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 .
DETAILED ACTION
This action is in response to the papers filed on July 31, 2024. Pursuant to amendment
filed on July 31, 2024, claim 6 is amended.
Therefore, claims 1-17 are under examination.
Priority
The present application is a 35 U.S.C. 371 national stage filing of the International Application No. PCT/JP2023/002794, filed January 30, 2023, which claims priority to Japanese Application No. JP2022-013646, filed on January 31, 2022, is acknowledged.
Acknowledgment is made of applicant's claim for foreign priority based on an application filed in Japan on January 31, 2022. Filing on July 31, 2024, of certified untranslated copy of the Japanese publication JP2022-013646 filed on January 31, 2022, is acknowledged. Should applicant desire to obtain the benefit of foreign priority under 35 U.S.C. 119(a)-(d) prior to declaration of an interference, a certified English translation of the foreign application must be submitted in reply to this action. 3 7 CPR 41. l 54(b) and 41.202(e). Failure to provide a certified translation may result in no benefit being accorded for the non-English application.
Thus, priority date is not yet perfected, and the earliest possible priority for the instant
application is January 31, 2022.
Claim objection
Claim 14 is objected to because three compositionally different media used in sequential culture stages are each identified as “the second medium.” Applicant may obviate the objection by clarifying that the recited media are respective sequential media used during step (2).
Claim 14 is additionally objected to because the method steps re designated using parenthetical identifiers “(2-1),” “(2-2),” and “(2-3).” Parenthetical notation is generally reserved for reference characters rather than method-step designations. Applicants may obviate this objection by presenting the method steps in conventional sequential format without using reference-character style identifies.
Claim 17 is objected to because the limitation “does not require a step of sorting NK cells” does not clearly exclude an optional sorting step. Applicant may obviate the objection by stating whether NK-cell sorting is excluded.
Claim Rejections - 35 USC § 112(a)
The following is a quotation of the first paragraph of 35 U.S.C. 112(a):
(a) IN GENERAL.—The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor or joint inventor of carrying out the invention.
The following is a quotation of the first paragraph of pre-AIA 35 U.S.C. 112:
The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor of carrying out his invention.
Claims 1-7 and 17 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, because the specification, while being enabling for the production of natural killer (NK) cells from a particular human induced pluripotent stem cell, (iPSC) cell line 06E (TC-1133HKK_06E_MCB), using a specifically disclosed three-stage differentiation protocol performed under defined perfusion culture conditions, does not reasonably provide enablement for the full scope of the claims without undue experimentation. In particular, the specification does not enable the broad genus of NK-cell production methods, encompassing any pluripotent stem cell, any differentiation media, and all cellular spheroid populations having an average particle size of at least 200 µm without undue experimentation. The specification therefore does not enable one of ordinary skill in the art to make and use the claimed invention commensurate in scope with these claims.
While determining whether a specification is enabling, one considers whether the claimed invention provides sufficient guidance to make or use the claimed invention, if not, whether an artisan would require undue experimentation to make and use the claimed invention and whether working examples have been provided. Factors to be considered in determining whether a disclosure meets the enablement requirement of 35 USC§ 112, first paragraph, have been described by the court in In re Wands, 8 USPQ2d 1400 (CA FC 1988).
Wands states on page 1404:
“Factors to be considered in determining whether a disclosure would require undue
experimentation have been summarized by the board in Ex parte Forman. They include
(1) the quantity of experimentation necessary, (2) the amount of direction or guidance
presented, (3) the presence or absence of working examples, (4) the nature of the
invention, (5) the state of the prior art, (6) the relative skills of those in the art, (7) the
predictability or unpredictability of the art, and (8) the breadth of the claims.”
In the instant application, the specification provides a working example using a single iPSC source, a particular sequence of differentiation media containing specific cytokines and signaling molecules, defined perfusion culture equipment, selected membrane pore sizes, and specific culture conditions to produce NK-cell populations. The specification therefore reasonably enables these disclosed embodiments (pg. 23-30).
However, claim 1 encompasses sustainably more than the disclosed embodiments. The claim broadly encompasses any pluripotent stem cell, any first, second, and third media capable of performing the recited differentiation, any perfusion culture system, any operating parameters for perfusion culture, and any sphere population having an average particle size of at least 200 µm. The claims are not limited to the disclosed cytokine combinations, membrane systems, cell densities, or pluripotent stem-cell lines demonstrated in the specification.
The differentiation of pluripotent stem cells into hematopoietic progenitor cells and subsequently into NK cells is a biological complex and relatively unpredictable process. Culture performance is known to depend upon numerous interacting variables, including the pluripotent stem-cell line, aggregate size, aggregate size distribution, cytokine identity and concentration, timing of media transitions, perfusion rate, oxygen transfer, nutrient availability, cell density, and 3D culture configuration. The specification does not provide sufficient guidance demonstrating that these variables may be modified across the full breath of the claims while still reliably producing the claimed cell populations.
Although the specification provides several preferred parameters, the working example is directed to a single experimental protocol and do not establish that the claimed invention may be successfully practice across the full genus encompassed by the claims. Practicing the invention throughout its entire scope would therefore require substantial empirical optimization and screening to identify suitable pluripotent stem-cell lines, differentiation media, culture parameters, perfusion conditions, and operating ranges capable of achieving the claimed differentiation sequence.
According, due to the breath of the claims, the limited number of representative working examples, the absence of guidance commensurate with the claimed genus, and the unpredictably of pluripotent stem-cell differentiation, one of ordinary skill in the art would be required to engage in undue experimentation to practice the full scope of the claimed invention.
Therefore, the scope of the claims exceeds the scope of enablement provided by the specification. While the specification reasonably enables the particular three-stage perfusion culture protocol disclosed therein, it does not enable the broader genus of methods encompassing any pluripotent stem cell, any differentiation media, any perfusion culture system, and all process capable of producing NK-cell populations, as recited in claims 1-7 and 17.
Claim Rejections - 35 USC § 112(b)
The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
Claim 1, and by dependence 2-17, are rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as failing to set forth the subject matter which the inventor or a joint inventor, or for applications subject to pre-AIA 35 U.S.C. 112, the applicant regards as the invention.
Claim 1 recites pluripotent stem-cell spheres having an “average particle size of not less than 200 µm.” The specification only states the microscopic photographs were used (pg. 24, lines 14-17), and does not identify the dimension being measured, the sampling procedure, the number of spheres measured, the averaging method, or the time at which the measurement is performed. Nor does the specification define how irregular or fused spheres are treated or whether the reported value is based on a number, area, or volume-weighted average.
As evidenced by the Anton Par (Particle size distribution) disclosure on measurements of particle size distribution, particle-size distributions are commonly characterized using several different statistical descriptors, including arithmetic mean diameter, volume-weighted mean diameter, surface-weighted mean diameter, mean diameter, percentile diameters, span, and other distribution parameters. Each of these descriptors characterizes the same particle population differently and can produce materially different numerical values for the same heterogenous sample.
For example, a sphere population may have a median diameter greater than 200 µm while its arithmetic mean diameter is less than 200 µm or may satisfy a volume-weighted mean diameter of greater than 200 µm while fewer than fifty percent of the individual spheres exceed 200 µm. Likewise, a population in which 90% of the spheres are smaller than 200 µm differs substantially from a population having an average diameter greater than 200 µm. Without identifying the statistical parameter being claimed, the scope of the limitation cannot be objectively determined. Since the specification fails to identify the measurement methodology or statistically definition corresponding to the claimed “average particle size,” one of the ordinary skill in the art would not be able to determine, with reasonable certainty, whether a particular sphere population falls within or outside the scope of the claims.
Claims 2-17 are rejected for being dependent from the rejected claims 1 and also for failing to further clarify the basis of the rejection.
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.
Claims 1-3 and 6-17 are rejected under 35 U.S.C. 103 as being unpatentable over Feng et al. (US 2021/0395684 A1, published Dec. 23, 2021), in view of STEMCELL Technologies technical manual “Expansion of Human Pluripotent Stem Cells as Aggregates in Suspension Culture Using mTeSR™3D”, Version 2, published September 2020, and Kropp et al. (Stem Cells Transl Med. 2016 Oct;5(10):1289-1301. Epub 2016 Jul 1.), and further in view of Valamehr et al (US 10,626,372 B1).
Regarding claim 1, Feng teaches methods for producing hematopoietic-lineage cells, including NK cells, from human pluripotent stem cells. Feng teaches initiating 3D suspension culture by seeding dissociated undifferentiated human iPSCs into a spinner flask in medium containing ROCK inhibitor, Y-27632, and culturing cells under agitation to form pluripotent stem-cell spheres ([0188-0190]; claim 1).
Feng further teaches differentiating 3D pluripotent stem cell spheres through hemogenic endothelial and hematopoietic progenitor stages. Feng teaches transitioning spheres into hematopoietic commitment and expansion medium containing SCF, Flt3L, IL-3, IL-6, TPO, and EPO, thereby inducing and expanding hematopoietic progenitor cells from the spheres ([0131-0132]). Feng characterizes the process as a scalable 3D system for producing hematopoietic and lymphoid lineages from human PSCs ([0165]).
Feng also teaches collecting hematopoietic progenitor cells produced by the sphere differentiation process and culturing those cells in 3D, ultralow attachment culture under conditions suitable for NK-lineage development. The NK cell promoting medium contains SCF, Flt3L, IL-7, and IL-15 and NK differentiation is confirmed by expression of CD56 and other NK-lineage markers ([0199]).
Feng does not expressly teach forming spheres having an average particles size of not less than 200 µm or performing each of the three stages by perfusion culture.
However, STEMCELL teaches expansion of human pluripotent stem cells, including ES and iPS cells, as spherical aggregates in dynamic 3D suspension culture (pg. 1-2). STEMCELL teaches representative pluripotent stem cell aggregates having diameters of 200-300 µm and identifies such aggregates as typical prior to passage (pg. 12, Fig, 1). STEMCELL further teaches that aggregate size is affected by seeding density, cell line, vessel, agitation, and passage schedule, and states that undifferentiated aggregates should generally not exceed 400 µm because larger aggregates may experience nutrient deficiency, differentiation, and loss of pluripotent stem cell markers (pg. 9 and 11-13).
Therefore, before the effective filing date, the ordinary artisan would have found it obvious to culture Feng’s pluripotent stem cell spheres to an average size of at least 200 µm, since STEMCELL expressly teaches viable pluripotent suspension aggregates in the 200-300 µm range. Where a variable is disclosed in a range in the prior art is taught, there exists a prima facie case of obviousness based on optimization where the variable was recognized in the prior art to be a result-effective variable. That is to say that the particular parameter was taught and known to affect the result. A person of ordinary skill in the art would have recognized aggregate size was a known process variable that could be controlled through ordinary adjustment of seeding density, culture duration, agitation, and passage schedule. The claimed lower limit of 200 µm therefore represents selection of a known aggregate size within a range expressly taught for human pluripotent stem cell suspension culture.
Furthermore, Feng teaches the utility of continuous 3D sphere culture and integration with several dissociation and reaggregation steps, where growth factors and small molecules can be added at different stages to induce differentiation ([0126]). Furthermore, the ordinary artisan would have recognized the additional applicability and benefit of perfusion culture techniques and staged differentiation protocols, as taught by Kropp and Valamehr. Kropp teaches matrix-free 3D suspension culture of human pluripotent stem-cell aggregates in stirred-tank bioreactors employing continuous perfusion through a cell retention filter to continuously replenish nutrients, remove metabolic wastes, maintain a homogenous culture environment, improve cell viability, increase cell density and yield, and facilitate scalable, automated manufacturing (Abstract; pg. 1289-1291). Valamehr additionally teaches sequential differentiation of pluripotent stem cells through mesoderm, definitive hemogenic endothelium, hematopoietic progenitor cells, NK progenitors, and mature NK cells using defined combinations of BMP4, GSK3β inhibitors, VEGF, TGFβ/Smad inhibitors, SCF, Flt3L, IL-7 and IL-15 (Abstract; columns 41-44, 63 and 76-77).
Therefore, before the effective filing date of the claimed invention, the person of ordinary skill in the art would have been motivated to modify Feng’s 3D differential process by implementing Kropp’s continuous perfusion culture system while employing the known stage specific differentiation conditions taught by Valamehr in order to provide controlled nutrient delivery, metabolite removal, improved culture homogeneity, reduced manual intervention, scalable manufacturing, and predictable progression through the hematopoietic and NK cell developmental pathway, with a reasonable expectation of successfully producing NK cells from pluripotent stem cell aggregates.
Regarding claim 2, the combined teachings of Feng, STEMCELL, Kropp, and Valamehr render claim 1 obvious. Further, STEMCELL teaches that hPSCs grow as spherical aggregates in suspension without the addition of matrices or microcarriers (pg. 2, para. 3). Additionally, Kropp similarly teaches hPSC expansion as “cell-only aggregates” and states that the method overcomes the need for matrices used in feeder-free adherent culture and microcarriers used in other suspension systems (Abstract; pg. 1290, column 1, para. 2). Therefore, the ordinary artisan would have found it obvious to perform the combined method without a 3D carrier or extracellular substrate to simplify manufacture, reduce culture components, support scale-up and automation, and obtain the matrix-free manufacturing advantages, as taught by STEMCELL and Kropp.
Regarding claim 3, the combined teachings of Feng, STEMCELL, Kropp, and Valamehr render claim 1 and 2 obvious. Kropp additionally teaches establishing perfusion using a porous filter having a pore size of 20-40 µm as a cell-retention device (pg. 1290, Section: Bioreactor System). STEMCELL additionally teaches the use of a 37 µm reversible strainer to retain and process hPSC aggregates, further demonstrating that a separation structure having pore size within the claimed range was known for separating pluripotent stem cell aggregates from non-aggregated cells or medium (pg. 15, para. 2-3).
Regarding claim 6, the combined teachings of Feng, STEMCELL, Kropp, and Valamehr render claim 1 obvious. Kropp additionally teaches perfusion as continuous medium replacement and teaches automated continuous medium exchange (pg. 1290, column 1, para. 4; Fig. 1).
Regarding claim 7, the combined teachings of Feng, STEMCELL, Kropp, and Valamehr render claim 1 obvious. Feng additionally teaches initiating 3D suspension culture in medium containing y-27632, a ROCK inhibitor ([0188]; [0190]). STEMCELL further teaches the initiation of cell culture with medium containing Y-27632 (pg. 1).
Regarding claim 8-9, the combined teachings of Feng, STEMCELL, Kropp, and Valamehr render claim 1 obvious. Additionally, Feng identifies BMP4, VEGF, bFGF, Y-27632, and the GSK3 inhibitor CHIR99201 among the reagents used in its 3D hematopoietic differentiation process ([0188]). Moreover, Valamehr teaches differentiating pluripotent cells toward mesoderm and definitive hemogenic endothelium using BMP activator, including BMP4, a Wnt-pathway activator or GSK3 inhibitor, VEGF, bFGF, and optionally a ROCK inhibitor (columns 41-44 and 76-77). The ordinary artisan would have found it obvious to use the taught combination during the early hematopoietic differentiation stage since the prior art taught those factors for mesodermal and hemogenic specification.
Regarding claims 10-11, the combined teachings of Feng, STEMCELL, Kropp, and Valamehr render claim 1 obvious. Feng teaches SCF, VEGF, bFGF, Y-27632, and the TGFβ/Smad-pathway inhibitor SB431542 among the reagents used in its hematopoietic differentiation process ([0188]). Moreover, Valamehr teaches use of a TGFβ-receptor/ALK inhibitor with VEGF and further teaches SCF, bFGF, and ROCK inhibitors for generation or expression of definitive hemogenic endothelium and hematopoietic progenitors (column 10-column bridging para through para. 5; column 41-44; column 76-77). The ordinary artisan would have found it obvious to employ these known factors during the corresponding hemogenic-endothelial stage because they were recognized for their utility in promoting hematopoietic specification, progenitor-cell survival, and expansion.
Regarding claims 12-13, the combined teachings of Feng, STEMCELL, Kropp, and Valamehr render claim 1 obvious. Feng teaches transitioning spheres into hematopoietic commitment and expansion medium containing SCF, Flt3L, and IL-3 ([0131]). Feng further teaches NK-lineage culture using SCF, Flt3L, IL-7, and in a serum-free embodiment, IL-3 ([0199]). Likewise, Valamehr teaches hematopoietic and NK-lineage media containing SCF, Flt3L, IL-3, IL-7, and ROCK inhibitors (column 39, 43, 63, and 76-77). The utility of these factors would have been obvious to the ordinary artisan because they were recognized to support hematopoietic progenitor expansion and lymphoid or NK-lineage commitment.
Regarding claim 14, the combined teachings of Feng, STEMCELL, Kropp, and Valamehr render claim 1 obvious. Feng teaches stepwise 3D differentiation of hPSC spheres using different growth factors and small molecules during successive developmental stages ([0131-0132]; [0188-0190]). Additionally, Valamehr teaches a corresponding staged developmental pathway in which pluripotent cells are induced toward mesoderm using a BMP activator and GSK3-pathway modulation, transitioned toward definitive hemogenic endothelium using VEGF and TGFβ receptor inhibition, and expanded as hematopoietic progenitors using SCF and Flt3L (column 41-44, 63, and 76-77). The ordinary artisan would have found it obvious to arrange these known factors in the recites sequence because the media correspond to known successive developmental transitions from pluripotency through mesoderm and hemogenic endothelium to hematopoietic progenitor expansion.
Regarding claims 15-16, the combined teachings of Feng, STEMCELL, Kropp, and Valamehr render claim 1 obvious. Feng expressly teaches NK-lineage differentiation of hematopoietic progenitor cells in medium comprising SCF, Flt3L, IL-7, and IL-15 ([0199]). Additionally, Valamehr similarly teaches NK-cell media containing SCF, Flt3L, IL-7, and IL-15 (columns 39, 63, and 76-77). The media taught by Valamehr as iNK media contains combinations of SCF, Flt3L, IL-7, and IL-15. Thus, the recited factor combination was taught for NK-lineage differentiation and the ordinary artisan would have found it obvious to employ this media during the NK cell differentiation stage.
Regarding claim 17, the combined teachings of Feng, STEMCELL, Kropp, and Valamehr render claim 1 obvious. Feng teaches collecting hematopoietic progenitor cells and directly culturing those cells in NK-promoting medium. Feng monitors the resulting populations for CD56 and other NK markers but does not require an NK-cell sorting step before or during the disclosed NK-lineage culture ([0199]). STEMCELL additionally teaches that suspension aggregate culture may be conducted without the manual selection and removal of differentiated cells (pg. 2). The ordinary artisan would have found it obvious to omit a sorting step where the differentiation population could be produced, expanded, and characterized without sorting. The omission of an unnecessary separation would predictably reduce handling, cell loss, processing time, cost, and manufacturing complexity.
Conclusion
Claims 1-17 are rejected. No claims are allowed.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to JOEL D LEVIN whose telephone number is (571)270-0616. The examiner be reached 8:00 am to 5:00 pm, Monday through Friday.
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If attempts to reach the examiner by telephone are unsuccessful, the examiner’s supervisor, Christopher Babic can be reached at (571) 272-8507. The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300.
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/J.D.L./Examiner, Art Unit 1633
/FEREYDOUN G SAJJADI/Supervisory Patent Examiner, Art Unit 1699
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