Notice of Pre-AIA or AIA Status
The present application is being examined under the pre-AIA first to invent provisions.
DETAILED ACTION
Claims 1-12 and 14-19 are pending and under examination herein.
Continued Examination Under 37 CFR 1.114
A request for continued examination under 37 CFR 1.114, including the fee set forth in 37 CFR 1.17(e), was filed in this application after final rejection. Since this application is eligible for continued examination under 37 CFR 1.114, and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114. Applicant's submission filed on April 23, 2026 has been entered.
Claim Rejections - 35 USC § 112
The following is a quotation of the first paragraph of 35 U.S.C. 112(a):
(a) IN GENERAL.—The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor or joint inventor of carrying out the invention.
The following is a quotation of the first paragraph of pre-AIA 35 U.S.C. 112:
The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor of carrying out his invention.
Claims 1-12 and 14-19 are rejected under 35 U.S.C. 112 (pre-AIA ), first paragraph, as failing to comply with the written description requirement. The claims contains subject matter which was not described in the specification in such a way as to reasonably convey to one skilled in the relevant art that the inventor or a joint inventor, or for applications subject to pre-AIA 35 U.S.C. 112, the inventors, at the time the application was filed, had possession of the claimed invention.
Applicant is referred to MPEP 2163(II)(A)(3)(a)(i and ii), which states that the written description requirement for a claimed genus may be satisfied through sufficient description of a representative number of species by actual reduction to practice, reduction to drawings, or by disclosure of relevant, identifying characteristics, i.e., structure or other physical and/or chemical properties, by functional characteristics coupled with a known or disclosed correlation between function and structure, or by a combination of such identifying characteristics, sufficient to show the applicant was in possession of the claimed genus. A "representative number of species" means that the species which are adequately described are representative of the entire genus. Thus, when there is substantial variation within the genus, one must describe a sufficient variety of species to reflect the variation within the genus. The disclosure of only one species encompassed within a genus adequately describes a claim directed to that genus only if the disclosure indicates that the patentee has invented species sufficient to constitute the genus. For inventions in an unpredictable art, adequate written description of a genus which embraces widely variant species cannot be achieved by disclosing only one species within the genus.
Claims 1-12 and 14-19 are drawn to a system comprising (a) a bioactive agent conjugated to a fluorophore; (b) a polynucleotide encoding: (i) a protein of interest and (ii) a luciferase; wherein the bioactive agent is capable of binding non-covalently to the protein of interest upon interaction therewith; and (c) a substrate for the luciferase. The claims encompass any bioactive agent conjugated to any fluorophore, and any polynucleotide encoding any protein of interest and a luciferase. The specification does not disclose a representative number of species of bioactive agents conjugated to a fluorophore, nor a representative number of species of proteins of interest.
The current specification identifies “a bioactive agent” as a small molecule, protein, nucleic acid, lipid, etc. (Specification p.10, last paragraph). The current specification identifies “a protein of interest” as a cellular target (Specification p.3, top paragraph). The current specification identifies a “cellular target” as comprising any suitable binding/interaction partner (e.g. receptor/enzyme) for a bioactive agent (e.g. small molecule, protein, nucleic acid, lipid, etc.) (Specification p.10, last paragraph). The current specification provides an example demonstrating PBI tracer conjugates in an example using conjugates of suberoylanilide hydroxamic acid (SAHA), an inhibitor of histone deacetylase 6 (HDAC6), and a PBI dye (SAHA-TOM (PBI-4968)) or standard dye (SAHA-TAMRA (PBI-4967)) in an intracellular BRET assay using a NANOLUC-HDAC fusion protein (SEQ ID NO: 3) (Specification p.18, Example 1). The current specification discusses an experiment measuring relative drug affinities using BIRB-796 or PBI-4835 (Specification p.21, Example 5). The specification discusses using specific NANOLUC™ substrates or enzyme components (Specification p.23, Example 7). The specification describes measuring the relative affinity of a fluorescently-labeled iBET compound for wild-type and mutant version of BRD4 protein (Specification p.32, Example 14). The specification describes specific bioactive agent-fluorophore conjugates of purvanol B conjugated to TOM dye (p.33-34, Example 16); drug-tracer combinations BIRB-TOM; PBI-4838 and Dasatinib-TOM; PBI5170 (p.34-35, Example 17);
The specification does not identify other fluorophores other than TOM or TAMRA, or provide any guidance as to any requirements of the fluorophore, such as excitation wavelength or emission wavelength. The specification does not provide any guidance on how to determine whether a bioactive agent is “capable of binding to the protein of interest upon interaction”. The specification does not identify any other bioactive agent and target proteins combinations other than suberoylanilide hydroxamic acid (SAHA) and histone deacetylase 6 (HDAC6). The specification fails to describe cellular targets. The specification does not teach how to form a system with any bioactive agent and fluorophore and any protein of interest.
The prior art does not provide any structure-function relationship of bioactive agents and proteins of interest so that one of ordinary skill in the art could envision a representative number of bioactive agents; bioactive agents or proteins of interest by disclosing structural or functional features of bioactive agents.
The disclosure of a single species of bioactive agent and target protein is not considered to constitute a representative number of species of the genus of bioactive agents, or the genus of proteins of interest, that are capable of binding noncovalently upon interaction, wherein the emission spectrum of the luciferase overlaps with the excitation spectrum of the fluorophore. Thus, one of ordinary skill in the art could not conclude that Applicant was in possession of the claimed system comprising a bioactive agent conjugated to a fluorophore, a polynucleotide encoding a protein of interest and a luciferase, and a substrate for the luciferase. This disclosure does not constitute a representative number of species of the genus in view of the potential breadth and variability in the genus, and so there is a failure to satisfy the written description requirement for the genus.
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.
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 argued that the §102 rejection is defective because Examiner relies on a secondary reference to supply a limitation that is concededly absent from the primary reference (See Remarks dated 4/23/2026, p.4 – Section A). Applicant argues that Su et al. does not provide evidence that the binding in Miller is non-covalent as required by the claims (See Remarks dated 4/23/2026, p.5 top paragraph).
Applicant’s arguments filed April 23, 2026 have been fully considered but they are not found persuasive. The claim requires that the bioactive agent “is capable of binding non-covalently to the protein of interest”. Miller does not identify whether the bioactive bis-arsenical sNIRF is “capable of binding non-covalently to a protein of interest”. The Su reference is relied upon as an evidentiary reference to support the idea that while Miller does not identify the type of binding, Su identifies that most bioactive compounds bind to their protein targets non-covalently, and therefore the Miller bioactive would necessarily be capable of binding non-covalently to a protein of interest.
Applicant argued that the “non-covalent binding” limitation is not merely a desired property, rather it defines a structural and functional relationship between two separately recited components of the claimed system: the bioactive agent of element (a) and the protein of interest encoded by element (b) (See Remarks dated 4/23/2026, p.5 – Section B). Applicant argues that Miller’s system does not meet the structural limitation because Miller’s fluorophore conjugate binds the tetracysteine tag on the luciferase, not a separate protein of interest; and the binding interaction is covalent not non-covalent as required by the claims (See Remarks dated 4/23/2026, p.5 – Section B).
Applicant’s arguments filed April 23, 2026 are not found persuasive. The limitation of claim 1 is attempting to claim a bioactive agent through its functionality, which is a property of the bioactive agent. As discussed above, the Su reference provides evidence that most bioactive compounds find to their protein targets non-covalently. Thus, although Miller does not disclose whether the bis-arsenical sNIRF binds non-covalently to a protein of interest, it would be inherent to Miller that binding to the protein of interest would be non-covalent.
Applicant argued that Miller’s central design principle is constitutive, covalent proximity, and the proximity that drives BRET in Miller is a structural property of the construct, not the result of a binding event between a ligand and a separate target protein (See Remarks dated 4/23/2026, p.6 – Section C.1). Applicant argues that in the claimed system, a BRET signal is generated only when the free bioactive agent-fluorophore conjugate encounters and non-covalently engages the protein of interest, which is a fundamentally different system architecture from Miller’s constitutive, intramolecular BRET (See Remarks dated 4/23/2026, p.6 – Section C.2).
Applicant argues that Miller teaches away from non-covalent, intermolecular BRET, and the teach-away argument is about the design principle that Miller endorses (See Remarks dated 4/23/2026, p.7 – Section D). Applicant further argues that the entire inventive thrust of Miller is to move away from non-covalent intermolecular proximity approach and toward constitutive intramolecular BRET, and is additional evidence that Miller does not anticipate the claimed system, which improves upon the very intermolecular approach that Miller rejected (See Remarks dated 4/23/2026, p.8 top paragraph).
Applicant's arguments filed April 23, 2026 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 function of the bioactive agent, but the limitation 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’s system operates differently than the instantly claimed system, Miller’s teaching does not teach away from the structural requirements of the instant claims.
Applicant argues that Claim 17 is independently not anticipated by Miller because it requires that BRET arise as a consequence of the bioactive agent binding the protein of interest (See Remarks dated 4/23/2026, p.8 – Section E). Applicant argues that Miller does not teach or suggest that BRET is generated as a result of binding of a bioactive agent to a protein of interest (See Remarks dated 4/23/2026, p.8 – Section E).
Applicant’s argument is not found persuasive. Claim 17 does not further limit the structure of the system required by Claim 1. Claim 17 describes a function of
Applicant argues that dependent claims 2-6, 10-11. 14-15 and 18-19 all depend from Claim 1, and because Miller does not anticipate Claim 1 for the reasons set forth above, the rejection of these dependent claims necessarily fails as well (See Remarks dated 4/23/2026, p.8 – Section F).
Applicant’s arguments are not found persuasive for the same reasons discussed above regarding claim 1.
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).
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.
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 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.
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 April 23, 2026. All rejections are maintained.
Conclusion
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/DEEPA MISHRA/Examiner, Art Unit 1657
/LOUISE W HUMPHREY/Supervisory Patent Examiner, Art Unit 1657
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