of Pre-AIA or AIA Status
The present application is being examined under the pre-AIA first to invent provisions.
DETAILED ACTION
An amendment was filed on 10/16/2025. Claim 13 is cancelled.
The previous rejection of claims 1-19 under 35 U.S.C. 112(b) as being indefinite is withdrawn in light of the cancellation of claim 13; the amendment of claim 1 to state “a polynucleotide encoding”, amendment correcting the dependency of claim 4, and clarifying which agent is cell-permeable in claim 11.
Claims 1-12 and 14-19 are pending and under examination herein.
Claim Rejections - 35 USC § 102
The following is a quotation of the appropriate paragraphs of pre-AIA 35 U.S.C. 102 that form the basis for the rejections under this section made in this Office action:
A person shall be entitled to a patent unless –
(a) the invention was known or used by others in this country, or patented or described in a printed publication in this or a foreign country, before the invention thereof by the applicant for a patent.
(b) the invention was patented or described in a printed publication in this or a foreign country or in public use or on sale in this country, more than one year prior to the date of application for patent in the United States.
Maintained rejection: Claims 1-6, 10-11, 14-15 and 17-19 are rejected under pre-AIA 35 U.S.C. 102(a) and 102(b) as being clearly anticipated by Miller (US 2008/0299592 A1, published December 4, 2008; previously cited). The rejection of claim 1 is further evidenced by Su et al. (“Target identification of biological active small molecules via in situ methods”, Current Opinion in Chemical Biology, 2013, Vol. 17, Issue 5, pp.768-775; previously cited).
Regarding claim 1, Miller teaches an isolated nucleic acid including a sequence of nucleotides that encode a modified luciferase polypeptide and a second nucleic acid molecule including a second sequence of nucleotides encoding a preselected protein (relevant to (b) a polynucleotide encoding a protein of interest and a luciferase) (description p.1, paragraphs [0007] – [0008]).
Miller teaches isolated polypeptides also include a protein of interest fused in frame with the luciferase and the tetracysteine tag or HaloTag™ protein (description p.1, paragraph [0012]). Miller further teaches using tetracysteine-tagged luciferase in combination with a bis-arsenical sNIRF (description p.4, paragraph [0051]), and identifies sNIRFs as small-molecule near-IR fluorophores (relevant to (a) a bioactive agent conjugated to a fluorophore).
Miller further teaches providing a cell expressing a modified luciferase polypeptide as described; contacting the cell with luciferin; (relevant to (c) a substrate for the luciferase) contacting the cell with a near-infrared acceptor dye; and detecting NIR emission from the NIR acceptor dye (description p.1, paragraph [0014]).
Miller is silent regarding whether the bioactive agent is “capable of binding non-covalently to the protein of interest”. However, as evidenced by Su et al., most bioactive compounds bind to their protein targets non-covalently (p.771, 2nd column last paragraph). Thus, Miller’s teaching of isolated peptides including a protein of interest fused with luciferase and the tetracysteine tag would be capable of binding non-covalently to the protein of interest encoded by the polynucleotide, as required by instant claim 1(b).
Regarding claim 2, Miller teaches tetracysteine-tagged luciferase (relevant to wherein the bioactive agent is a peptide) (description p.4, paragraph [0054]).
Regarding claims 3 and 4, Miller teaches nucleic acids encoding polypeptides, and vectors comprising such nucleic acids (relevant to claim 3: wherein the nucleic acid is RNA; relevant to claim 4: wherein the nucleic acid is DNA) (description p.5, paragraph [0060]).
Regarding claim 5, Miller teaches chloroalkyl-tethered small molecule fluorophores (description p.5, paragraph [0056]; FIG. 4).
Regarding claim 6, Miller teaches sulforhodamine 101 [0058], tetramethylrhodamine and Rhodamine B (relevant to wherein the small molecule fluorophore is a carboxy rhodamine analog) (description p.5, paragraph [0067]).
Regarding claim 10, Miller teaches that the ability of bis-arsenical or chloroalkyl dyes to red-shift the light output of cells expressing the tetracysteine-tagged or a HaloTag protein-luciferase was evaluated by imaging tagged-luciferase constructs in mammalian cells and in blood with or without the addition of the fluorophore (relevant to wherein the conjugate of the bioactive agent and fluorophore is added extracellularly and enters the cell) (description p.11, paragraphs [0122]-[0123]).
Regarding claim 11, Miller teaches cell-permeable near-IR dyes include near-IR oxazine dyes that can be readily attached to a chloroalkyl group (relevant to wherein the bioactive conjugated to the fluorophore is cell permeable) (description 8, paragraph [0094]).
Regarding claim 14, Miller teaches HaloTag™ protein-luciferase fusion proteins, comprising HaloTag™ proteins which are haloalkane dehydrohalogenase mutant that form a covalent attachment to chloroalkane-tethered small molecule fluorophores that are cell permeable (relevant to wherein the bioactive agent is produced by non-natural chemical synthesis) (description p.4-5, paragraph [0056]).
Regarding claim 15, Miller teaches resonance energy transfer from firefly luciferase to ReAsH, using firefly luciferase fusion proteins with an optimized tetracysteine tag (TC) that were cloned and expressed in E. coli as GST fusions (description p.9, paragraph [0114]). Miller teaches that treatment of TCLuc1 or TCLuc2 with ReAsH resulted in a dramatic shift in the emission wavelength maximum, with better BRET efficiency seen with the N-terminal TC tag (description p.9, paragraph [0115]). Miller teaches that the emission spectrum was sharpened, such that the maximum of 608nm is actually higher than the 560 nm peak of luciferase (relevant to claim 15: wherein said luciferase has a first emission spectrum with a first peak emission; said fluorophore has an excitation spectrum that overlaps with said first emission spectrum, and said fluorophore has a second emission spectrum with a second peak emission, said second peak emission being separated from said first peak emission) (description p.9, paragraph [0115]; FIG. 3(c)).
Regarding claim 17, Miller teaches providing a cell expressing the polypeptide of claim 27; contacting the cell with luciferin; contacting the cell with a near-infrared (NIR) acceptor dye that binds to the polypeptide and undergoes intramolecular biofluorescence resonance energy transfer (BRET) with the modified luciferase polypeptide; and detecting the NIR emission from the NIR acceptor dye (relevant to claim 17: wherein upon binding of the bioactive agent to the protein of interest, conversion of the substrate to a reaction product by the luciferase results in excitation of the fluorophore by bioluminescence )
Regarding claims 18 and 19, Miller teaches the reporter construct can be integrated into the genome of a cell or non-human animal or can be independently replicating, e.g. on a plasmid vector (relevant to claim 18: wherein the polynucleotide is expressed from a vector; relevant to claim 19: wherein the vector is a plasmid) (description p.8, paragraph [0102]).
Response to Arguments
Applicant argues that the present technology relates to a system comprising a bioactive agent conjugated to a fluorophore and a protein of interest fused to a luciferase, and non-covalent binding of the bioactive agent to the protein of interest brings the fluorophore and luciferase within sufficient proximity to detect a BRET signal in the presence of a substrate for the luciferase (See Remarks dated 10/16/2025, p.5, Novelty). Applicant argues that Miller provides systems and methods for covalently tethering a small molecule near-IR fluorophore to a luciferase with the aim of shifting the light output of the luciferase to the near-IR by resonance transfer energy, and refers to two exemplary systems from Miller (See Remarks dated 10/16/2025, p.6 top paragraph). Applicant argues that Miller does not teach or suggest non-covalent binding of a fluorophore-conjugated bioactive agent to a luciferase-fused protein of interest, and further argues that Miller teaches away from the use of non-covalent interactions, stating that “the observed energy transfer was weak” (See Remarks dated 10/16/2025, p.6 2nd paragraph).
Applicant's arguments filed October 16, 2025 have been fully considered but they are not persuasive. The system of instant claim 1 requires (a) a bioactive agent conjugated to a fluorophore; (b) a polynucleotide encoding (i) a protein of interest and (ii) a luciferase; and (c) a substrate for the luciferase. The limitation “wherein the bioactive agent is capable of binding non-covalently to the protein of interest upon interaction therewith” is a desired property of the bioactive agent, but does not structurally differentiate the bioactive agent from what is taught by the prior art. As discussed in the rejection above, Miller teaches isolated polypeptides include a protein of interest fused in frame with the luciferase and the tetracysteine tag or HaloTag™ protein (description p.1, [0012]), and using tetracysteine-tagged luciferase in combination with a bis-arsenical sNIRF (description p.4, paragraph [0051]). Miller identifies sNIRFs as small-molecule near-IR fluorophores; the bis-arsenic molecule is a bioactive agent; thus the bis-arsenical sNIRF meets the structural limitations of claim 1(a) “a bioactive agent conjugated to a fluorophore”. Miller teaches an isolated nucleic acid including a sequence of nucleotides that encode a modified luciferase polypeptide and a second nucleic acid molecule including a second sequence of nucleotides encoding a preselected protein (relevant to (b) a polynucleotide comprising a protein of interest and a luciferase) (description p.1, [0007] – [0008]). Miller further teaches providing a cell expressing a modified luciferase polypeptide as described; contacting the cell with luciferin; (relevant to (c) a substrate for the luciferase) contacting the cell with a near-infrared acceptor dye; and detecting NIR emission from the NIR acceptor dye (description p.1, [0014]).
While Applicant argues that Miller teaches that energy transfer was weak, the claims do not require any particular level of energy transfer, and Miller’s teaching does not teach away from the structural requirements of the instant claims.
Claim Rejections - 35 USC § 103
The following is a quotation of pre-AIA 35 U.S.C. 103(a) which forms the basis for all obviousness rejections set forth in this Office action:
(a) A patent may not be obtained though the invention is not identically disclosed or described as set forth in section 102, if the differences between the subject matter sought to be patented and the prior art are such that the subject matter as a whole would have been obvious at the time the invention was made to a person having ordinary skill in the art to which said subject matter 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 pre-AIA 35 U.S.C. 103(a) 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 under pre-AIA 35 U.S.C. 103(a), the examiner presumes that the subject matter of the various claims was commonly owned at the time any inventions covered therein were made absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and invention dates of each claim that was not commonly owned at the time a later invention was made in order for the examiner to consider the applicability of pre-AIA 35 U.S.C. 103(c) and potential pre-AIA 35 U.S.C. 102(e), (f) or (g) prior art under pre-AIA 35 U.S.C. 103(a).
Maintained rejection: Claims 7-8 are rejected under pre-AIA 35 U.S.C. 103(a) as being unpatentable over Miller (US 2008/0299592 A1, published December 4, 2008; previously cited) as applied to claim 1 above, and further in view of Encell et al. (US 2010/0281552 A1, published on Nov. 4, 2010; previously cited).
The teachings of Miller are discussed above.
Regarding claims 7 and 8, Miller teaches imaging cells or animals that express a mutated luciferase, and adding sufficient amounts of luciferin to the cells (description p.8, [0098]).
Miller does not teach wherein the luciferase comprises a polypeptide with at least 70% sequence identity to SEQ ID NO:1 (claim 7) or wherein the substrate for luciferase is coelenterazine or a coelenterazine derivative (claim 8).
However, Encell teaches SEQ ID NO:13, an amino acid sequence for a modified luciferase polypeptide derived from a wild-type Oplophorus luciferase with 171 amino acids that has 100% sequence homology with instant SEQ ID NO:1 (relevant to claim 7) (FIG. 31). Encell teaches that the mechanism underlying the luminescence of Oplophorus involves the oxidation of Oplophorus luciferin (coelenterazine) with molecular oxygen (relevant to claim 8) (description paragraph [0004]). Encell further teaches that the luciferase secreted from the deep-sea shrimp Oplophorus gracilirostris has been shown to possess many interesting characteristics, such as high activity, high quantum yield, and broad substrate specificity (coelenterazine, coelenterazine analogs) (description p.9, paragraph [0105]).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify the teaching of Miller to replace the mutated luciferase/luciferin enzyme/substrate combination taught by Miller with the enzyme/substrate combination of Oplophorus luciferase having the amino acid sequence identified as SEQ ID NO:13 and Oplophorus luciferin (coelenterazine) taught by Encell to arrive at the claimed invention, because Encell teaches luciferase secreted from Oplophorus gracilirostris has high activity, high quantum yield and broad substrate specificity. Each of Miller and Encell teach the use of a luciferin substrate with a luciferase enzyme to cause luminescence. One of ordinary skill in the art would have found it beneficial to select the Oplophorus luciferase having SEQ ID NO:13 taught by Encell and its substrate Oplophorus luciferin (coelenterazine) as the luciferase/luciferin combination in the system taught by Miller, because Encell teaches Oplophorus luciferase has high activity, binds to Oplophorus luciferin (coelenterazine), and the amino acid sequence of Oplophorus luciferase identified as SEQ ID NO:13, which has 100% homology to instant SEQ ID NO:1, was known in the art at the time of invention.
Maintained rejection: Claims 9 and 16 are rejected under pre-AIA 35 U.S.C. 103(a) as being unpatentable over Miller (US 2008/0299592 A1, published December 4, 2008; previously cited) in view of Encell et al. (US 2010/0281552 A1, published on Nov. 4, 2010; previously cited) as applied to claims 1, 8 and 15 above, and further in view of Hall et al. (“Engineered luciferase reporter from a deep sea shrimp utilizing a novel imidazopyrazinone substrate”, ACS Chemical Biology, Aug. 15, 2012, Vol. 7, Issue 11, pp.1848-1857; previously cited).
The teachings of Miller and Encell are discussed above.
Regarding claim 9, Miller teaches adding sufficient amounts of luciferin and an appropriate fluorophore to cells that express a mutated luciferase (description p.8, paragraph [0098]).
Miller does not teach wherein the substrate is 2-furanylmethyl-deoxy-coelenterazine.
Encell teaches that the mechanism underlying the luminescence of Oplophorus involves the oxidation of Oplophorus luciferin (coelenterazine) with molecular oxygen (relevant to claim 8) (description p.1, paragraph [0004]). Encell further teaches that the luciferase secreted from the deep-sea shrimp Oplophorus gracilirostris has been shown to possess many interesting characteristics, such as high activity, high quantum yield, and broad substrate specificity (coelenterazine, coelenterazine analogs) (description p.9, paragraph [0105]).
Encell is silent as to whether coelenterazine analogs include 2-furanylmethyl-deoxy-coelenterazine.
However, Hall teaches using a small luciferase subunit from the deep sea shrimp Oplophorus gracilirostris which improves luminescence expression in mammalian cells ~2.5 million-fold by merging optimization of protein structure with development of a novel imidazopyrazinone substrate (furimazine) (abstract). Hall teaches that the substrate producing the brightest luminescence signal with this enzyme variant (~25-fold over coelenterazine) was 2-furanylmethyl-deoxy-coelenterazine (furimazine; Figure 1). Hall further teaches that furimazine was found to be more stable in cell culture media and produced lower autoluminescence than coelenterazine or coelenterazine h (p.1850, 1st column, 2nd paragraph).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to replace the coelenterazine substrate taught by Encell with the furimazine substrate taught by Hall in the system of Miller and Encell, because Hall teaches that furimazine produced the brightest luminescence signal with the enzyme variant Oplophorus luciferase taught by Encell and Hall. One of ordinary skill in the art would have found it beneficial to use a substrate that produced the brightest luminescent signal, and also was more stable in cell culture and produced lower autoluminescence in a BRET assay that requires luminescence detection.
Regarding claim 16, Miller teaches the emission of TCLuc1 and TCLuc2 was centered at 560nm, whereas treatment of TCLuc1 and TCLuc2 with ReAsh shifted the emission wavelength maximum to 608nm, with a corresponding increase in light emitted in the 600-700nm range.
Miller and Encell do not explicitly teach wherein said second emission peak is separated from said first emission peak by at least 80nm.
However, Hall teaches spectral emission peaks for Nluc (460nm), Rluc (480nm), Fluc (565nm) and CBR (605nm) (p.1851, Figure 2). Hall teaches that the spectrum of Nluc is 20nm blue-shifted relative to that of Rluc, and about 20% narrower, making it well suited for applications involving multiplexing with longer wavelength luminescence reporters, providing dual luciferase assays with well separated spectra to support greater composite dynamic range and sensitivity (p.1850, 2nd column last paragraph – p.1851 1st column top paragraph). The difference between Nluc (460nm) and Miller’s TCLuc1 (560nm) is 100nm, and Miller’s TCLuc1 with ReAsh at 608nm is a shift of 148nm, which are greater than 80 nm separation required by the claim.
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to replace the luciferase used by Miller with Nluc taught by Hall, because Hall teaches that Nluc emission max is 460nm, which makes is compatible with other luciferase with emissions greater than 600nm. Each of Miller and Hall teach the excitation and emission of luciferase using a substrate and measuring luminescence using BRET. One of ordinary skill in the art would reasonably expect that selecting dyes having emission peaks with the desired peak separation would predictably result in visualizing emission spectrum that contained at least 80nm separation between the two peak emissions to visualize each signal peak separately.
Maintained rejection: Claim 12 is rejected under pre-AIA 35 U.S.C. 103(a) as being unpatentable over Miller (US 2008/0299592 A1, published December 4, 2008; previously cited) as applied to claims 1 and 11 above, and further in view of Farquhar et al. (“Protein Kinase A-Dependent Step(s) in Hepatitis C Virus Entry and Infectivity”, Journal of Virology, 2008, Vol. 82, No.17, pp.8797-8811; previously cited).
The teachings of Miller are discussed above.
Regarding claim 12, Miller is silent on the use of a permeabilization agent to potentiate the entry of the bioactive agent conjugated to the fluorophore into the cell.
However, Farquhar teaches studying the role of protein kinase-A by BRET using Renilla luciferase (p.8798, 2nd column – BRET assay to investigate PKAI and PKAII dynamics in living cells). Farquhar also teaches cells were permeabilized for 30 min, washed three times before the addition of the relevant secondary Alexa Fluor-conjugated antibodies in PBS-saponin-BSA (p.8798, 2nd column, last paragraph).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify the method of Miller to add a permeabilization step as taught by Farquhar to allow the bioactive conjugate to enter the cell. Each of Miller and Farquhar teach methods of luminescent assays in live cells. One of ordinary skill in the art would reasonably expect that permeabilizing the cell as taught by Farquhar would predictably result in improving entry of the Alexa Fluor-conjugated antibody (i.e. bioactive-fluorophore conjugate) into the cell, because it was known in the art at the time of invention that cells could be permeabilized to increase the entry of fluorophore conjugates into cells and improve fluorophore uptake and signal.
Response to Arguments
Applicant did not argue against the above rejections in their response filed October 16, 2025. All rejections are maintained.
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 mailing date of this final action.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to DEEPA MISHRA whose telephone number is (571) 272-6464. The examiner can normally be reached Monday - Friday 9:30am - 3:30pm EST.
Examiner interviews are available via telephone, in-person, and video conferencing using a USPTO supplied web-based collaboration tool. To schedule an interview, applicant is encouraged to use the USPTO Automated Interview Request (AIR) at http://www.uspto.gov/interviewpractice.
If attempts to reach the examiner by telephone are unsuccessful, the examiner’s supervisor, Louise W. Humphrey can be reached on (571) 272-5543. The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300.
Information regarding the status of published or unpublished applications may be obtained from Patent Center. Unpublished application information in Patent Center is available to registered users. To file and manage patent submissions in Patent Center, visit: https://patentcenter.uspto.gov. Visit https://www.uspto.gov/patents/apply/patent-center for more information about Patent Center and https://www.uspto.gov/patents/docx for information about filing in DOCX format. For additional questions, contact the Electronic Business Center (EBC) at 866-217-9197 (toll-free). If you would like assistance from a USPTO Customer Service Representative, call 800-786-9199 (IN USA OR CANADA) or 571-272-1000.
/LOUISE W HUMPHREY/Supervisory Patent Examiner, Art Unit 1657
/DEEPA MISHRA/Examiner, Art Unit 1657