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and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114.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 06/11/2026 has been entered.
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.
Claims 1, 7, 9, 15, 50, and 51 are rejected under 35 U.S.C. 103 as being unpatentable over Kim et al. (Application of single-cell RNA sequencing in optimizing a combinatorial therapeutic strategy in metastatic renal cell carcinoma, 2016) in view of Kazuo et al. (JP 2014-176353 A) and Boj et al. (Organoid Models of Human and Mouse Ductal Pancreatic Cancer, 2015)
Regarding Claims 1 and 15: Kim teaches use of single cell RNA sequencing (RNA-seq) to compare the expression profiles from biopsied metastatic renal cell carcinoma (mRCC), patient-derived xenografts (PDX) and primary renal cell carcinoma (pRCC) (pg 5, Single-cell RNA sequencing and quality assessment for expression profiling) and used single-cell dissociation of xenografted tumor specimens for both single-cell RNA sequencing (pg 11, Single-cell RNA sequencing and processing) and for short-term culture (pg 12, Primary in vitro short-term culture) This reads on the claimed method of generating an ex-vivo cell based system from a single-cell dissociated tissue sample obtained from a subject and performing single-cell RNA sequencing on said tissue sample. This further reads on the claimed method of the original tissue sample being a tumor or metastatic tumor tissue sample. Lastly, this reads on the method of claim 15 of an ex-vivo cell-based system derived by the method of claim 1.
Kim further teaches use of basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) in the use of their xenograft-based short term culture (pg 12, Primary in vitro short-term culture) which reads on the claimed method of use of the addition of growth factors or cell signaling molecules in the media of the ex vivo cell-based model. Kim fails to teach the claimed method of use of WNT7B or WNT10A in the media in addition to co-culture with fibroblasts.
Kazuo et al. teaches a method of expanding human hematopoietic stem cells (0001) with use of a medium comprising growth or expansion factors, chemokines, cytokines, and other factors to promote cell survival and proliferation. (0033) Kazuo further specifies use of Wnt7b as a factor used to increase proliferation and cell survival (0033) which reads on use of Wnt7b or Wnt10A or a combination thereof in the culture media. Both Kim and Kazuo fail to teach use of the harvest of PDAC tumors.
Boj et al. teaches a method of isolating murine primary pancreatic ductal tumors and establishing an organoid model in culture of said tumors to show in vitro how pancreatic ductal adenocarcinoma progresses. (Pg 327, Tumor-Derived Organoids Provide a Model for Murine PDA Progression)
It would have been obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to combine the teaching of Boj of use of murine PDA tumors in primary culture with the single cell dissociation method taught by Kim and the media factors and co-culture method as taught by Kauzo to create a culture method with cells derived from a PDAC tumor. One would have had motivation and a reasonable expectation of success based on the teachings of Boj, who detail a method of extraction of primary murine PDAC tumors which were used to establish organoid models in culture.
It further would have been obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to combine the sequence-based analysis and culture method of Kim with the culture media factors and co-culture method as taught by Kauzo to create an ex vivo cell culture model comprising Wnt7Bb or Wnt10A in the media comprising co-culturing the tissue-based cell sample with fibroblasts. One would have been motivated and had a reasonable expectation of success at doing so based on the teachings of Kazuo, who state that the addition of growth factors can increase cell proliferation, amplification and survival of cells in culture and that co-culture can aid in proliferation.
Regarding Claims 7 and 9: Kim et al. teaches use of xenograft tumor specimens being dissociated into a single cell suspension and being cultured in neurobasal media supplemented with N2, bFGF, EGF, neuregulin 1, and insulin-like growth factor. (Pg 12, Primary in vitro short-term culture) This reads both on the medium further comprising the addition of one or more additional growth factors or cell signaling molecules (claim 7) and the method of the medium not comprising a TGFb inhibitor (claim 9).
Regarding claim 50: Kim teaches use of both primary and metastatic sites in the harvest of renal cell carcinoma. (Pg 1, Abstract)
Regarding claim 51: Kim teaches use of fibroblast growth factor in the primary culture media. (Pg 12, Methods)
Claim 4 is rejected under 35 U.S.C. 103 as being unpatentable over Kim et al. (Application of single-cell RNA sequencing in optimizing a combinatorial therapeutic strategy in metastatic renal cell carcinoma, 2016) in view of Kazuo et al. (JP 2014-176353 A) and Agbunag et al. (Oncogenic K-ras Drives Cell Cycle Progression and Phenotypic Conersion of Primary Pancreatic Duct Epithelial Cells, 2004)
The teachings of Kim and Kazuo are described above. Both Kim and Kazuo fail to teach manipulation of the media or culture conditions to revert the phenotype of the cells in culture.
Regarding Claim 4: Kim teaches use of single-cell analysis and transcriptome profiling to examine the growth inhibitory effects of afatinib and dasatinib on cells in culture. (Pg 6, Evaluation of the single-cell analysis-driven therapeutic strategy) This reads on use of single-cell RNA analysis performed before and after establishing the cells of the claimed invention in an ex vivo cell-based system. After these results were obtained, a combination therapy was tried and it was found that the combination treatment of afatinib and dasatinib was much more effective than individual treatment, which had a more efficient inhibition of downstream AKT and ERK phosphorylation in PDX-mRCC cells. (Pg 6-7, Evaluation of the single-cell analysis-driven therapeutic strategy) Kim fails to teach modification of the cell culture media to make the cellular phenotype revert.
Agbunag et al. teaches a method of modeling pancreatic ductal adenocarcinoma (PDA) and that k-ras mutations are one of the first genetic changes to be detected with early PDA formation. (Pg 5659, Introduction) To model this, primary pancreatic duct epithelial cells (PDEC) were established in culture and said cells were introduced to k-ras, causing a significant increase in cell size, mimicking the phenotype of PDA. (Pg 5659-5661, Results and Discussion) PDEC cells were then microinjected with both k-ras and a P13K inhibitor, which failed to lead to an increase in cell size. (Pg 5661, Results and Discussion) This reads on the claimed method of modifying the medium or conditions to revert the expression space between the current phenotype (enlarged k-ras exposed PDEC which mimic PDA) to non-enlarged PDEC cells (with the addition of the P13K inhibitor).
It would have been obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to use the condition manipulation of the addition of a P13K inhibitor to cause a phenotype change to PDEC cells as taught by Agbunag with the single cell dissociation method taught by Kim and the media factors and co-culture method as taught by Kauzo to use single-cell RNA sequencing to determine the phenotype of cells in culture before and after a phenotypic change in the culture has been observed with the phenotypic change being caused by manipulation of the cell culture media or conditions. One would have been motivated to do so and had a reasonable expectation of success based on the teachings of Agbunag which detail use of a P13K inhibitor microinjected into the cells in culture with k-ras which result in the inhibition of the phenotypic change caused by just k-ras of increased cell size.
Claim 10 is rejected under 35 U.S.C. 103 as being unpatentable over Kim et al. (Application of single-cell RNA sequencing in optimizing a combinatorial therapeutic strategy in metastatic renal cell carcinoma, 2016) in view of Kazuo et al. (JP 2014-176353 A), Boj et al. (Organoid Models of Human and Mouse Ductal Pancreatic Cancer, 2015), Mullenders et al. (Mouse and human urothelial cancer organoids: A tool for bladder cancer research, 2019), and Yu et al. (Ex vivo culture of circulating breast tumor cells for individualizing testing of drug susceptibility, 2014)
The teachings of Kim and Kazuo are described above. Both Kim and Kazuo fail to teach use of a pancreatic ductal adenocarcinoma (PDAC) tumor, breast tumor, and bladder tumor as the source for the method of culture of claim 1.
Regarding claim 10: Boj et al. teaches a method of isolating murine primary pancreatic ductal tumors and establishing an organoid model in culture of said tumors to show in vitro how pancreatic ductal adenocarcinoma progresses. (Pg 327, Tumor-Derived Organoids Provide a Model for Murine PDA Progression) Boj fails to teach use of breast or bladder tumors in the method of claim 1.
Mullenders teaches the generation of a living biobank created from over 50 patient samples that have been characterized histologically and functionally. (Pg 1, Abstract) Mullenders further performed RNA sequencing to better understand the transcriptional patterns in said bladder organoids. (Pg 3, Results) The goal of the biobank created by Mullenders was to capture the diversity of bladder cancer on the molecular level (Pg 4, Results) and allows for functional studies to be performed on said organoids. (Pg 6, Discussion) Mullenders fails to teach use of breast tumors.
Yu teaches analysis of circulating single cell breast cancer tumors which were cultured in vitro post harvest and had RNA sequencing performed on them. (Pg 2-3) The availability of cultures generated from single cell circulating tumor cells allowed for a comprehensive mutational analysis which allows for the tailoring of therapeutic targeting to the individual patient based on said sequencing results. (Pg 4) This proof-of-concept study allows for the study of patterns of drug susceptibility on a patient by patient basis. (Pg 6)
It would have been obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to combine the teaching of Boj of use of murine PDA tumors in primary culture with the single cell dissociation method taught by Kim and the media factors and co-culture method as taught by Kauzo to create a culture method with cells derived from a PDAC tumor. One would have had motivation and a reasonable expectation of success based on the teachings of Boj, who detail a method of extraction of primary murine PDAC tumors which were used to establish organoid models in culture.
It would have further been obvious to combine the teachings of Yu and Mullenders of the culture of ex vivo tumor cells (breast and bladder, respectively) and perform RNA sequencing on said tumor cells as is taught in the combined method of Kim and Kazuo. One skilled in the art would have been motivated and had a reasonable expectation of success based on the teachings of Mullenders of the generation of a living biobank cataloguing the phenotypic diversity of bladder cancer, allowing for functional studies on said organoids to be carried out and the teachings of Yu of use of RNA sequencing to generate a comprehensive mutational analysis of breast circulating tumor cells, allowing for tailored therapeutic targeting on an individual patient level.
Claim 14 is rejected under 35 U.S.C. 103 as being unpatentable over Kim et al. (Application of single-cell RNA sequencing in optimizing a combinatorial therapeutic strategy in metastatic renal cell carcinoma, 2016) in view of Kazuo et al. (JP 2014-176353 A) and Castro (Interferon-Gamma at the Crossroads of Tumor Immune Surveillance or Evasion, 2018)
The teachings of Kim and Kazuo are disclosed above. Both Kim and Kazuo fail to teach use of IFNγ in their culture media.
Regarding Claim 14: Castro et al. teaches that IFNγ plays a role in the tumor microenvironment in that it is antiproliferative, anti-angiogenic, and has pro-apoptotic effects and may favor tumor immune invasion. (pg 6, IFNγ Actions on Transformed cells and the Tumor microenvironment)
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to combine the teaching of Castro of use of IFNγ in the culture media with the sequence-based analysis and culture method of Kim with the culture media factors and co-culture method as taught by Kauzo to create an ex vivo cell culture model comprising IFNγ in the culture media. One would have been motivated and had a reasonable expectation of success at doing so based on the teaching of Castro, who states that IFNγ is antiproliferative, anti-angiogenic, and may favor tumor immune invasion.
Response to Arguments
Applicant has argued that neither Kim nor Kazuo teach a method of maintaining the in vivo phenotype of a PDAC tumor in single-cell culture regarding claims 1 and 15. As necessitated by amendment, the teachings of Boj et al. have been incorporated to the rejection of claims 1 and 15 as Boj teaches the harvest of a PDAC tumor, as discussed above. In addition, Kim teaches a method of dissociating xenograft tumor specimens into a single cell suspension and placing them into a neurobasal media with the intent to run drug sensitivity assays on said cultures. (pg 12, Primary in vitro short term culture) In order to be able to run drug sensitivity assays and gain meaningful results, it is obvious that the primary xenograft tumor specimens in culture would have to maintain their in vivo phenotype.
Applicant further argues that a person of ordinary skill in the art would have no motivation to combine the teachings of Kazuo of the addition of WNT7B and WNT10A “among numerous factors” with the teachings of Kim to create a media comprising WNT7B and/or WNT10A. This is unpersuasive because Kazuo teaches that WNT7B, among other factors, is known to promote cell survival, maintenance, proliferation, amplification, and self-replication. (0033) This taken in the context of Kim of the teaching of a short-term culture of primary xenografted tumor cells would result in the formation of a media which is better suited to long-term culture, as the factors taught by Kazuo are stated to promote cell survival, proliferation, and maintenance. Furthermore, per MPEP 2141.II.C "A person of ordinary skill in the art is also a person of ordinary creativity, not an automaton."KSR, 550 U.S. at 421, 82 USPQ2d at 1397. "[I]n many cases a person of ordinary skill will be able to fit the teachings of multiple patents together like pieces of a puzzle."Id. at 420, 82 USPQ2d at 1397. Office personnel may also take into account "the inferences and creative steps that a person of ordinary skill in the art would employ."Id. at 418, 82 USPQ2d at 1396.
Conclusion
Any inquiry concerning this communication or earlier communications from the examiner should be directed to HANNA M THUESON whose telephone number is (571) 272-3680. The examiner can normally be reached M-F 7:30-5 EST. Examiner interviews are available via telephone, in-person, and video conferencing using a USPTO supplied web-based collaboration tool. To schedule an interview, applicant is encouraged to use the USPTO Automated Interview Request (AIR) at http://www.uspto.gov/interviewpractice.
If attempts to reach the examiner by telephone are unsuccessful, the examiner's supervisor, Tracy Vivlemore, can be reached on (571) 272-2914. The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300. Information regarding the status of published or unpublished applications may be obtained from Patent Center. Unpublished application information in Patent Center is available to registered users. To file and manage patent submissions in Patent Center, visit: https://patentcenter.uspto.gov. Visit https://www.uspto.gov/patents/apply/patent-center for more information about Patent Center and https://www.uspto.gov/patents/docx for information about filing in DOCX format. For additional questions, contact the Electronic Business Center (EBC) at 866-217-9197 (toll-free). If you would like assistance from a USPTO Customer Service Representative, call 800-786-9199 (IN USA OR CANADA) or 571-272-1000.
/HANNA MARIE THUESON/Examiner, Art Unit 1638
/Tracy Vivlemore/Supervisory Primary Examiner, Art Unit 1638
Prosecution Insights