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 .
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and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114.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 6/9/2026 has been entered.
Application Status
This action is written in response to applicant’s correspondence received on 6/9/2026. 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 Claims 25-40 have been withdrawn.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.
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).
Furthermore, Yi teaches that their fluorescently tagged proteins are responsive to AR stimulation by R1881 and repression by AIL, as the introduction of such compounds affects the localization of the AR (see Figure 3C). Thus, Yi teaches evidence that the fluorescent tagging of AR does not interfere with the ability of the AR to localize in response to exogenous signals (Figure 3C). Yi therefore teaches predictability with respect to the functionality of an AR protein and its interaction with stimulants and repressors of androgen when it is tagged with a fluorescent reporter (Figure 3C).
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, where the tagged androgen receptor retains comparable activity with a native, untagged protein.
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).
Furthermore, Leonetti teaches that:
“Tagging proteins with a functional sequence is a powerful way to access function, and inserting tags at endogenous genomic loci allows the preservation of a near-native cellular background. To characterize the cellular role of human proteins in a systematic manner and in a native context, we developed a method for tagging endogenous human proteins with GFP that is both rapid and readily applicable at a genome-wide scale. Our approach allows studying both localization and interaction partners of the protein target,” (page 1, “Significance”).
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). Leonetti taught that their method is useful to preserve native cellular background, and to study both the localization and interaction partners of endogenous proteins (page 1, “Significance”). Thus Leonetti teaches that the function of tagged proteins are not hindered by their method of tagging, where the natural state of the cell is preserved and the interaction partners of a given protein remain intact (“Significance,” page 1). Leonetti therefore teaches the high predictability of a tagged protein retaining its functional interactions as compared with an untagged protein, as this is a key advantage of using their tagging system as discussed above.
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” and “the androgen receptor in the fusion protein responds to stimulus by an androgen, repression by an antiandrogen, and repression by an androgen receptor degrader, binds to an androgen response element, and regulates AR target gene expression in the prostate cancer cell comparably to untagged endogenous 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, where Leonetti teaches that such tagging methods are highly predictable and allow for native protein interactions (Discussion, first paragraph, and “Significance,” page 1).
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. Yi also teaches LNCap cells as known prostate cancer cell lines, but reduced to practice PC3 cells as opposed to LNCap cells using their fluorescent tagging system (page 20, final paragraph). Yi teaches that LNCap cells are AR-positive cells, and therefore express the androgen receptor (page 27, first paragraph).
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. Furthermore, the practitioner would be motivated to apply the teachings of an endogenous tagging system such as those taught by Leonetti, as a practitioner of ordinary skill in the art would understand that AR-positive cells such as LNCap endogenously express AR; therefore, to study/tag AR in LNCap cells, a practitioner would be motivated to use the seamless, effective, and simple tagging system taught by Leonetti (above) to LNCap cells to study these known model cells.
Thus the invention as a whole was prima facie obvious.
Response to Arguments
The Applicant’s arguments submitted 6/9/2026 have been considered but are not persuasive.
The Applicant argues that their amendments to the claims are sufficient to overcome the rejection. This argument is not persuasive. As an initial matter, the rejection using the original art is maintained, where the Applicant’s amendments are not sufficient to overcome the original art rejection (103 rejection, above).
Regarding the Applicant’s amendments, the Applicant has amended claim 16 to include features of the androgen fusion protein, where the features presently recited in claim 16 (e.g., response to stimulant, repression, degradation, gene regulation, etc.) are recited to be comparable to an untagged AR protein. The inclusion of such inherent features of the AR-fusion protein, in the sense that it functions comparably with an untagged AR protein, does not overcome the art rejection which is based upon motivational teachings to apply the split fusion protein to AR, which itself has already been tagged fluorescently for study as taught by the prior art. Furthermore, when tagging a protein, a practitioner has a reasonable expectation of success where the tagged protein retains interaction partners, localization, and functionality as taught in the art by Leonetti (page 1, Significance, as discussed in the 103 rejection, above). Given the motivational teaching of the art to tag AR, where such strategies of tagging and the tags themselves are already known in the art, the prior art teaches sufficient motive with a reasonable expectation of success to arrive at the claimed invention, where the new amendments are only drawn to inherent characteristics of the AR-tagged fusion protein rendered obvious by the prior art.
The Applicant argues that they have performed testing to show that tagged AR receptor behaves indistinguishably from native, untagged AR, as mRNA and CHiP assays have demonstrated that mRNA and binding associations are comparable between tagged-AR and untagged AR. Applicant argue that the reporter system allows measurement of endogenous AR protein levels and localization, and that the cited prior art does not show that such a reporter system would not perturb or interfere with AR function. This argument is not persuasive.
As an initial matter, the prior art references supply ample motivational teachings for their combination, where prior art such as Leonetti teaches that endogenous tagging of proteins offers several advantages including enhanced understanding of native proteomics, simplicity, and the ability to study proteomics/genetics in the native context of the cell (Abstract, Discussion first paragraph, “Significance”). Even if it were argued that the Applicant has discovered a novel property of tagging AR proteins, where the novel property is that the tag does not interfere with the native protein’s interactions with other molecules, which the Office does not concede, the fact that the inventor is claiming to have recognized another advantage which would flow naturally from following the suggestion of the prior art cannot be the basis for patentability when the differences would otherwise be obvious. See Ex parte Obiaya, 227 USPQ 58, 60 (Bd. Pat. App. & Inter. 1985).
Furthermore, the art in fact teaches that there is high predictability of such endogenous tagging methods, where the native protein retains its functionality. For instance, Leonetti teaches that:
Tagging proteins with a functional sequence is a powerful way to access function, and inserting tags at endogenous genomic loci allows the preservation of a near-native cellular background. To characterize the cellular role of human proteins in a systematic manner and in a native context, we developed a method for tagging endogenous human proteins with GFP that is both rapid and readily applicable at a genome-wide scale. Our approach allows studying both localization and interaction partners of the protein target,” (page 1, “Significance”).
Thus, not only does Leonetti teach a strong motivational teaching to tag endogenous proteins, they also teach that doing so yields proteins which can be studied both in the context of their localization and their ability to interact with other targets (above). Thus, Leonetti teaches that their reporter method allows for “precise measurement of dynamic endogenous protein levels and localization,” contrary to the Applicant’s assertion because Leonetti teaches that their method is predicable and useful. There is no evidence to suggest the AR-tagged protein would not function comparably to an untagged protein. To the contrary, there is strong evidence given by Leonetti to suggest that such reporter systems are specifically designed to yield functional proteins where their interactions, binding partners, and cellular localization can be studies in a near-native cellular context (above).
Furthermore, 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 to include the newly recited claim limitations, namely, that the tagged and untagged AR protein function comparably.
Regarding claim 24, the Applicant argues that Yi reduced to practice PC3 cells, which are AR-negative, and does not teach LNCap cells. This argument is not persuasive. As an initial matter, Yi does teach LNCap cells as known cell models, but reduced to practice their fluorescent system in AR-negative PC3 cells. However, this does not teach away from or preclude a practitioner from studying other well-known prostate cancer cell models such as LNCap cells, or adopting the teachings of the art to such LNCap cells. Given that prior art references such as Leonetti teach that there are numerous advantages to using endogenous tagging, and that Yi teaches that LNCap cells are AR-positive (i..e,. express AR endogenously), and further that Campana teaches that LNCap cells are known prostate carcinoma cell lines useful for studying/screening androgen ligands, the practitioner would certainly be motivated to use an endogenous tagging system for such cells as LNCap cells because they would understand that LNCap cells endogenously express AR, and would therefore apply the techniques of Leonetti and the above cited art references. In other words, the AR-positive LNCap cells comprise endogenous AR, and so an endogenous tagging system would be used as opposed to the reporter constructs of PC3.
The Applicant argues that Campana does not reliably predict whether endogenous tagging of AR would yield functionally comparable cells as untagged cells, as Campana uses a different tagging strategy. This argument is not persuasive. Campana is relied upon simply to teach that LNCap cells are known cell models useful for androgen ligand screening. Leonetti is relied upon for teaching techniques such as endogenous tagging, where furthermore Leonetti teaches a high degree of predictability with their method, apart from teaching ample motivation to apply their method “genome-wide” which would include the known AR protein.
Furthermore, Yi teaches that their fluorescently tagged proteins are responsive to AR stimulation by R1881 and repression by AIL, as the introduction of such compounds affects the localization of the AR (see Figure 3C). Thus, Yi teaches evidence that the fluorescent tagging of AR does not interfere with the ability of the AR to localize in response to exogenous signals (Figure 3C). Yi therefore teaches predictability with respect to the functionality of a fluorescently tagged AR and its response to stimulants and repressors (R1881 and AIL, Figure 3C).
Finally, regarding the argument in general that the prior art does not teach that a tagged AR would functional comparably with an untagged AR, this argument is not persuasive. MPEP 2143.02 teaches that obviousness does not require absolute predictability, but only a reasonable expectation of success is required. Leonetti and the prior art cited supply a reasonable prediction of success, as discussed above with regards to endogenous tagging of proteins and whether or not such tagging will interfere or perturb their ability to localize or act as binding partners (above, Leonetti “Significance”).
Conclusion
Any inquiry concerning this communication or earlier communications from the examiner should be directed to DOUGLAS CHARLES RYAN whose telephone number is (571)272-8406. The examiner can normally be reached M-F 8AM - 5PM.
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/D.C.R./Examiner, Art Unit 1635
/RAM R SHUKLA/Supervisory Patent Examiner, Art Unit 1635
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