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 .
Priority
This application was filed April 11, 2024, and is a 371 application of PCT/US2022/046436 filed on Oct. 12, 2022, which claims benefit to the provisional application 63/325,070 filed on March 29, 2022, and the provisional application 63/254,920 filed on Oct. 12, 2021.
Applicants claim for priority to a provisional application is acknowledged. The effective filing date for claims is Oct. 12th, 2021.
Claim Status
In the response filed Dec. 10, 2024, Applicant has amended claims 3, 8-11, 15-16, 32, 37, and 40, and canceled claims 12, 18-31, 33-36, 38, and 41-100.
Currently, claims 1-11, 13-17, 32, 37, 39-40 are pending in this application and are under consideration in this office action.
Information Disclosure Statement
Applicant is reminded of 37 CFR §1.56, which details Applicant's duty to disclose all information known to be material to patentability.
The information disclosure statements (IDS) submitted on 01/02/2026 are in compliance with the provisions of 37 CFR 1.97. Accordingly, the information disclosure statements are being considered by the examiner.
Notably, the disclosure statements filed lists a “Search Reports”. The listing of the references cited in a Search Report itself is not considered to be an information disclosure statement (IDS) complying with 37 CFR 1.98. 37 CFR 1.98(a)(2) requires a legible copy of: (1) each foreign patent; (2) each publication or that portion which caused it to be listed; (3) for each cited pending U.S. application, the application specification including claims, and any drawing of the application, or that portion of the application which caused it to be listed including any claims directed to that portion, unless the cited pending U.S. application is stored in the Image File Wrapper (IFW) system; and (4) all other information, or that portion which caused it to be listed. In addition, each IDS must include a list of all patents, publications, applications, or other information submitted for consideration by the Office (see 37 CFR 1.98(a)(1) and (b)), and MPEP § 609.04(a), subsection I. states, "the list ... must be submitted on a separate paper." Therefore, the references cited in the Search Report have not been considered. Applicant is advised that the date of submission of any item of information or any missing element(s) will be the date of submission for purposes of determining compliance with the requirements based on the time of filing the IDS, including all "statement" requirements of 37 CFR 1.97(e). See MPEP § 609.05(a).
Note: If copies of the individual references cited on the Search Report are also cited separately on the IDS (and these references have not been lined-through) they have been considered.
Specification
The disclosure is objected to because it contains an embedded hyperlinks, located on para. 229 (e.g. www.ebi.ac.uk/Tools/psa/emboss_needle/nucleotide.html). Applicant is required to delete the embedded hyperlink and/or other form of browser-executable code; references to websites should be limited to the top-level domain name without any prefix such as http:// or other browser-executable code. See MPEP § 608.01.
Claim Objections
Claim 8 is objected to because of the following informalities: grammar. Claim 8 recites the phrase “wherein the enucleated cell fraction produced by performing the continuous flow centrifugation once comprises more than or equal to about” (lines 1-3). The claim appears to be missing a verb (e.g. “is”) between “cell fraction” and “produced” and a coordinating conjunction (e.g. “and”) between “one” and “comprises”. Appropriate correction is suggested.
Claim 32 and 40 is objected to under 37 CFR 1.75(c) as being in improper form because a multiple dependent claim “the method of any one of claims 1-39”. Further, it is noted that claims 12, 18-31, 33-36, 38 have been canceled. See MPEP § 608.01(n). For compact prosecution the claim 40 will be examined as being dependent on claim 1.
Claim 37 is objected to because of the following informalities: repetition. Claim 37 recites “wherein the enucleated cells lack a nucleus.” It is suggested to amend the claim and remove the phrase “lack a nucleus” because the definition of enucleated cells is lacking a nucleus. Appropriate correction is required.
Claim Rejections - 35 USC § 112
(scope of enablement)
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 1-4, 8, 10, 14-17, 37, 39-40 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:
a method for stem cell, mesenchymal stromal cells, or immune cell processing, the method comprising: a) providing a composition consisting of a high density polysaccharide gradient with Cytochalasin B (CytoB) and mammalian nucleated cells; and b) enucleating a portion of the mammalian nucleated cells to produce an enucleated mammalian cell fraction using continuous flow centrifugation,
does not reasonably provide enablement for a method for any cell processing, the method comprising: a) providing any composition comprising any nucleated cells; and b) enucleating a portion of the nucleated cells to produce an enucleated cell fraction using only continuous flow centrifugation.
The specification does not enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the 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 and 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. When determining whether a specification meets the enablement requirements, some of the factors that need to be analyzed are: the breadth of the claims, the nature of the invention, the state of the prior art, the level of one of ordinary skill, the level of predictability in the art, the amount of direction provided by the inventor, the existence of working examples, and whether the quantity of any necessary experimentation to make and use the invention based on the content of the disclosure is “undue”.
Nature of the Invention: Claims are drawn to a method of cell processing, the method comprising: a) providing a composition comprising a nucleated cells; and b) enucleating a portion of the nucleated cells to produce an enucleated cell fraction using continuous flow centrifugation (see claim 1).
Breadth of the Claims: The claims do not specify the type of cell or the type of composition in the claim’s method of cell processing to produce an enucleated cell fraction using continuous flow centrifugation. As such, the breadth of the claims encompasses a method for any cell type for processing, the method comprising: a) providing any type of composition comprising any type of nucleated cells; and b) enucleating a portion of the nucleated cells to produce an enucleated cell fraction using only continuous flow centrifugation. As such, the breadth of the claims encompasses producing any type of enucleated cell by introducing any type of composition (e.g. media, agent, or inhibitor) with any type of nucleated cell (e.g. animal, plant, or bacteria cell) to produce an enucleated cell fraction using only continuous flow centrifugation. As such, the claim is broad encompassing embodiments contemplated and not contemplated by the specification.
Specification Guidance/Working Examples: The claims broadly encompass a method for any type of cell for cell processing, the method comprising: a) providing any composition comprising any nucleated cells; and b) enucleating a portion of the nucleated cells to produce an enucleated cell fraction using only continuous flow centrifugation (see claim 1),
the specification solely provides guidance to:
The enucleation efficiency and recovery rate of various types of mammalian cells (e.g., mesenchymal stem cells, neutrophils, fibroblast, and natural killer cells) was determined. After removal of the mammalian cells from the cell culture plates, the mammalian cells were enucleated by density gradient centrifugation using discontinuous Ficoll gradients, high-speed centrifugation. Table 1 summarizes the results of enucleation using a suspension protocol. Enucleation efficiency and cell viability was the highest in both hTERT transformed and primary mesenchymal stem cells (MSCs), as well as in fibroblasts and neutrophils. (See Spec. para. 371, Example 1, Fig. 4A-Fig. 4C, Table 1).
Accordingly, the specification solely enables mammalian cell enucleation, which is specific to the cell type of stem cell, mesenchymal stromal cells, or immune cells. Further, the specification uses a suspension protocol consisting of density gradient centrifugation with discontinuous Ficoll gradients, and high-speed centrifugation. Thus, the specification fails to provide any cell type or any method for producing enucleated cells.
Further, the specification solely provides guidance to:
Enucleation of mammalian cells using “Ficoll gradients, where 2X Cytochalasin B (CytoB) was added to 50% Ficoll aliquots at 1:1 dilution to make 25% Ficoll stock concentration. Next, 17%, 16%, 15% and 12.5% Ficoll were made by diluting 25% Ficoll with the appropriate volume of IX MEM buffer (2X MEM containing Cytochalasin B added to ultrapure water at 1: 1 dilution). The dilutions were equilibrated in a CO2 incubator for at least 1 hour covered with loose cap. The Ficoll gradients were then poured into 13.2mL ultra-clear tubes (Beckman, 344059), and incubated overnight (6-18 hours) in the CO2 incubator” (see Spec. page 109, Example 5. Generating the Enucleated cells).
Accordingly, the specification enables the enucleation of mammalian cells in a method with a Ficoll gradient and Cytochalasin B (CytoB) as a composition added to the nucleated cells, which is then incubated overnight. Thus, the specification fails to provide any other type of composition in the method for producing enucleated mammalian cells.
Further, the specification discloses that on the day of enucleation, 12-25M mesenchymal stem cells (MSC) (ideally 20M) were collected into each tube. When 90% of the cells were detached, 5mL full MSC media was added, and the cells were collected into 50ml tubes (3-4 plates/tube). The tubes were then centrifuged at 1, 200 rpm for 5 minutes. The pellet was resuspended in 10 mL PBS. Cells were counted, pelleted, and re-suspended with 12.5% Ficoll. Next, the cell-Ficoll mixture was dropwise passed through a 40 μm cell strainer (Falcon 352340) into a new 50 mL tube. Using a syringe, 3.2mL of cell suspension was slowly loaded onto the pre-made gradients. One mL of 1X MEM buffer was added at the final (top) layer with syringe. The tubes were then loaded into rotor buckets, balanced, and run in the ultracentrifuge (Beckman, L8M) for 60 minutes, 26,000 rpm, 31 °C, Accel 7, Deccel 7. At the end of the centrifugation, there were three layers: one near the top of the 12.5% (cytoplasts and debris), one near the 12.5/15% interface (cytoplasts), and a pellet at the bottom of the 25% (karyoplasts). (see Spec. page 109, Example 5. Generating the Enucleated cells).
Thus, the specification solely enables enucleation of mammalian cells using a composition consisting of a high density polysaccharide (i.e. Ficoll) gradient and Cytochalasin B (CytoB) with continuous flow centrifugation to produce an enucleated cell fraction from a portion of nucleated mammalian cells. Thus, the specification fails to provide a method for producing enucleated cells only using continuous flow centrifugation.
State of the Art: At the time of effectively filing, the prior art teaches that enucleation by centrifugation is highly unpredictable. The prior art of Shay et al., (Exp Cell Res. 1975 Aug;94(1):47-55, hereinafter as “Shay”) discloses Chinese hamster ovary cells (i.e. CHO cells) were subjected to centrifugal force without cytochalasin B were not enucleated, and that those cells were not different in morphology from the cells of control cultures, which were not subjected to centrifugal force (see e.g. abstract). Thus, Shay provides robust evidence that it was well-known in the art that having only the step of centrifugation for enucleation of mammalian cells without cytochalasin B results in an unpredictable method that results in cells that were not enucleated. Additionally, the prior art of Wigler, et al., (Methods in Cell Biology. Vol. 14. Academic Press, 1976. 87-93, cited IDS 1/2/2026, published 1976), discloses that any cell lacking a cell wall can be applied the enucleation method involving isopycnic centrifugation (i.e. Ficoll density gradients) in the presence of cytochalasin B and yield high efficiency and yield (see e.g. page 88). Thus, Wigler provides robust evidence that it was well-known in the art that not any cell or any type of composition with any methos would yield a high efficiency enucleations. Further, Wigler discloses the importance of having isopycnic centrifugation (i.e. Ficoll density gradients) in the presence of cytochalasin B for enucleation of mammalian cells. Furthermore, the prior art of Tesarik et al., (Fertility and sterility 79: 677-681, published 2003), which discloses that it was well known that the effect of oocyte exposure to the microfilament disrupting agent Cytochalasin B and D and latrunculin were most commonly used for nuclear transfer in human oocytes (see e.g. abstract, pages 679-680). Further, Tesarik discloses that “the exposure to cytochalasin B was clearly beneficial when nuclear transfer was performed at the germinal vesicle stage, because an overwhelming majority of germinal vesicle oocytes undergoing enucleation in the absence of cytochalasin B did not survive the manipulation, and only 1 of 50 oocytes forming this group incorporated the foreign nucleus” (see e.g. page 679), which teaches that the cell type, stage, and timing of the microfilament disrupting agents being applied to for enuculation is important. Thus, Tesarik provides robust evidence that not any type of cell can be used for enuculation methods, and that the microfilament disrupting agent CytoB is an important factors when enucleating a cell. Additionally, the prior art of Degaetano, and Schindler (Journal of cellular physiology 130.2: 301-309, cited IDS 1/2/2026, published 1987, hereinafter as “Degaetano”) discloses that the extent of enucleation as a function of centrifugal force is unpredictable resulting in 50% enucleation between different cell lines whereas using centrifugation with cytochalasin B or D were far more effective in enucleation (see e.g. abstract, page 302-303 and Fig. 1-2). Thus, Degaetano provides robust evidence that it was well known in the art that centrifugal force in enucleation is a supporting factor when enucleating cells with cytochalasin (see page 302-303 of Degaetano) and cannot substitute for the actin disruption that is provided with cytochalasin.
Thus, the breadth of the claims encompasses any type of cell, any type of composition, and only using continuous flow centrifugation for the process of enucleation. However, the prior art teaches that enucleation of mammalian cells with exposure to the microfilament disrupting agent Cytochalasin with a high density gradient using continuous flow centrifugation was known in the prior art. As discussed above, the art does not provide specific guidance to supplement the obstacles to enucleating any type of cell with any type of composition only using continuous flow centrifugation, which is not taught by the specification. As such, the art also fails to provide predictable enabling guidance to enucleating any type of cell with any type of composition only using continuous flow centrifugation.
Amount of Experimentation: Since the specification provides guidance to enucleation of mammalian cells, such as stem cell, mesenchymal stromal cells, or immune cells using a composition consisting of density gradients and Cytochalasin B (CytoB) with continuous flow centrifugation in order to produce an enucleated cell fraction from a portion of mammalian nucleated cells and the art at the time of the invention provide guidance to density gradients and Cytochalasin B (CytoB), discovery experimentation would need to be provided to determine IF any other enucleated cell fraction can be developed that will predictably produce an enucleated cell fraction. This level of discovery experimentation goes beyond routine optimization into experimentation to determine IF the method is enabled for any other enucleation cell fraction method. Therefore, the amount of experimentation needed would be considered undue.
Thus, in conclusion, the breadth of the claims encompassing any cell processing comprising any composition comprising nucleated cells and enucleated a cell fraction solely through continuous flow centrifugation is not enabled. The specification solely provides specific guidance to a method of enucleating mammalian cells (i.e. stem cell, mesenchymal stromal cells, or immune cell processing) using a composition consisting of Ficoll gradients and Cytochalasin B (CytoB) with continuous flow centrifugation in order to produce an enucleated cell fraction from a portion of mammalian nucleated cells, and fails to describe any other structural means of enucleating mammalian cells. Further, the state of the art does not provide additional guidance to another predictable means of processing mammalian nucleated cells to produce an enucleated cell fraction.
It is further noted that the claim 1 recites, “a) providing a composition comprising any nucleated cells;” (claim 1, line 2). The specification states that “In some embodiments, the continuous flow centrifugation generates a density gradient that separates the enucleated cell fraction from the nucleated cells in the composition. In some embodiments, the density gradient comprises a polysaccharide density gradient” (see Spec. para. 2). Thus, the specification and the above discussion of a composition comprising a Ficoll gradients and Cytochalasin B (CytoB) with continuous flow centrifugation that separates the enucleated cell fraction from the nucleated cells is consistent with the art established means of enucleating a portion of mammalian nucleated cells.
Therefore, at the time of filing the skilled artisan would need to perform an undue amount of experimentation without a predictable degree of success to implement the invention as claimed.
Claim Rejections - 35 USC § 112
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.
Claims 13 and 37 are rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim 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.
Regarding claim 13, recites wherein “at least one density fraction is obtained from the density gradient, wherein the at least one density fraction comprises a mixed population of a subset of nucleated and enucleates cells of the enucleated cell fraction”. However, it is unclear how a mixed population of cells is obtained from the enucleated cell fraction if the enucleated cell fraction is recited as only have enucleated cells. Thus, a person of ordinary skill in the art would not know that the enucleated cell fraction contains a mixed cell population of nucleated and enucleated cells because it recited as the enucleated cell fraction. Therefore, it is unclear what fraction is being obtained in the density gradient (i.e. a mixed population of cells or the enucleated cell fraction). For compact prosecution the claim will be interpreted as the broadest reasonable interpretation as any enucleated cell fraction.
Regarding claim 37, recites “the method of claim 1, wherein the enucleated cells lack a nucleus and comprise one or more intracellular organelles for synthesis or secretion of an exogenous polypeptide in the absence of the nucleus”. The claim limitation of "wherein the enucleated cells” in line 2 has insufficient antecedent basis because claim 1 only recites a “enucleated cell fraction”. Therefore, it is unclear what enucleated cells are being referred to since claim 1 does not recite producing enucleated cell population but rather a fraction of the nucleated cells have been enucleated. For compact prosecution the claim will be interpreted as the reciting “the enucleated cells from the enucleated cell fraction”.
The prior art rejection below are directed to the scope that is enabled.
Claim Rejections - 35 USC § 102
The following is a quotation of the appropriate paragraphs of 35 U.S.C. 102 that form the basis for the rejections under this section made in this Office action:
A person shall be entitled to a patent unless –
(a)(1) the claimed invention was patented, described in a printed publication, or in public use, on sale, or otherwise available to the public before the effective filing date of the claimed invention.
Claims 1-2, 4-7, 10-11, 16, 32, 37, and 39-40 are rejected under 35 U.S.C. 102(a)(1) as being anticipated by Klemke, Richard; Wang, Huawei (WO2019/032628 A1, The Reagents Of The University of California, published the 14th of February 2019; hereinafter as “Klemke”).
Claim Interpretation: The specification does not recite a special definition for continuous flow centrifugation. The specification does recite that in some embodiments continuous flow centrifugation is “using the continuous flow centrifugation in the method is performed using an ultracentrifuge” and “in some embodiments, enucleating the portion of the nucleated cells to produce the enucleated cell fraction using the continuous flow centrifugation is performed using fixed angle centrifugation or swinging bucket centrifugation. (see e.g. Spec. para 9, 141, 147, and fig. 12). Therefore, the broadest reasonable interpretation of continuous flow centrifugation is ultracentrifuge, fixed angle centrifugation, or swinging bucket.
Regarding claim 1, 4, and 32, Klemke discloses a method for cell processing (i.e. mesenchymal stem cells (MSC))(see e.g. Example 1-5, fig. 1 and table 1), the method comprising: a) providing a composition (i.e. high density Ficoll gradient with Cytochalasin B) comprising nucleated cells (e.g. MSC); and b) enucleating a portion of the nucleated cells to produce an enucleated cell fraction (i.e. cytoplasts)(see e.g. claim 1, Example 1-5, fig. 1, table 1) using continuous flow centrifugation (i.e. ultracentrifuge)(see e.g. Example 5, page 76).
Regarding claim 2, Klemke discloses wherein the portion of the nucleated cells comprises greater than or equal to about 95% of the nucleated cells (see e.g. page 35, Example 1-5, fig. 1 and table 1-2).
Regarding claim 4, Klemke discloses wherein the continuous flow centrifugation generates a density gradient that separates the enucleated cell fraction from the nucleated cells in the composition (see e.g. page 76, Example 1-5, fig. 1-2 and table 1).
Regarding claim 5, Klemke discloses wherein the density gradient comprises a polysaccharide density gradient (i.e. Ficoll gradient)(see e.g. Example 1-5, fig. 1-2, and table 1).
Regarding claim 6, Klemke discloses wherein the density gradient comprises at least two or three ranges (i.e. layers) of the density gradient (see e.g. page 76, Example 1-5, fig. 1-2 and table 1).
Regarding claim 7, Klemke discloses wherein the polysaccharide density gradient comprises about 15% polysaccharide (i.e. Ficoll). (see e.g. page 76, Example 1-5, fig. 1-2 and table 1).
Regarding claim 10, as states supra, Klemke discloses wherein the continuous flow centrifugation generates a density gradient that separates the enucleated cell fraction from the nucleated cells in the composition (see e.g. page 76, Example 1-5, fig. 1-2 and table 1).Thus, Klemke inherently discloses wherein continuous flow centrifugation generates zonal centrifugation for separating at least one enucleated cell from the nucleated cells (see e.g. page 76, Example 1-5, fig. 1-2 and table 1).
Regarding claim 11, Klemke discloses wherein the zonal centrifugation separates the at least one enucleated cell from the nucleated cells based on size of the at least one enucleated cell (see e.g. page 72, fig. 15 page 16-18, Example 1-5, fig. 1-2 and table 1).
Regarding claim 16, Klemke discloses wherein the nucleated cells comprise a heterologous polynucleotide (e.g. human telomerase reverse transcriptase (hTERT) adipose derived human mesenchymal stem cells or HSV-1716 (i.e. oHSV))(see e.g. page 11, 67, Example 1-5, fig. 1-2 and table 1).
Regarding claim 32, Klemke discloses wherein the nucleated cells comprise stem cells, mesenchymal stromal cells, or immune cells (see e.g. Example 1-5, fig. 1-2 and table 1).
Regarding claim 37, Klemke discloses wherein the enucleated cells lack a nucleus and comprise one or more intracellular organelles for synthesis or secretion of an exogenous polypeptide in absence of the nucleus (i.e. cytoplast or recombinant cytoplast)(see e.g. page 28, 32-34, 37, Example 1-5, fig. 1-2 and table 1).
Regarding claim 39, Klemke discloses The method of claim 37, wherein the exogenous polypeptide comprises a therapeutic agent (see e.g. page 34, 38-40, 57, claims 1-2, 8-10, 13, 15 and 17; Example 1-5, fig. 1-2 and table 1).
Regarding claim 40, Klemke discloses wherein the enucleated cells comprise at least one targeting moiety (e.g. CXCR4, pages 52) and/or immune evasion moiety (see e.g. CD47, page 57)(see e.g. pages 3, 11, 57-60, claims 6-7, 12; Example 1-5, fig. 1-2 and table 1).
Thus, Klemke anticipates the instant claims.
The following is a quotation of the appropriate paragraphs of 35 U.S.C. 102 that form the basis for the rejections under this section made in this Office action:
A person shall be entitled to a patent unless –
(a)(2) the claimed invention was described in a patent issued under section 151, or in an application for patent published or deemed published under section 122(b), in which the patent or application, as the case may be, names another inventor and was effectively filed before the effective filing date of the claimed invention.
Claims 1-2, 4-5, 10-11, 16-17, 32, 37, and 40 are rejected under 35 U.S.C. 102(a)(2) as being anticipated by Klemke, Richard; Wang, Huawei; Pi, Willie (WO 2021/158991 A1, The Reagents Of The University of California, published August 8, 2021, cited IDS 01/02/2026; hereinafter as “Wang”).
The applied reference has a common inventor with the instant application. Based upon the earlier effectively filed date of the reference, it constitutes prior art under 35 U.S.C. 102(a)(2). This rejection under 35 U.S.C. 102(a)(2) might be overcome by: (1) a showing under 37 CFR 1.130(a) that the subject matter disclosed in the reference was obtained directly or indirectly from the inventor or a joint inventor of this application and is thus not prior art in accordance with 35 U.S.C. 102(b)(2)(A); (2) a showing under 37 CFR 1.130(b) of a prior public disclosure under 35 U.S.C. 102(b)(2)(B) if the same invention is not being claimed; or (3) a statement pursuant to 35 U.S.C. 102(b)(2)(C) establishing that, not later than the effective filing date of the claimed invention, the subject matter disclosed in the reference and the claimed invention were either owned by the same person or subject to an obligation of assignment to the same person or subject to a joint research agreement.
Regarding claim 1, 4, and 32, Wang discloses a method for cell processing (e.g. mesenchymal stem cells, MSC)(see e.g. Fig. 1), the method comprising: a) providing a composition (i.e. cytochalasin) comprising nucleated cells (i.e. MSC)(see e.g. page 12-22, fig. 1, Example 1, claims 1-20); and b) enucleating a portion of the nucleated cells to produce an enucleated cell fraction (i.e. cargocytes) using continuous flow centrifugation (i.e. ultracentrifugation)(see e.g. Example 1, page 21-22).
Regarding claim 2, Wang discloses wherein the portion of the nucleated cells comprises greater than or equal to about 95% of the nucleated cells (see e.g. page 15-20).
Regarding claim 4, Wang discloses wherein the continuous flow centrifugation generates a density gradient that separates the enucleated cell fraction from the nucleated cells in the composition(see e.g. page 12-22, fig. 1, Example 1, claims 1-20).
Regarding claim 5, Wang wherein the density gradient comprises a polysaccharide density gradient (i.e. Ficoll)(see e.g. page 12-22, fig. 1-3, Example 1, claims 1-20).
Regarding claim 10, as states supra, Wang discloses wherein the continuous flow centrifugation generates a density gradient that separates the enucleated cell fraction from the nucleated cells in the composition (see e.g. page 12-22, fig. 1, Example 1, claims 1-20). Thus, Wang inherently discloses wherein continuous flow centrifugation generates zonal centrifugation for separating at least one enucleated cell from the nucleated cells (see e.g. page 12-22, fig. 1, Example 1, claims 1-20)
Regarding claim 11, Wang discloses wherein the zonal centrifugation separates the at least one enucleated cell from the nucleated cells based on size of the at least one enucleated cell (see e.g. pages 19, fig. 1, Example 1, claims 1-20).
Regarding claim 16, Wang discloses wherein the nucleated cells comprise a heterologous polynucleotide (see e.g. page 28-31).
Regarding claim 17, Wang discloses wherein the method comprises inducing cell death (i.e. suicide switch) of the nucleated cells that are not enucleated after b), wherein the cell death is induced by expressing a heterologous gene product encoded by the heterologous polynucleotide in the nucleated cells (see e.g. page 17).
Regarding claim 32, as stated supra, Wang discloses wherein the nucleated cells comprise stem cells, mesenchymal stromal cells, or immune cells (see e.g. pages 19, fig. 1, Example 1, claims 20-21)
Regarding claim 37, Wang discloses wherein the enucleated cells lack a nucleus and comprise one or more intracellular organelles for synthesis or secretion of an exogenous polypeptide in absence of the nucleus (see e.g. pages 19-20, fig. 1, Example 1, claims 1-20).
Regarding claim 40, Wang discloses wherein the enucleated cells comprise at least one targeting moiety (e.g. biomolecule)(see e.g. page 22), fusogenic moiety (see e.g. peptide)(page 12, 17,19), and/or immune evasion moiety (e.g. cytokine) (see e.g. page 23).
Thus, Wang anticipates the instant claims.
Claim Rejections - 35 USC § 103
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 3, 8-9, and 15 are rejected under 35 U.S.C. 103 as being unpatentable over Klemke, Richard; Wang, Huawei (WO2019/032628 A1, The Reagents Of The University of California, published the 14th of February 2019; hereinafter as “Klemke”), as applies to claims 1-2, 4-7, 10-11, 16, 32, 37, and 39-40 above, Wigler, Michael H., and I. Bernard Weinstein. (Biochemical and biophysical research communications 63.3: 669-674, published 1975; cited IDS 01/02/2026, herein after as “Wigler”) as evidence by Beckman Coulter Inc. (Ultracentrifugation: Product selection guide (BR-8101J-2), Beckman Coulter, Inc., published n.d., date accessed May 2026, hereinafter as “Beckman Coulter Inc.”).
Claim Interpretation: The specification does not recite a special definition for continuous flow centrifugation. The specification does recite that in some embodiments continuous flow centrifugation is “using the continuous flow centrifugation in the method is performed using an ultracentrifuge” and “in some embodiments, enucleating the portion of the nucleated cells to produce the enucleated cell fraction using the continuous flow centrifugation is performed using fixed angle centrifugation or swinging bucket centrifugation. (see e.g. Spec. para 9, 141, 147, and fig. 12). Therefore, the broadest reasonable interpretation of continuous flow centrifugation is ultracentrifuge, fixed angle centrifugation, or swinging bucket.
The teachings of Klemke apply here as indicated above.
Regarding claim 3 and 9, Klemke discloses methods for large scale in vitro production of enucleated cells (i.e. cytoplasts)(see e.g. page 33 and 70; claims 1-2, 8-10, 13, 15 and 17; Example 1-5, fig. 1-2 and table 1). Further, Klemke discloses the enucleation method wherein the ultracentrifuge (Beckman, L8M) was performed at 26,000 rpm (see e.g. page 76, Example 1-2).
Klemke does not explicitly state wherein the composition has a volume comprising between more than or equal to about 500 mL to about 10,000 mL and does not explicitly state wherein the continuous flow centrifugation is a maximum centrifugal force of between about 30,000 RCF to about 200,000 RCF.
However, Klemke does discloses using the ultracentrifuge of Beckman, L8M (i.e. continuous flow and zonal rotors), and as evidence by Beckman Coulter Inc. catalog the ultracentrifuge is able to have a composition volume that is more than or equal to about 500 mL (i.e. 50mL to more than 1,000 mL)(see e.g. page 1-5, 1-6, 1-7, 1-37, 1-40), and a maximum centrifugal force of between about 102,000 RCF to about 172,000 RCF (i.e. between about 30,000 RCF to about 200,000 RCF)(see e.g. page 1-7).
Further, the following is noted from the MPEP: MPEP 2144.05: “In the case where the claimed ranges ‘overlap or lie inside ranges disclosed by the prior art’ a prima facie case of obviousness exists. In re Wertheim, 541 F.2d 257, 191 USPQ 90 (CCPA 1976); In re Woodruff, 919 F.2d 1575, 16 USPQ2d 1934 (Fed. Cir. 1990).” MPEP 2144.05(I) teaches “a prima facie case of obviousness exists where the claimed ranges or amounts do not overlap with the prior art but are merely close.” Titanium Metals Corp. of America v. Banner, 778 F.2d 775, 227 USPQ 773 (Fed. Cir. 1985).” In regards to overlapping ranges, MPEP 2144.05(I) states, “In the case where the claimed ranges ‘overlap or lie inside ranges disclosed by the prior art’ a prima facie case of obviousness exists. In re Wertheim, 541 F.2d 257, 191 USPQ 90 (CCPA 1976); In re Woodruff, 919 F.2d 1575, 16 USPQ2d 1934 (Fed. Cir. 1990)”, continuing in regards to ranges are close, “Similarly, a prima facie case of obviousness exists where the claimed ranges or amounts do not overlap with the prior art but are merely close. Titanium Metals Corp. of America v. Banner, 778 F.2d 775, 783, 227 USPQ 773, 779 (Fed. Cir. 1985)”.
In the instant case, the specification teaches that ultracentrifuge was performed using the Beckman, L8M at 26,000 rpm (see specification, para. 385; page 109), which is the same ultracentrifuge (i.e. Beckman, L8M) and force (i.e. 26,000 rpm) in the enucleation method as taught by Klemke (see e.g. page 76, Example 1-2). Therefore, the composition volume and centrifuge force reads on the claimed composition volume range and maximum centrifugal force range.
Accordingly, it would have been obvious for a person of ordinary skill in the art to have modified the methods of Klemke and incorporated the composition volume range and maximal centrifuge force range as evidence by Beckman Coulter Inc. catalog which discloses that the Beckman L8M (i.e. continuous flow and zonal rotor) model can spin all of Beckman’s compatible rotors (see e.g. page 1-5). Thus, it would have been obvious for one of ordinary skill in the art to have optimized the volume range comprising between more than or equal to about 500 mL to about 10,000 mL and optimize the continuous flow centrifugation force range to have a maximum centrifugal force of between about 30,000 RCF to about 200,000 RCF with a reasonable expectation of success. Further, the instant specification discloses using the same ultracentrifuge and force (i.e. Beckman, L8M at 26,000 rpm (see specification, para. 385; page 109) as taught by Klemke (see e.g. page 76, Example 1-2). Thus, it would have been obvious to combine prior art elements according to known methods to yield predictable results with a reasonable expectation of success.
Regarding claim 8, as state supra, Klemke discloses methods for large scale in vitro production of enucleated cells (i.e. cytoplasts)(see e.g. page 33 and 70). Further, Klemke discloses using more than 30-40 million (i.3. 30-40M) cells for each enucleation tube (see e.g. page 80).
Klemke does not explicitly state wherein the enucleated cell fraction comprises more than or equal to about: 6 x 107 (i.e. 60 million (60M)) of enucleated cells to 250 x 107 (2.5 billion) of enucleated cells.
However, the prior art of Wigler discloses that “5 x 108 cytoplasts can be obtained with a single preparation” (i.e. 500 million)(see e.g. page 674).
Accordingly, it would have been obvious for a person of ordinary skill in the art to have modified the methods of Klemke to incorporate obtaining 5 x 108 enucleated cells as taught by Wigler because both Klemke and Wigler teach methods for obtaining enucleated cells using continuous flow centrifugation (see e.g. Examples 1 and pages 670-674, respectively). As Klemke teaches large scale production of enucleated cells using continuous flow centrifugation (see e.g. Example 1-2) and Wigler teaches obtaining 5 x 108 cytoplasts with a single preparation (see e.g. page 674), it would have been obvious for a person of ordinary skill in the art to have obtained more than or equal to about 6 x 107 (i.e. 60 million (60M)) of enucleated cells with a reasonable expectation of success. Thus, a person of ordinary skill in the art would have combined known prior art elements according to known methods to yield predictable results with a reasonable expectation of success.
Regarding claim 15, as stated supra, Klemke discloses methods for large scale in vitro production of enucleated cells (i.e. cytoplasts) by using continuous flow centrifugation (i.e. Beckman, L8M) (see e.g. page 33, 70, and 76; claims 1-2, 8-10, 13, 15 and 17; Example 1-5, fig. 1-2 and table 1).
Klemke does not explicitly discloses wherein the continuous flow centrifugation increases a yield of obtaining enucleated cells from nucleated cells by at least 0.1 fold or more fold compared to a method of obtaining the enucleated cells from the nucleated cells by a method without using the continuous flow centrifugation.
However, MPEP 2111.04 states “The broadest reasonable interpretation of a method (or process) claim having contingent limitations requires only those steps that must be performed and does not include steps that are not required to be performed because the condition(s) precedent are not met.” Ex parte Marhold, 231 USPQ 904, 905 (Bd. Pat. App. & Int. 1986) relying on In re Sussman, 141 F.2d 267, 269-70, 60 USPQ 538, 540-41 (CCPA 1944) provides "that since the steps are the same, the results must inherently be the same unless they are due to conditions not recited in the claims." In the instant case, the claims are drawn to an invention employing the same process steps but the product(s) is(are) alleged to be different. Applicant is required to recite the missing steps to form the alleged different product(s) in view of the above-cited decision.
In the instant case, the specification teaches that ultracentrifuge was performed using the Beckman, L8M at 26,000 rpm (see specification, para. 385; page 109), which is the same ultracentrifuge (i.e. Beckman, L8M) and force (i.e. 26,000 rpm) in the enucleation method as taught by Klemke (see e.g. page 76, Example 1-2). Therefore, the continuous flow centrifugation as taught by Klemke would also increase the yield of obtaining enucleated cells from nucleated cells by at least 0.1 fold or more fold compared to a method of obtaining the enucleated cells from the nucleated cells by a method without using the continuous flow centrifugation.
Accordingly, it would have been obvious for a person of ordinary skill in the art to have the methods of Klemke and have an increase in the yield of obtaining enucleated cells from nucleated cells by at least 0.1 fold or more fold compared to a method of obtaining the enucleated cells from the nucleated cells by a method without using the continuous flow centrifugation because Klemke uses continuous flow centrifugation and zonal rotors as evidence by Beckman Coulter Inc. (see e.g. page 1-7). As discussed above, it would have been obvious for a person of ordinary skill in the art to have the predictable results with a reasonable expectation of success because Klemke discloses using the Beckman, L8M (i.e. continuous flow and zonal rotor) model, which Beckman Coulter Inc. discloses that the model can spin all of Beckman’s compatible rotors (see e.g. page 1-5). Thus, a person of ordinary skill in the art with a reasonable expectation of success would optimize and obtain enucleated cells from nucleated cells by at least 0.1 fold or more fold compared to a method of obtaining the enucleated cells from the nucleated cells by a method without using the continuous flow centrifugation. Further, the instant specification discloses using the same ultracentrifuge and force (i.e. Beckman, L8M at 26,000 rpm)(see specification, para. 385; page 109) as taught by Klemke (see e.g. page 76, Example 1-2). Thus, it would have been obvious to combine prior art elements according to known methods to yield predictable results with a reasonable expectation of success.
Hence, the claimed invention as a whole was prima facie obvious in the absence of evidence to the contrary.
Claims 13 and 14 are rejected under 35 U.S.C. 103 as being unpatentable over Klemke, Richard; Wang, Huawei (WO2019/032628 A1, The Reagents Of The University of California, published the 14th of February 2019; hereinafter as “Klemke”), as applies to claims 1-2, 4-7, 10-11, 16, 32, 37, and 39-40 above, Wigler, Michael H., and I. Bernard Weinstein. (Biochemical and biophysical research communications 63.3: 669-674, published 1975; cited IDS 01/02/2026, herein after as “Wigler”) as evidence by Alarcon, Christina Nicole. (Therapeutic Functions of Bioengineered, Enucleated Cells. University of California, San Diego, 2020, hereinafter as “Alarcon”; cited IDS 01/02/2026).
The teachings of Klemke apply here as indicated above.
Regarding claim 13 and 14, Klemke discloses enucleation efficiency was greater than 70% of enucleated cells (see e.g. page 68, Example 1). Further, Klemke discloses that Ficoll fractions were made at 25%, 17%, 16%, 15% and 12.5% (bottom to top fractions)(see e.g. page 75, Example 5). Further, Klemke discloses obtaining “three layers: one near the top of the 12.5% (cytoplasts and debris), one near the 12.5/15% interface (cytoplasts), and a pellet at the bottom of the 25% (karyoplasts)” (see e.g. page 68-69, Example 1 and 5),
Klemke does not explicitly state a mixed population of at least 70% of enucleated cells were obtained.
However, the prior art of Wigler discloses that 70% of the cytoplasts were recovered from the 15-17% Ficoll region (see e.g. page 670).
Accordingly, it would have been obvious for a person of ordinary skill in the art to have modified the methods of Klemke and incorporate a density fraction comprising a mixed population of cells as taught by Wigler because Wigler and Klemke both disclose methods for obtaining at least 70% of enucleated cells (see e.g. page 670 and page 68-69, respectively). A person of ordinary skill in the art would have a reasonable expectation of success because Wigler discloses that 50-70% of the cells were ultimately recovered in the 15-17% Ficoll region (see e.g. page 670). Thus, a person of ordinary skill in the art would have had a reasonable expectation of success because both Klemke and Wigler disclose obtaining fractions that encompass the 15% Ficoll region. Furthermore, the prior art of Alarcon, discloses a mixed cell population comprising of cytoplasts (i.e. enucleated cells) aggregating in the 15-17% fraction, and karyoplasts (i.e. nucleated cells) banded at the 17-25%. Therefore, a person of ordinary skill in the art would have expected to obtain a mixed cell population when incorporating the 15-17% Ficoll region as taught by Wigler as evidence by Alarcon, which suggests to one of ordinary skill in that art that has a method with the fractions lower than 25% (i.e. 17%, 16%, 15% and 12.5%) of Ficoll would have obtained a mixed cell population. Thus, a person of ordinary skill in the art would have been able to have obtained a mixed cell population that comprises at least 70% enucleated cells with predictable results and a reasonable expectation of success.
Hence, the claimed invention as a whole was prima facie obvious in the absence of evidence to the contrary.
Claims 16-17 is rejected under 35 U.S.C. 103 as being unpatentable over Klemke, Richard; Wang, Huawei (WO2019/032628 A1, The Reagents Of The University of California, published the 14th of February 2019; hereinafter as “Klemke”), as applies to claims 1-2, 4-7, 10-11, 16, 32, 37, and 39-40 above, and further view of Wang, Huawei, et al. ("Cargocyte Biofactories: A Novel Platform for Delivering Oncolytic Viruses to Treat Metastatic Cancer." Molecular Therapy, Vol. 27. No. 4. 50: Cell press, published 2019).
The teachings of Klemke apply here as indicated above.
Regarding claim 16-17, as stated supra, Klemke discloses methods that producing therapeutic enucleated cells (i.e. cytoplasts)(see e.g. page 28). Further, Klemke teaches wherein the mesenchymal stem cells (i.e. nucleated cells) comprise a heterologous polynucleotide, such as human telomerase reverse transcriptase (hTERT) adipose derived human mesenchymal stem cells (MSC) that may be infected with a virus (see e.g. page 4, 11, 67, 77-78; Example 1-5, fig. 21), such as an oncolytic herpes simplex virus (i.e. HSV-1716, oHSV)(see e.g. page 4, 78, Example 1-5, fig. 21). Further, Klemke teaches that the hTERT-MSC-derived cytoplasts can be loaded with the oncolytic viruses (i.e. oHSV) and can be deliver to tumors (see e.g. page 30 and 77, Fig. 21)
Klemke does not explicitly state wherein the method comprises inducing cell death of the nucleated cells that are not enucleated after b), wherein the cell death is induced by expressing a heterologous gene product encoded by the heterologous polynucleotide in the nucleated cells (see e.g. page 27).
However, the prior art of Wang discloses that when obtaining nucleated and enucleated hTERT-MSCs with an oncolytic virus the nucleated MSCs will have an oncolytic viral infection that will activate the antiviral response mediated by NF-kB and transcription of interferon α/β genes, whereas the enucleated cell will already have the nucleus removed, therefore the enucleated cells will have no new gene transcription, which leads to enhanced virus propagation (see e.g. section A and Fig.1), corresponding to the claim limitation of inducing cell death of the nucleated cells that are not enucleated after b), wherein the cell death is induced by expressing a heterologous gene product encoded by the heterologous polynucleotide in the nucleated cells (see e.g. fig. 1 of Wang).
Accordingly, it would have been prima facie obvious to one of ordinary skill in the art to use an oncolytic virus as taught by Wang in a method of producing therapeutic enucleated cells (i.e. cytoplasts) as taught by Klemke because Klemke and Wang both use oncolytic viruses for producing therapeutic enucleated cells (i.e. cytoplasts)(see e.g. page 28 and section A, respectively). Further, a person of ordinary skill in the art would have done so because Wang teaches that since the nucleus has already been removed there will be no new gene transcription resulting in an improved systemic delivery of oncolytic viruses and targeting them to tumors in a safe manner (see e.g. section background and conclusions). Thus, the use of an oncolytic virus such as the oncolytic vesicular stomatitis virus (i.e. oVSV) as taught by Wang in place of the oncolytic virus (i.e. HSV) taught by Kleme would have been considered obvious since it is prima facie obvious to substitute one known element (i.e. oncolytic virus) for another to obtain predictable results with a reasonable expectation of success. In the instant case, as discussed above, both Klemke and Wang use enucleated cell methods with MSCs and oncolytic viruses for obtaining therapeutic enucleated cells, therefore one of ordinary skill could have substituted one element for another with predictable results, since both are readily available and are deliverable using similar techniques.
Hence, the claimed invention as a whole was prima facie obvious in the absence of evidence to the contrary.
Conclusion
No claim is allowed.
The prior art made of record and not relied upon is considered pertinent to applicant's disclosure.
Watson, F., Wang, H., Alarcón, C. N., Liu, B., & Klemke, R. L. (2018, December). Enhancing Chemotaxis of Enucleated Cells by Genetic Engineering. In Conference (poster)-Molecular Biology of the Cell (Vol. 29, p. 26).
Klemke, Richard, et al. "Cargocyte (TM) Biofactories: A New Versatile Cell Therapy Platform for Delivery of a Wide Range of Biologics." MOLECULAR THERAPY. Vol. 27. No. 4. 50 HAMPSHIRE ST, FLOOR 5, CAMBRIDGE, MA 02139 USA: CELL PRESS, 2019.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to JOSEPHINE GONZALES whose telephone number is (571)272-1794. The examiner can normally be reached M-Th: 9AM - 5:00PM (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, Doug Schultz can be reached at 571-272-0763. 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.
JOSEPHINE GONZALES
Examiner
Art Unit 1631
/JOSEPHINE GONZALES/Examiner, Art Unit 1631
/JAMES D SCHULTZ/Supervisory Patent Examiner, Art Unit 1631
Prosecution Insights