DETAILED ACTION
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and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114.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 03/11/2026 has been entered.
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 .
Status of the Claims
Claims 1, 6-15, 31-36, and 38-40 are pending (claim set as filed on 03/10/2026).
Applicant’s election without traverse of Group I in the reply filed on 09/21/2022 is acknowledged. Claims 9-11, 13-15, and 31-34 are withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected invention, there being no allowable generic or linking claim. Therefore, only claims 1, 6-8, 12, 35-36, and 38-40 are under examination.
Priority
This application is a 371 of PCT/JP2018/044894 filed on 12/06/2018 which has foreign applications to: JP 2018-195647 filed on 10/17/2018 and JP 2017-236374 filed on 12/08/2017.
Withdrawal of Rejections
The response and amendments filed on 03/10/2026 are acknowledged. Any previously applied minor objections and/or minor rejections, not explicitly restated herein for brevity, have been withdrawn necessitated by Applicant’s formal corrections and/or amendments. For the purposes of clarity of the record, the reasons for the Examiner’s withdrawal, and/or maintaining if applicable, of the essential claim rejections are detailed below in the Examiner’s response to arguments section.
The following rejections and/or objections are either reiterated or newly applied. They constitute the complete set presently being applied to the instant application.
New Grounds of Rejection Necessitated by Amendment
Claim Rejections - 35 USC §103, Obviousness
The text of those sections of Title 35, U.S. Code not included in this action can be found in a prior Office action.
Claims 1, 6-8, 12, 35-36, and 38-40 are rejected under 35 U.S.C. 103 as being unpatentable over Yamazaki (US 2013/0288248 A1 - newly cited) in view of Clevers (US 2014/0243227 - cited in the IDS filed on 11/18/2021) and Hu (US 2019/0161734 A1 with a PCT filing date of 06/10/2016).
Yamazaki’s general disclosure relates to a cancer stem cell population and a production method thereof; using a non-human animal model transplanted with the cancer stem cell population or a culture system of the cancer stem cell population under in vitro conditions (see ¶ [0001], [0013]).
Regarding the culturing, Yamazaki teaches “the cancer stem cell population of any one of [1] to [7], which is produced by a method comprising the step of adherently culturing a cell group containing cancer stem cells; (1) transplanting a cell group containing cancer stem cells into a non-human animal that belongs to the same or different species to produce a cancer cell mass; (2) fragmenting the produced cancer cell mass; and (3) adherently culturing the cell population obtained in step (2) in a stem cell medium … wherein the cell group containing cancer stem cells is allowed to proliferate before performing adherent culture; [15] the method of [14], wherein the cell group containing cancer stem cells is allowed to proliferate by spheroid culture; [16] the method of [14], wherein the cell group is proliferated by being transplanted to and passaged in a non-human animal; [17] the method of any one of[l 1] to [16], wherein the cancer stem cells are derived from a human tumor tissue; [18] the method of [17], wherein the human tumor tissue is derived from epithelial cancer” (see ¶ [0020], [0083], [0091]-[0092], [0098], [0151], Examples 1 and 3). Yamazaki teaches “Matrigel basement membrane matrix, diluted to 50% with Hank’s Balanced Salt Solution, was used to prepare” cell solutions (see ¶ [0136], [0141], [0149], [0153]).
Regarding the medium, Yamazaki teaches “culture solutions that can be used in the present invention are not particularly limited as long as they can be used to culture cancer stem cells. For example, a conventionally-known basal culture solution supplemented with EGF, bFGF … or a mixture of these can be used as a culture solution. The concentration of EGF is not
particularly limited but is 0.1-100 ng/mL, preferably 0.5-50 ng/mL, and more preferably 1-20 ng/mL. The concentration of bFGF is not particularly limited but is 0.1-100 ng/mL, preferably 0.5-50 ng/mL” (see ¶ [0088]).
Regarding the cell or tissue source, Yamazaki teaches “the source of the cell group is not particularly limited, and it is possible to use those derived from mammals such as human” and “such cell lines can be established by physically mincing surgically-resected human colon cancer” (see ¶ [0084]-[0085]). Yamazaki further teaches “cancer stem cells obtained by this method were stably maintained for over a month without phenotypic change by repeated passage of the adherent culture using a serum-free stem cell medium. These cells expressed various colon cancer stem cell markers” (see ¶ [0018]). Yamazaki teaches the cancer stem cell population wherein the cancer stem cells are derived from a human tumor tissue; wherein the human tumor is derived from epithelial cancer (see ¶ [0020], [0065]). Yamazaki teaches colon cancer cell lines (see ¶ [0021]-[0028], Figures, & Example 1).
Regarding the total amount of cells, Yamazaki teaches “the cancer stem cells can be prepared in large quantities. In principle, a desired number of cancer stem cells can be obtained by increasing flasks for adherent culture. For example, when a T150 flask is used, 4x107 or more cells can be generally obtained when cultured to confluence, and therefore 2x108 or more cells can be prepared by using five flasks. Thus, a desired number of cells can be prepared in the present invention” (see ¶ [0077]-[0078]).
Regarding claim 8 pertaining to serum, Yamazaki teaches in vitro cancer cell lines were cultured according to a standard method (e.g., at 37°C and in the presence of 5% CO2) in a medium comprising fetal bovine serum (see ¶ [0134], [0139]).
Regarding claim 12 pertaining to a xenograft, Yamazaki teaches producing a non-human animal model by transplanting the cancer stem cell population of any one of [1] to [10] to a non-human animal (see ¶ [0020], [0132]) and further teaches xenograft tumors (see ¶ [0041]-[0043], [0059], [0154], [0158], [0177]).
Regarding claim 39, Yamazaki teaches “colon cancer specimens were obtained from patients … Tumors were minced with scissors and transplanted into the flanks of NOG mice. Human colon cancer xenografts were maintained through passage in NOG mice” and “colon cancer cell lines established in NOG or SCID mice were subcutaneously transplanted into NOG mice to generate cancer cell masses. The cancer cell masses were excised, fixed for 16-24 hours in 4% paraformaldehyde at 4° C., and embedded by the AMeX method to prepare sliced tissue specimens” (see ¶ [0132]-[0133]).
However, Yamazaki does not teach: a ROCK inhibitor, a TGF-β inhibitor, or an agent selected from NECA, Y27632, or SB431542 (claims 1’s limitations and 6-7); or the cell population is continuously cultured until a total amount of cells forming the cancer spheroid is at least 1x105 to 1x107 prior to storing (claim 1’s last limitation); or wherein the cancer spheroid has a diameter of 0.01 to 2 mm (claim 36).
Clevers’ general disclosure relates to culture media and methods for expanding and differentiating populations of stem cells and for obtaining organoids (see abstract & ¶ [0002]). Clevers discloses “investigated signaling pathways that are known to be subverted in certain cancers e.g., colorectal cancer. They hypothesized that these pathways, which affect cell fate in cancer, may also play a role in determining cell fate in culture conditions” (see ¶ [0010]).
Clevers teaches “a method for expanding a single stem cell or a population of stem cells, preferably to generate an organoid, wherein the method comprises culturing the single stem cell or population of stem cells in a culture medium according to the invention, wherein the method comprises: culturing the stem cell, population of stem cells or tissue fragments in a first expansion medium; continuing to culture the stem cell, population of stem cells or tissue fragments and replenishing the medium with a differentiation medium” (see ¶ [0303]-[0305]). Clevers teaches “Expanding organoids may generally be more appropriate for other uses, such as (but not limited to) regenerative medicine and drug screening, for example for cancer or cystic fibrosis. Expanding organoids generally have more growth potential (and thus greater longevity) than differentiated organoids. In some embodiments, the colon, liver and pancreatic organoids are not further differentiated” (see ¶ [0307]). Clevers teaches “for example, an organoid according to the present invention may comprise a population of cells of at least 1x103 cells, at least 1x104 cells, at least 1x105 cells, at least 1x106 cells, at least 1x107 cells or more” (see ¶ [0310]-[0313]) and further teaches the invention provides an organoid or population of cells which is frozen and stored in liquid nitrogen (claim 40) (see ¶ [0316], [0466], [0562]).
Regarding the basement membrane matrix, Clever teaches a culture medium of the invention may be diffused into an extracellular matrix (ECM); ECM is composed of a variety of polysaccharides, water, elastin, and glycoproteins, wherein the glycoproteins comprise collagen, entactin (nidogen), fibronectin, and laminin; said ECM is commercially provided and examples of commercially available extracellular matrices are extracellular matrix proteins (Invitrogen) and basement membrane preparations from Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells (e.g. Matrigel) (see ¶ [0129]).
Regarding claim 6 pertaining to the ROCK inhibitor, Clevers teaches “small molecule inhibitors related to relevant signaling pathways, such as ERK, p38, JNK, PTEN, ROCK, and Hedgehog, were tested” (see ¶ [0011], [0082]) and further teaches “ROCK inhibitors such as Y27632 can be included in any of the media described, in particular in the first few days of culture before performing cell sorting experiments, because it is known to avoid anoikis (a form of programmed cell death which is induced by anchorage-dependent cells detaching from the surrounding extracellular matrix)” (see ¶ [0099]-[0100]) and the optional additional components may be added for optimization of the culture medium for culturing cells originating from particular tissues (see ¶ [0083]).
Regarding claim 7 pertaining to the TGF-β inhibitor, Clevers teaches “two small molecule inhibitors, A83-01 (Alk4/5/7 inhibitor; nM) and SB202190 (p38 inhibitor; 10 uM) significantly improved the plating efficiency. Other TGF-beta receptor 1 (ALK 5) inhibitors that were also tested and showed the same results as A83-01 were LY364947, SB431542, SB50512” (see ¶ [0718]).
Regarding claim 8 pertaining to serum, Clevers teaches the “culture medium of the invention may contain serum. Serum obtained from any appropriate source may be used, including fetal bovine serum (FBS), goat serum or human serum. Preferably, human serum is used” (see ¶ [0121]).
Regarding claim 36 pertaining to the diameter, Clevers teaches the organoids can have a diameter of up to 1 mm (see ¶ [0337]).
Hu teaches a “growth factor is thyroid hormone. In some embodiments, the thyroid hormone is selected from the group consisting of 3,3'-5-triiodo-l-thyronine (T3), (5)-thyroxine (T4), and hormones that stimulate cAMP, such as parathyroid hormone (PTH), the adenosine A2 receptor agonist 5'-(N-ethylcarboxamido)-adenosine (NECA), prostaglandin E2, or forskolin” (see ¶ [0202]). Hu discloses thyroid hormone (such as T3) as an agent that can stimulate the cell to release cAMP and increase the cell’s metabolic rate (see ¶ [0204]).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to employ or add the ingredients of: a ROCK inhibitor, TGF-β inhibitor, and/or serum such as taught by Clevers in the culture medium of Yamazaki. The ordinary artisan would have been motivated to do so because Clevers teaches their individual functions and suggests their addition for optimization of the culture medium for culturing cells organoids. The MPEP at 2141 provides for exemplary rationales that may support a conclusion of obviousness include: (a) combining prior art elements according to known methods to yield predictable results. The ordinary artisan would have had a reasonable expectation of success because both references are directed to cell culture of spheroids/organoids and culture media components therewith.
Furthermore, it would have been secondly obvious to one of ordinary skill in the art to employ or add an adenosine receptor agonist of NECA such as taught by Hu in the method of Yamazaki and Clevers. The ordinary artisan would have been motivated to do so is because Hu suggests that as an agent that can stimulate the cell to release cAMP and increase the cell’s metabolic rate. The ordinary artisan would have had a reasonable expectation of success because the references are directed to cell culture methods and ingredients thereof.
Regarding claim 1’s last limitation, it would have been obvious to continuously culture until a total amount of cells is at least 1x105 to 1x107 prior to storing such as taught by Clevers (at ¶ [0307] and [0310]-[0313]) because it was suggested that expanding organoids may generally be more appropriate for other uses, such as (but not limited to) regenerative medicine and drug screening, for example for cancer or cystic fibrosis. Expanding organoids generally have more growth potential (and thus greater longevity) than differentiated organoids. Moreover, in the cell cultivation arts, the continuous culture of cells to reach a desired quantity is within the purview of the ordinary artisan and routinely performed or considered a common practice. As noted by Clevers, the expansion may be dependent upon the intended application of the organoids. Yamazaki also teaches a desired number of cells can be prepared in the present invention (see Yamazaki at ¶ [0077]-[0078]).The MPEP 2141(III) states that “Prior art is not limited just to the references being applied, but includes the understanding of one of ordinary skill in the art”.
Regarding claims 35 and 38, claim interpretation: a wherein or whereby clause in a method claim is not given weight when it simply expresses the intended result of a process step positively recited (MPEP 2111.04(I)). Moreover, to one of ordinary skill in the art, there is a reasonable expectation that the addition of each individual ingredient would be additively or collectively beneficial for cell growth and thus, having three the claimed agents (EGF, bFGF, and NECA) would be expected to have an increased growth of cells as compared to only two agents.
Conclusion
No claims were allowed.
Correspondence Information
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/NGHI V NGUYEN/Primary Examiner, Art Unit 1653
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