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and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114.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 01/26/2026 has been entered.
Claim 13 has been amended. Claim 14 has been newly canceled and no claims have been newly added.
Claims 13, 15-20 and 22 are currently pending.
Claim 16 is withdrawn from further consideration pursuant to 37 CFR 1.142(b), as being drawn to a nonelected specie (skeletal muscle myoblasts-SMBs), there being no allowable generic or linking claim. Applicant timely traversed the restriction (election) requirement in the reply filed on 10/14/2024.
Claims 13, 15, 17-20 and 22 have been examined on their merits.
Rejections and/or objections not reiterated from previous office actions are hereby withdrawn due to amendment. The following rejections and/or objections are either reiterated or newly applied. They constitute the complete set presently being applied to the instant application.
Claim Rejections - 35 USC § 103
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. Considering objective evidence present in the application indicating obviousness or nonobviousness.
This application currently names joint inventors. In considering patentability of the claims the examiner presumes that the subject matter of the various claims was commonly owned as of the effective filing date of the claimed invention(s) absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and effective filing dates of each claim that was not commonly owned as of the effective filing date of the later invention in order for the examiner to consider the applicability of 35 U.S.C. 102(b)(2)(C) for any potential 35 U.S.C. 102(a)(2) prior art against the later invention.
Claim(s) 13, 15 and 17-20 are rejected under 35 U.S.C. 103 as being unpatentable over Rhee et al (BMC Biotechnology 2009) in view of Teng et al (eLIFE 2016, from IDS filed 07/07/2023), Sadick et al (Scientific Reports 2016), Walev et al (PNAS 2001-from IDS filed 07/07/2023) and Tsuruo et al (US 2003/0022878-newly cited).
Regarding claims 13, 15, 17-20, Rhee teach a method of detecting protein stem markers in live stem cells, specifically human induced pluripotent stem cells, and isolating these cells from a mixed population by targeting intracellular stem cell markers with a combination of molecular beacons and dye-labeled antibodies through flow cytometry (abstract, pages 6-7 Discussion and Figure 5). Although they use carcinoma cells as a model, the same approach is taught to be applicable to detecting iPS (induced pluripotent stem cells) (page 6 Discussion). Detection of stem cell markers in undifferentiated cells is performed by treating the cells with streptolysin O (SLO) and then treating them with fluorescent dye-conjugated antibodies followed by flow cytometric analysis (page 4, column 2, page 5, column 2-page 6, Figure 5). FACS is a specific type of flow cytometry used for the analysis (page 9 column 2). Detecting multiple stem cell markers for the isolation of iPS cells is suggested as necessary and includes Oct-4 and Nanog (page 7 column 1).
Rhee do not specifically exemplify their method using a primary antibody against a target intracellular protein of the target cells and a secondary antibody that is fluorescent and dye-conjugated. Rhee are silent with regard to the size of the fluorescent dye-conjugated antibody and do not teach the claimed fluorescent dye.
Teng disclose a method for labeling proteins inside living mammalian cells using external fluorophores and Streptolysin O. The Streptolysin O forms temporary pores in the cell membrane and allows delivery of virtually any fluorescent probes ranging from labeled IgGs to small ligands, with high efficiency, and with probes ranging in size from small molecules to large proteins up to 150 kDa (abstract, pages 1-2 Introduction). Alexa and FITC are both indicated as fluorescent dyes that are suitable for use (page 4 and page 6). This general method allows for the targeting of intracellular proteins (page 2). A variety of cell viability tests are performed after treatment with SLO to ensure that the antibody labeled cells have intact membranes, are able to divide, respond normally to signaling molecules and maintain healthy organelle morphology (abstract).
Sadick disclose methods for protein characterization of intracellular target-sorted cell populations and prepare samples for FACS by fluorescently labeling intracellular proteins that are characteristic of the target cell type. This allows subpopulations to be targeted more specifically with a broad array of available antibodies (page 2 first paragraph). Immunolabeling and sorting based on intracellular markers provides investigators with vastly more cell type specific targets and by taking advantage of commercially available antibodies this can be applied to any cell or animal model of interest (paragraph spanning pages 5-6). While mRNA abundance has been used as a proxy for protein abundance, which assumes that mRNA presence is the major factor determining the amount of protein made, overall mRNA abundance has a low correlation with protein synthesis. Thus, protein characterization provides the most concrete representation of cell phenotype (page 6). Suitable antibodies for sorting studies include those with a secondary antibody conjugated to Alexa Fluor 488 (page 8, Materials and Methods).
One of ordinary skill in the art would have been motivated to use antibody labeled probes to detect intracellular proteins in the method of Rhee because Teng and Sadick teach and suggest that it is beneficial to detect intracellular proteins in a desired cell type using antibody probes. Teng suggest that doing this in a manner that allows the cells to remain viable is beneficial and desirable and does not require the fixation step that was previously thought to be required. Sadick specifically points out the benefits and advantages of detecting intracellular protein rather than mRNA as protein characterization provides the most concrete representation of cell phenotype (page 6). The use of a secondary antibody conjugated with a fluorescent dye up to about 150 kD, such as Alexa Fluor 488, would have been motivated by Sadick as they teach that these are suitable antibodies for sorting with FACS and Teng also suggest that Alexa dyes are beneficial for use with fluorescent antibody probes up to 150 kD when used with SLO and FACS as well. One of ordinary skill in the art would have had a reasonable expectation of success because Teng teach that variety of cell viability tests are performed after treatment with SLO to ensure that the antibody labeled cells have intact membranes, are able to divide, respond normally to signaling molecules and maintain healthy organelle morphology (abstract).
One of ordinary skill in the art would have been motivated with a reasonable expectation of success to use the method of Rhee to detect and isolate human induced pluripotent stem cells (iPS cells) because Rhee specifically state that although they use carcinoma cells as a model, that the same approach is applicable to detecting iPS (induced pluripotent stem cells) (page 6 Discussion). The use of an antibody probe to labeled to detect Nanog would have been obvious as suggested above by Teng and Sadick and because this is the protein expressed by iPS cells.
Rhee do not teach wherein streptolysin O is used at a treatment concentration of 2.5 U/mL or more and 5 U/mL or less.
Walev teach a method of delivering proteins into living cells by reversible membrane permeabilization with streptolysin-O (SLO) to deliver biologically active molecules to intracellular targets (Title, page 3185, column 2, Materials and Methods- page 3186-column 1, second paragraph). Walev teach that low doses of SLO are used to provide efficient membrane repair in cells in order that the cells remain viable (live)(page 3186, column 1). Walev teach that the goal is to identify the concentration that effects permeabilization of 60-80% of the cells within 10-15 minutes and that the required concentration varies depending on cell target and must be determined by titration (page 3186, column 1). Resealing of the cells after treatment with SLO is by addition of medium with Ca2+ (page 3186, column 2).
Tsuruo disclose a method of permeabilizing cells with 5 U/mL to enable desired substances to be incorporated into cells (page 4 para 57).
One of ordinary skill in the art would have been motivated to optimize the concentration of SLO in the method of Rhee with a reasonable expectation of success using the value provided by Tsuruo as a starting point to arrive at the claimed concentrations because Walev teach that the optimal SLO concentration varies depending on cell target and must be determined by titration.
Generally, differences in concentration or temperature will not support the patentability of subject matter encompassed by the prior art unless there is evidence indicating such concentration or temperature is critical. “[W]here the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimum or workable ranges by routine experimentation.” In re Aller, 220 F.2d 454, 456, 105 USPQ 233, 235 (CCPA 1955) (MPEP 2144.05).
The selection of specific concentrations clearly would have been a routine matter of optimization and experimentation on the part of the artisan of ordinary skill, said artisan recognizing that the amount and viability of the cells permeabilized would have been affected by these concentrations as suggested by Walev.
Therefore, the combined teachings of Rhee et al, Teng et al, Sadick et al, Walev et al and Tsuruo et al render obvious Applicant’s invention as claimed.
Claim(s) 22 is rejected under 35 U.S.C. 103 as being unpatentable over Rhee et al (BMC Biotechnology 2009) in view of Teng et al (eLIFE 2016, from IDS filed 07/07/2023), Sadick et al (Scientific Reports 2016), Walev et al (PNAS 2001-from IDS filed 07/07/2023) and Tsuruo et al (US 2003/0022878-newly cited) as applied to claims 13, 15 and 17-20 above, and further in view of Sayre et al (US 2005/0170506-from IDS filed 07/07/2023).
Regarding claim 22, the combined teachings of Rhee, Teng, Sadick, Walev and Tsuruo render obvious the claimed method as described above, but do not specifically include wherein the target cells are suspended in calcium chloride-added cell culture to regenerate the cell.
Sayre teach wherein cells treated with SLO are then resuspended in a solution of calcium chloride in preparation media (culture media) (page 11-12 para 132).
One of ordinary skill in the art would have been motivated to resuspend the SLO- treated cells of Rhee with a culture media containing calcium chloride because Sayre indicate that this is suitable and beneficial for the culture of SLO-treated cells. One of ordinary skill in the art would have had a reasonable expectation of success because Teng specifically indicate that a complete supplemented medium can be used to reseal cells treated with SLO (page 2) and Rhee indicate that it is desirable to isolate the iPS cells in a live, viable form. Additional motivation and a reasonable expectation of success are provided by Walev as they also disclose resealing of cells after treatment with SLO is by addition of medium with Ca2+ (page 3186, column 2).
Therefore, the combined teachings of Rhee et al, Teng et al, Sadick et al, Walev et al, Tsuruo et al and Sayre et al render obvious Applicant’s invention as claimed.
Response to Arguments
Applicant's arguments filed 01/26/2026 have been fully considered but they are not persuasive. Applicant’s arguments have been addressed in so far as they relate to the rejections above.
Applicant argues that a skilled artisan would not have any motivation to combine the teachings of Rhee, Teng and Sadick to arrive at the presently claimed invention. Applicant asserts that excellent effects are obtained when SLO is treated at a concentration of 2.5 U/mL or more and 5 U/mL or less. Applicant asserts that at concentrations exceeding 5 U/mL (10 U/mL and 25 U/mL) it was observed that cell death occurs abruptly or increases significantly and point to figures 2A and 2B as evidence. Applicant points to figures 3A and 3B as evidence that when SLO is treated at 5 U/mL that target cells could be isolated with efficiency equivalent to that obtained in cases where cells were killed prior to isolation.
This is not found persuasive. The obviousness rejection has been modified to include the teachings of Walev and Tsuruo which render obvious the optimization of the SLO concentration and demonstrate that SLO concentrations that fall within Applicant’s claimed concentration are well known in the prior art.
Applicant argues that in contrast with the present invention that Teng teaches that the working concentration of SLO is 25-200 U/mL which corresponds to a markedly higher concentration compared to the present invention. Applicant also argues that Rhee discloses a lower SLO concentration compared to the present invention of 0.2-2 U/mL. Applicant asserts that according to the present invention that at concentrations of 2.5 U/mL or less that it is not possible to achieve a degree of permeability sufficient to allow antibodies to permeate.
This is not found persuasive. The test for obviousness is not whether the features of a secondary reference may be bodily incorporated into the structure of the primary reference; nor is it that the claimed invention must be expressly suggested in any one or all of the references. Rather, the test is what the combined teachings of the references would have suggested to those of ordinary skill in the art. See In re Keller, 642 F.2d 413, 208 USPQ 871 (CCPA 1981).
The obviousness rejection is based on the modification of the Rhee method which does not require high SLO concentrations. In addition the teachings of Walev and Tsuruo provide the motivation to optimize the SLO concentrations to around 5 U/mL as described above.
Applicant argues that even when Rhee and Teng are considered in combination that it is by no means obvious whether cells can be maintained in a living state without affecting cell physiology. Applicant asserts that it is not obvious without experimentation whether cells can be maintained in a living state without affecting cell physiology. Rhee uses Triton-X to deliver antibodies into cells and not SLO and it is impossible to expect that cells could be isolated in a living state. Teng is not drawn to the claimed invention and in contrast to the claimed invention.
This is not found persuasive. In response to applicant's arguments against the references individually, one cannot show nonobviousness by attacking references individually where the rejections are based on combinations of references. See In re Keller, 642 F.2d 413, 208 USPQ 871 (CCPA 1981); In re Merck & Co., 800 F.2d 1091, 231 USPQ 375 (Fed. Cir. 1986).
In the current case the motivation to modify the method of Rhee is provided by the teachings of Sadick, Walev, Tsuruo as well as Teng. The effects of SLO on the permeabilization of a living cell is well known in the art as shown by the teaching of Walev.
Applicant argues that Sadick does not remedy the deficiencies Of Rhee and Teng as Sadick does not describe SLO at all.
This is not found persuasive because the new rejections provided above now include the teachings of Walev and Tsuruo which address the optimization of the concentration of SLO used in methods of permeabilizing cells as described above.
Applicant argues that because Sayre includes no experiments in which SLO is treated on iPS cells and does not disclose or suggest at all the delivery of antibodies into cells other than cell extracts that it is not easy to expect that cells could be isolated in a living state. Applicant asserts there are too many things that can affect the results and thus the results are difficult to predict in advance and can only be confirmed through actual experimentation. Applicant points to example 7 in Sayre that includes a high SLO concentration as evidence that Sayre does not teach the claimed SLO concentration.
This is not found persuasive. The test for obviousness is not whether the features of a secondary reference may be bodily incorporated into the structure of the primary reference; nor is it that the claimed invention must be expressly suggested in any one or all of the references. Rather, the test is what the combined teachings of the references would have suggested to those of ordinary skill in the art. See In re Keller, 642 F.2d 413, 208 USPQ 871 (CCPA 1981). In the current case, Sayre discloses the beneficial use of calcium chloride to reseal cells that have been treated with SLO as described above.
Applicant argues that Rhee only teaches the use of SLO to enable entry of the molecular beacon, a double labeled antisense oligonucleotide probe, into the cells, and there is no indication that SLO can be used for visualizing antibodies which differ in size and structure from molecular beacons.
This is not found persuasive. As described above, the obviousness rejection also includes the teachings of Teng and Sadick. Teng discloses the use of SLO for the labeling of proteins inside living cells using external fluorophores and Sadick discloses the benefits of intracellular protein with labeled antibodies over the reliance on mRNA alone as described above.
Applicant argues that even if a molecular beacon has a sequence that is specific to a target it may not effectively bind to the target mRNA in the actual cellular environment and point to Figure 3A in Rhee as evidence where 13 molecular beacons were designed and tested with only weak signals for MB1 and MB5-MB13. Applicant asserts that these beacons failed to access the target sequences and therefore a person of ordinary skill in the art reviewing Rhee would recognize that designing and optimizing molecular beacons is highly challenging and not easy to extend to antibodies and expect positive results.
This is not found persuasive. As described above, the obviousness rejection now includes the teachings of Teng and Sadick. Teng discloses the use of SLO for the labeling of proteins inside living cells using external fluorophores and Sadick discloses the benefits of intracellular protein with labeled antibodies over the reliance on mRNA alone as described above.
Applicant argues that Rhee does not conduct any experiments on induced pluripotent stem cells (iPSCs) or suggest that antibodies can be successfully delivered into iPSCs which are larger than molecular beacons.
This is not found persuasive. Although Rhee uses carcinoma cells as a model, the same approach is taught to be applicable to detecting iPS (induced pluripotent stem cells) (page 6 Discussion) and Sadick discloses the benefits of intracellular protein with labeled antibodies over the reliance on mRNA alone as described above.
In view of the foregoing, when all of the evidence is considered, the totality of the rebuttal evidence of nonobviousness fails to outweigh the evidence of obviousness.
Conclusion
No claims are allowed.
The prior art made of record and not relied upon is considered pertinent to applicant's disclosure.
Klaus et al., “Identification of Human Cytomegalovirus Genes Involved in Down-Regulation of MHC Class I heavy Chain Expression”, WO 9804285 A1, (specifically page 16 lines 1-3 which discloses SLO concentration of 4 U/mL).
Any inquiry concerning this communication or earlier communications from the examiner should be directed to LAURA J SCHUBERG whose telephone number is (571)272-3347. The examiner can normally be reached 8:30-5:00 EST.
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LAURA J. SCHUBERG
Primary Examiner
Art Unit 1631
/LAURA SCHUBERG/Primary Examiner, Art Unit 1631