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 .
Application Status
This action is written in response to applicant’s correspondence received on 2/17/2026. Claims 16 and 20-40 are pending. Claim 16 has been amended. Claims 1-15 and 17-19 have been cancelled. Claims 25-40 have been withdrawn. Claims 16 and 20-24 are currently under examination.
Any rejection or objection not reiterated herein has been overcome by amendment. Applicant’s amendments and arguments have been thoroughly reviewed, but are not persuasive to place the claims in condition for allowance for the reasons that follow. This Office Action is Final.
Claim Rejections - 35 USC § 103 -Maintained/Updated in Response to Amendment
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.
Claim(s) 16 and 20-23 are rejected under 35 U.S.C. 103 as being unpatentable over Yi et al (CA 2947802, of record) in view of Cabantous et al (Nature Biotech. 23(1): 102-107, 2005, of record), Leonetti et al (Proc. Nat. Acad. Sci. USA. 113(25):E3501-8, 2016, of record), and Feng et al (Nature Comm. 8:371, 11 pages, 2017, of record).
Yi taught an androgen receptor-fluorescent reporter system which was a fusion protein comprising AR and green fluorescent protein (GFP). Yi measured the effect of a drug on androgen receptor (AR) localization in PC3 prostate cancer cells by administering the drug to cells expressing the AR-GFP fusion protein. The fusion protein was produced by transfecting the PC3 cells with a recombinant expression construct (see Example 4, Fig. 3c, and brief description of Fig. 3 on pages 20-21).
Yi did not teach a split monomeric NeonGreen fluorescent protein where one component of the split protein was fused to the N-terminus of an androgen receptor in a genetic construct in the genome of a cell and a second component of the split protein was encoded in a vector.
Cabantous taught that GFP tagging of proteins was a powerful method for protein detection and localization but that it had drawbacks because such fusion proteins can misfold or exhibit altered processing (see e.g. abstract). Cabantous taught that an ideal protein tag would be genetically encoded, would work both in vivo and in vitro, would provide a sensitive analytical signal and would not require external chemical reagents or substrates (abstract). Cabantous developed a system in which engineered soluble, self-associating fragments of GFP are used to tag and detect either soluble or insoluble proteins in living cells or cell lysates. One fragment corresponded to the 11th beta strand of GFP and was fused to a target protein to be detected. The other fragment comprised GFP beta strands 1-10 and was expressed from a separate vector independently of the fusion protein (see first sentence of “In vivo whole-cell plate complementation assays” on page 107). Cabantous taught that the split GFP system is simple and does not change fusion protein solubility (abstract). An in vivo colony fluorescence assay using the system reported total protein in agreement with SDS-PAGE (Fig. 3b) and page 104, left column, second full paragraph).
It would have been obvious to one of ordinary skill in the art prior to the effective filing date of the claims to have modified the system Yi by replacing the AR-GFP fusion construct of Yi with the split protein GFP system of Cabantous. Doing so would have been no more than the application of a known technique to a known method ready for improvement to yield predictable results.
The combination of Yi and Cabantous does not account for prostate cancer cell with a modified genome comprising a nucleic acid encoding fusion of the 11th beta strand of GFP to the N-terminus of AR.
However, Leonetti taught an application of the split GFP system in which a nucleic acid encoding the 11th beta strand of GFP is knocked into endogenous genomic copies of genes of interest in a human cell line to produce modified genes encoding fusion proteins comprising an N-terminal domain that is the 11th strand of GFP and a C-terminal domain that is the protein of interest. The second component of the system (GFP beta strand 1-10) was supplied by transduction of the cells with a separate lentiviral expression vector (see last full sentence on page E3502).
Furthermore, Leonetti taught the broad applicability of their methods to endogenous genes within a genome (Abstract and Discussion). Specifically, Leonetti teaches that:
“our results establish GFP11 RNP knockin as a powerful strategy for the fast and efficient generation of endogenously tagged human cell lines. Our approach has several key advantages. First, contrary to designs that require the multistep preparation of HDR targeting vectors, all of the protocols we describe require no molecular cloning and can be carried out very rapidly and in large-scale format. Second, Cas9 RNP electroporation and ssDNA templates enable very high knockin efficiency while minimizing off-target cleavage or nonspecific tag integration (14). Third, the GFP11 system provides a simple solution for the study of low-abundance proteins because knockin of GFP11 repeats increases fluorescence signal. Fourth, GFP is a particularly versatile tool that enables the study of both protein localization and protein–protein interactions. Finally, the utility of endogenously tagged cell lines is evident, allowing the function of a protein to be characterized under the control of native regulators of gene expression and without disturbing endogenous interaction stoichiometry. In this respect, the small size of the GFP11 cassette is advantageous because its introduction into a locus of interest is relatively seamless, minimizing perturbation of the surrounding genomic structure. Together, the methods presented here provide scalability, specificity, versatility, and selectability and pave the way for the genome-scale construction of human cell lines tagged with GFP at endogenous loci,” (Discussion, first paragraph).
Thus, Leonetti teaches ample motivation to tag endogenous proteins using split fluorescent reporter systems, and furthermore teaches the feasibility and predictability of doing so (Discussion, first paragraph).
It would have been obvious to one of ordinary skill in the art prior to the effective filing date of the claims to have used the approach of Leonetti in modifying the PC3 cell-based system of Yi. As above, doing so would have been no more than the application of a known technique to a known method ready for improvement to yield predictable results. This would have led to a system comprising a PC3 prostate cancer cell comprising a modified genomic AR gene with a nucleic encoding the 11th beta strand of GFP fused to the 5’-end of an endogenous AR gene to encode an AR with the GFP beta strand fused to the AR N-terminus, and a lentiviral vector comprising an expression cassette for the remaining beta strands 1-10 of GFP. The system rendered obvious by the combined references is structurally indistinguishable from that which is claimed, therefore it is considered to inherently comprise the same performance characteristics (“produce a detectable fluorescent signal indicative of the level of the androgen receptor in the prostate cancer cell”). Furthermore, a practitioner would be highly motivated to use the endogenous tagging system of Leonetti given Leonetti’s strong motivational teachings with regards to the utility, ease, feasibility, and research implications of using endogenous loci (Discussion, first paragraph).
This combination does not account for a reporter system using a split monomeric NeonGreen fluorescent protein.
Feng taught that self-complementing split fluorescent proteins (FPs) are split FP constructs in which the two fragments can associate by themselves to form a fully functional FP without the assistance of other protein–protein interactions. By fusing one fragment on a target protein and detecting its association with the other fragment, these constructs have demonstrated powerful applications in the visualization of subcellular protein localization, quantification of protein aggregation, detection of cytosolic peptide delivery, identification of cell contacts and synapses, as well as scaffolding protein assembly. See abstract and first sentence on page 2. Such split FP systems included GFP1–10/11 (the system of Cabantous and Leonetti) where the split system relies on fusing the GFP 11th beta strand to a target protein and coexpressing a construct comprising the first 10 GFP beta strands in order to provide complementation and fluorescence. Feng disclosed a new yellow–green-colored mNeonGreen21–10/11 (mNG2) that has an improved ratio of complemented signal to the background of FP1–10-expressing cells as compared to GFP1–10/11. See sentence bridging pages 2 and 5. Furthermore, Feng teaches that their split yellow green are monomeric, and therefore teaches split monomeric NeonGreen fusion proteins (page 5, left column, third paragraph).
It would have been obvious to one of ordinary skill in the art prior to the effective filing date of the claims to have substituted the mNeonGreen21–10/11 (mNG2) split yellow-green fluorescence system of Feng, which is a split monomeric NeonGreen fluorescent protein, for the split GFP system used by Cabantous and Leonetti to modify the PC3 system of Yi. One would have been motivated to do so in order to obtain the advantage of the improved ratio of complemented signal to background disclosed by Feng.
Claim 24 is rejected under 35 U.S.C. 103 as being unpatentable over Yi et al (CA 2947802), Cabantous et al (Nature Biotech. 23(1): 102-107, 2005), Leonetti et al (Proc. Nat. Acad. Sci. USA. 113(25):E3501-8, 2016) and Feng et al (Nature Comm. 8:371, 11 pages, 2017), as applied to claims 16 and 20-23 above, and further in view of Campana et al (Semin Reprod Med. 2015 May;33(3):225-34. doi: 10.1055/s-0035-1552989. Epub 2015 Jun 2).
The teachings of Yi, Cabantous, Leonetti, and Feng are summarized above an render obvious instant claim 23 wherein the prostate cancer cell of the androgen receptor-fluorescent protein reporter system is a PC3 cell.
These references did not teach an LnCaP prostate cancer cell.
Campana reviewed cell based assays for screening androgen receptor ligands. Campana taught that yeast cell and mammalian immortalized cell line-based bioassays existed, and the mammalian systems are relatively easy to culture and maintain, and show higher sensitivity than the yeast system. Numerous mammalian cell lines, including prostate carcinoma cells such as LNCaP and PC3 have been used. See last full paragraph on page 7.
It would have been obvious to one of ordinary skill in the art prior to the effective filing date of the claims to have substituted LNCaP prostate cancer cells for the PC3 cells of Yi in the method of Yi as modified. Doing so would have been no more than the simple substitution of one known element for another to obtain predictable results in view of the fact that both the PC3 and LNCaP prostate cancer cell lines were known to be useful in assays of androgen receptor ligand binding.
Thus the invention as a whole was prima facie obvious.
Response to Arguments
The Applicant’s arguments submitted 2/17/2026 have been considered but are not persuasive. As an initial matter, the Applicant argues that the incorporation of previous claims 18-19 into claim 1 obviate the original rejection because claim 19 was not part of the original rejection. This argument is not persuasive because claim 19 was originally rejected in the non-final action mailed 11/18/2025, as were its limitations. Thus, incorporation of the phrase “monomeric NeonGreen” in place of “split yellow green” is not sufficient to obviate the rejection.
To further elaborate on this point, Feng is cited as teaching a monomeric mNeonGreen in the 103 rejection (see above). Thus, Feng does teach a monomeric NeonGreen fluorescent protein, as discussed in the rejection mailed 11/18/2025 (“mNeonGreen2”). Furthermore, even though the “yellow green” limitation is presently deleted from the claim set, claim 16 is now broadly drawn to “a split monomeric NeonGreen fluorescent protein,” which would also include the yellow green monomeric NeonGreen fluorescent protein taught by Feng, as previously cited in the rejection mailed 11/18/2025. Thus, claim 19 was addressed in the rejection, and its incorporation into claim 16 does not obviate the rejection.
Additionally, the specification does not clearly define “monomeric NeonGreen” fluorescent protein (for instance, the term “monomeric” does not appear to be recited in the specification, and is only previously recited in claim 19). When turning to the specification, the Applicant presents, for instance, Example 2 (see page 44 of specification), which recites “mNeonGreen2,” which appears to be the same fluorescent protein taught by Feng (“mNG2” in Feng, e.g., see the Caption under Figure 1, which describes a split “mNeonGreen2,” or”mNG2”). Additionally, the specification directly references Feng (paragraph 171)., and thus appears to recite the same “mNeonGreen2” protein of Feng. Thus, the teachings of Feng regarding the split monomeric NeonGreen fluorescent protein read on instant claim 16, as previously discussed in the non-final rejection mailed 11/18/2025, as the Applicant appears to be reciting the same protein as Feng (i.e., “mNeonGreen2” as taught by Feng, above, and in Example 2 of instant specification).
The Applicant argues that the use of LNCaP cells as taught by Campana is unpredictable with the reporter system. This argument is not found to be persuasive. Firstly, the Applicant is making arguments of counsel which are not supported by actual evidence. MPEP 716.01(c) makes clear that arguments of counsel cannot take the place of evidence in the record. Secondly, Campana teaches that numerous cell lines have been developed and tested for androgen reporter assays, including LNCaP, where LNCaP cells have been used by numerous groups for numerous purposes (see page 7, final two paragraphs). Thus, Campana teaches that LNCaP was a known cell line used for modeling. The evidence on record therefore favors predictability when using an established cell model such as LNCaP, where the Applicant offers no argument except the argument of record.
The Applicant argues that they extensively characterized the cells and reporter assay. However, the mere fact that the Applicant characterized a system rendered obvious by the prior art in a known cell line does not rise to the level of invention. For instance, the Applicant’s characterization of the system and the cell line do not support the claim that there is unpredictability when using LNCaP cells.
The Applicant argues that Campana teaches that ARE, and not AR, is tagged as a reporter system. This argument is not persuasive because Campana is not relied upon for teaching AR-tagging, as Yi has already taught such AR-tagging (see 103 rejection of claim 16). The fact that Campana teaches additional tagging is therefore irrelevant given that Campana is not relied upon for teaching the tagging of the AR receptor.
Conclusion
Applicant's amendment necessitated the new ground(s) of rejection presented in this Office action. Accordingly, THIS ACTION IS MADE FINAL. See MPEP § 706.07(a). 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.
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/D.C.R./Examiner, Art Unit 1635
/KIMBERLY CHONG/Primary Examiner, Art Unit 1636