DETAILED ACTION
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
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and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114.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 May 27, 2026, has been entered.
Claims 2-4, 6, 7, and 13-17 are canceled.
Claims 1, 5, 8-12, 18, and 19 are pending. Claims 9-12, 18, and 19 are withdrawn.
Claims 1, 5, and 8 are examined on the merits.
Claim Objections
Claims 1, 5, and 8 are objected to because of the following informalities:
Claim 1 is objected to because the comma after “first differentiation medium” in line 8 should be deleted.
Claim 1 is objected to because the parentheses around “IL-12” in line 12 should be deleted. Line 12 is the second line of the step starting with “further culturing for at least about 10 days in a second differentiation medium.”
Also, claim 1 is objected to because two consecutive commas are recited in lines 18-19. The two consecutive commas precede the recitation of “with the proviso that the expansion medium…” One of the two commas should be deleted.
Since claim 1 is objected to, then its dependent claims, claims 5 and 8, are objected to.
Appropriate correction is required.
Notice Re: Prior Art Available Under Both Pre-AIA and AIA
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.
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.
Claims 1, 5, and 8 are rejected under 35 U.S.C. 103 as being unpatentable over Murphy (US 2018/0008637. Previously cited) in view of Roeven March 2014 (Bone Marrow Transplantation. March 2014. 49(Suppl. 1): ppS50. Previously cited), and Spanholtz `118 (WO 2013/119118. Previously cited).
Murphy discloses methods of producing natural killer (NK) cells from a population of hematopoietic stem or progenitor cells using a three-stage expansion and differentiation method with media comprising stem cell mobilizing factors (abstract; paragraph [0002]). In certain embodiments, the hematopoietic cells are CD34+ cells (paragraph [0300]), and Murphy refers to CD34+ stem cells or progenitors cells as an example of the hematopoietic stem cells or progenitor cells when setting forth the steps of the method (paragraph [0312]). Additionally, Murphy discloses that in one embodiment, the hematopoietic cells are obtained from umbilical cord blood (paragraph [0297]). Moreover, Murphy discloses in Example 3 cultivating UCB (umbilical cord blood)) CD34+ cells to produce NK cells (paragraph [0632]). Therefore, Murphy discloses practicing their invention with umbilical cord blood-derived CD34-positive cells as the cells being cultured, thereby meeting a limitation of instant claim 1.
The method of Murphy comprises cell expansion, during which a plurality of hematopoietic cells within the hematopoietic cell population differentiate into NK cells (paragraph [0312]). The NK cells are CD56+, CD3-, and at least 70% of the natural killer cells are viable with certain embodiments (paragraph [0312]).
In one embodiment, the method comprises:
culturing hematopoietic stem cells or progenitor cells, e.g., CD34+ stem cells or progenitors cells, in a first medium comprising a stem cell mobilizing agent and thrombopoietin (Tpo) to produce a first population of cells, wherein the first medium further comprises one or more of a group of 7 components that include Flt-3L, SCF, and IL-7 (paragraph [0317};
subsequently culturing the cells in a second medium comprising a stem cell mobilizing agent and interleukin-15 (IL-15) and lacking Tpo to produce a second population of cells, wherein the second medium further comprises one or more of 7 components that include Flt-3[L], SCF, and IL-7 (paragraph [0318]);
subsequently culturing the cells in a third medium comprising IL-2 and IL-15, and lacking a stem cell mobilizing agent and LMWH (low-molecular weight heparin; see page 1, paragraph [0009]) to produce a third population of cells, e.g., natural killer cells, wherein the third medium further comprises one or more of 5 components that include SCF and IL-7 (paragraph [0319]).
In one embodiment, the stem cell mobilizing compound is an aryl hydrocarbon receptor inhibitor, e.g., an aryl hydrocarbon receptor antagonist (paragraph [0368]). Thus Murphy teaches an embodiment of the above method in which the first medium and the second medium each comprise an aryl hydrocarbon receptor antagonist, and the third medium lacks an aryl hydrocarbon receptor antagonist (as it lacks a stem cell mobility agent according to paragraph [0009], which encompasses an aryl hydrocarbon receptor antagonist). These meet limitations of the claimed expansion medium, first differentiation medium, and second differentiation medium, respectively.
There is no mention of Interleukin-12 (IL-12) in the second medium of Murphy (paragraph [0318]). Moreover, IL-12 is not recited at any instance in Murphy. Therefore, it is obvious that the second medium of Murphy, meeting limitations of the claimed ‘first differentiation medium,’ does not comprises IL-12, thereby rendering obvious a limitation of the claimed ‘first differentiation medium.’
In performing the method of Murphy, it would have been prima facie obvious to the skilled artisan to include any combination of the additional components (7 components for the first and second mediums; 5 components for the third medium) in the three mediums since Murphy teaches “one or more of” the list of additional components for inclusion in each of the three mediums (paragraphs [0317]-[0319]). Therefore, it would have been prima facie obvious to select Flt-3L, SCF, and IL-7 as the additional components of the first and second mediums of Murphy for the predictable result of culturing the cells to produce a first and second populations of cells in the respective steps. Further still, it would have been prima facie obvious to select SCF and IL-7 as the additional components of the third medium of Murphy for the predictable result of culturing the cells to finally produce a third population of cells, specifically of NK cells. The first medium rendered obvious by Murphy meets limitations of the claimed ‘expansion medium,’ the second medium rendered obvious by Murphy meets limitations of the claimed ‘first differentiation medium,’ and the third medium rendered obvious by Murphy meets limitations of the claimed ‘second differentiation medium.’
Additionally, since glycosaminoglycans are not disclosed for inclusion in the three mediums of Murphy, then Murphy meets the claimed proviso of mediums that do not comprise a glycosaminoglycan.
Further still, Murphy teaches that the cells are cultured in the first medium (meeting limitations of the claimed ‘expansion medium’) for a range of days, including 9 and 10 days, before culturing in the second medium (paragraph [0221]). This meets the limitation of instant claim 1 of culturing for about 9 to 10 days in an expansion medium. Additionally, the cells are cultured in the second medium (meeting limitations of the claimed ‘first differentiation medium’) for a range of days, including 4, 5, and 6 days, before culturing in the third medium (paragraph [0221]), which meets the limitation of instant claim 1 of culturing in a first differentiation medium from about day 9 to 10 (as would follow according to the teaching regarding the length of time for the first step) to about day 14 to 15 (15-9 days = 6 days maximum; 14-10 days = 4 days minimum). Furthermore, the cells can be cultured in the third medium (meeting limitations of the claimed ‘second differentiation medium’) for a range of days, including 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days, or for more than 30 days (paragraph [0221]), meeting the limitation of instant claim 1 of culturing in a second differentiation medium for at least about 10 days. Since the third medium of Murphy does not comprise an aryl hydrocarbon receptor antagonist (see discussion above regarding paragraph [0009]), then the culturing in the third medium of Murphy (meeting limitations of the claimed ‘second differentiation medium’) for 10 days or more, which includes days beyond day 21, meets the limitation ‘while not comprising any aryl hydrocarbon antagonist after day 21’ of instant claim 1. It would have been within the purview of the skilled artisan to perform the culturing of Murphy according to a combination of these teachings, thereby rendering obvious the culturing days in each of an expansion medium, a first differentiation medium, and a second differentiation medium as recited in instant claim 1.
In sum, Murphy renders obvious a method meeting limitations of the claimed invention since Murphy renders obvious a method for the ex vivo production of a population of NK cells from CD34+ cells comprising culturing umbilical cord blood-derived CD34+ cells in a first medium (meeting limitations of the claimed ‘expansion medium’) and subsequently culturing the cultured CD34+ cells in a second medium (meeting limitations of the claimed ‘first differentiation medium’ which is directed to a ‘differentiation medium’), comprising:
culturing for about 9 to 10 days in the first medium rendered obvious by Murphy comprising IL-7, SCF, TPO, Flt3L, and an aryl hydrocarbon receptor antagonist (meets limitations of the claimed ‘expansion medium’);
culturing from about day 9 to 10 to about day 14 to 15 in the second medium rendered obvious by Murphy comprising IL-7, SCF, Flt3L, IL-15, and an aryl hydrocarbon receptor antagonist, but not comprising TPO and IL-12 (meets limitations of the claimed ‘first differentiation medium’); and
further culturing for at least about 10 days in the third medium rendered obvious by Murphy comprising IL-7, SCF, and IL-15 (meets limitations of the claimed ‘second differentiation medium’), while not comprising an aryl hydrocarbon antagonist after day 21,
with the proviso that the first, second, and third mediums do not comprise a glycosaminoglycan.
Murphy differs from the claimed invention in that Murphy does not expressly disclose:
the first, second, and third mediums do not contain G-CSF, GM-CSF, and IL-6, and the third medium comprises Interleukin 12 (IL-12); and
at least about 1E+9 ‘highly functional’ and ‘highly pure’ CD56-positive, Perforin-positive and Eomesodermin (EOMES)-positive NK cells are produced from a single donor, wherein ‘highly pure’ is defined as the amount of CD3-positive cells in the population of NK cells being at most about 0.1% and wherein ‘highly functional’ is defined as having at least 10% CD56-positive cells being capable of secreting IFN-gamma upon stimulation with K562 target cells.
Regarding difference (1) (the first, second, and third mediums do not contain G-CSF, GM-CSF, and IL-6, and the third medium comprises Interleukin 12 (IL-12)):
Roeven March 2014 discloses ex vivo generation of NK cells from bone marrow (BM) or mobilized peripheral blood (PB) derived stem cells (“Introduction” paragraph). The culture process comprises expanding CD34+ cells in a medium containing SCF, Flt3L, TPO, IL-7, IL-15, and StemReginin1 (SR1) (“Materials (or patients) and Methods” paragraph). It is noted that Murphy recognizes SR1 as an aryl hydrocarbon receptor inhibitor (paragraph [0022]). Subsequently, the expanded cells were differentiated into NK cells using IL-15 (“Materials (or patients) and Methods” paragraph).
Spanholtz `118 discloses a method for producing natural killer (NK) cells comprising (i) providing a sample of human CD34 positive cells; (ii) expanding said CD34 positive cells ex vivo; and (iii) culturing CD34 positive cells obtained in step ii ex vivo in an NK-cell differentiation medium, wherein the NK-differentiation medium comprises IL-12 (page 2, line 26 through page 3, line 4). Spanholtz `118 discloses that IL-12 modulates ex vivo NK cell differentiation (abstract) and provides the finished NK cell product with new and/or enhanced properties (page 2, third paragraph). In particular, Spanholtz `118 discloses an example of the NK-cell differentiation medium comprising IL-7, IL-15, SCF, and FLT-3L (page 8, lines 25-28). Further still, that example of the NK-cell differentiation medium further comprises IL-12 for the purpose of the Spanholtz `118 invention (page 9, lines 1-3).
As discussed above, it would have been prima facie obvious to select Flt-3L, SCF, and IL-7 for inclusion in the first medium of Murphy which meets limitations of the claimed ‘expansion medium.’ In making that selection, then the first medium of Murphy does not comprise G-CSF, GM-CSF, and IL-6. Moreover, before the effective filing date of the claimed invention, it would have been obvious to select Flt-3L, SCF, and IL-7 as the additional components, excluding IL-6, G-CSF, and GM-CSF as additional components, in the first medium of Murphy because Roeven March 2014 teaches a medium that contains the same components of the first medium rendered obvious by Murphy (SCF, Flt-3L, TPO, IL-7, and an aryl hydrocarbon receptor antagonist) for initial culturing of CD34+ cells for the purpose of obtaining NK cells after differentiation, while not requiring IL-6, G-CSF, and GM-CSF. There would have been a reasonable expectation in producing NK cells by the first medium rendered obvious by Murphy that does not include IL-6, G-CSF, and GM-CSF because Roeven March 2014 demonstrates that such a medium is suitable for initial culturing of CD34+ cells for NK cells production.
Additionally, as discussed above, it would have been prima facie obvious to select Flt-3L, SCF, and IL-7 for inclusion in the second medium of Murphy (meeting limitations of the claimed ‘first differentiation medium’) and selecting SCF and IL-7 for inclusion in the third medium of Murphy (meeting limitations of the claimed ‘second differentiation medium’). In making those selections, the second and third mediums of Murphy do not comprise G-CSF, GM-CSF, and IL-6. Moreover, before the effective filing date of the claimed invention, it would have been obvious to select Flt-3L, SCF, and IL-7 as the additional components, excluding IL-6, G-CSF, and GM-CSF as additional components, in the second medium of Murphy, as well as selecting SCF and IL-7 as the additional components, excluding IL-6, G-CSF, and GM-CSF as additional components, in the third medium of Murphy because Spanholtz `118 teaches an NK-cell differentiation medium comprising IL-7, IL-15, SCF, and FLT-3L for a process in which CD34+ cells are differentiated into NK cells, while not requiring IL-6, G-CSF, and GM-CSF. There would have been a reasonable expectation in producing NK cells using the second and third mediums rendered obvious by Murphy that do not include IL-6, G-CSF, and GM-CSF because Spanholtz `118 teaches that a medium comprising IL-7, IL-15, SCF, and FLT-3 (some of the same components as Murphy’s second and third mediums) that does not include IL-6, G-CSF, and GM-CSF is suitable for the differentiation of CD34+ cells into NK cells.
Further still, before the effective filing date of the claimed invention, it would have been obvious to include IL-12 in the third medium when performing the method of Murphy. One of ordinary skill in the art would have been motivated to do this because IL-12 modulates ex vivo NK cell differentiation and provides an NK cell product with new and/or enhanced properties. There would have been a reasonable expectation of producing NK cells as sought by Murphy by this modification because Spanholtz `118 teaches the inclusion of IL-12 in culture medium for differentiation of CD34+ cells into NK cells.
As such, Murphy in view of Roeven March 2014 and Spanholtz `118 renders obvious a first medium that comprises the same materials as the claimed ‘expansion medium,’ a second medium that comprises the same materials as the claimed ‘first differentiation medium,’ and a third medium that comprises the same materials as the claimed ‘second differentiation medium.’ The instant specification states, “An expansion medium according to the invention and a differentiation medium according to the invention comprise a basic medium supplemented with further components as defined herein” (page 7, lines 6-8 of substitute specification filed June 16, 2022). Since Murphy in view of Roeven March 2014 and Spanholtz `118 renders obvious a first medium comprising IL-7, SCF, TPO, Flt3L, and an aryl hydrogen receptor antagonist which are the same materials comprised by the claimed ‘expansion medium’ and identical to the expansion medium of Roeven March 2014, then the first medium rendered obvious by Murphy, Roeven March 2014, and Spanholtz `118 is directed to an ‘expansion medium’ and the culturing in the first medium results in obtaining expanded CD34+ cells (hematopoietic cells which are umbilical cord blood-derived CD34+ cells). Moreover, there would have been a reasonable expectation of obtaining expanded CD34+ cells from the culturing in the first medium rendered obvious by Murphy in view of Roeven March 2014 and Spanholtz `118 because Roeven March 2014 demonstrates that a medium comprising the same materials is directed to an expansion medium and culturing in that expansion medium results in expanding CD34+ cells.
Likewise, since Murphy in view of Roeven March 2014 and Spanholtz `118 renders obvious a third medium comprising IL-7, SCF, IL-12, and IL-15 which are the same materials comprised by the claimed ‘second differentiation medium’ and comprises some of the same materials as the NK-differentiation medium of Spanholtz `118, then the third medium rendered obvious by Murphy in view of Roeven March 2014 and Spanholtz `118 is directed to a ‘differentiation medium.’
Regarding difference (2) (Murphy does not disclose that at least about 1E+9 ‘highly functional’ and ‘highly pure’ CD56-positive, Perforin-positive and Eomesodermin (EOMES)-positive NK cells are produced from a single donor, wherein ‘highly pure’ is defined as the amount of CD3-positive cells in the population of NK cells being at most about 0.1% and wherein ‘highly functional’ is defined as having at least 10% CD56-positive cells being capable of secreting IFN-gamma upon stimulation with K562 target cells):
Spanholtz `118 teaches that early studies indicated the potency of IL-12 to modulate the differentiation towards a cytotoxic and IFN-gamma producing NK cell (page 29, lines 23-24). In an experiment, the killing efficiency against K562 cells of the IL-12 induced ex vivo generated NK cells was tested (page 26, lines 12-19). See Figure 7 and page 17, lines 10-15. The results in Figure 7 show an enhanced cytotoxic activity of the IL-12 induced ex vivo differentiated NK cells against the K562 cells.
Since Murphy in view of Roeven March 2014 and Spanholtz `118 renders obvious the same steps of the claimed invention, then the same effects, including obtaining at least about 1E+9 NK cells from a single donor that are ‘highly functional’ defined as having at least 10% CD56 positive cells being capable of secreting IFN-gamma upon stimulation with K562 target cells, are ‘highly pure’ defined as the amount of CD3-positive cells in the population of NK cells being at most about 0.1%, and are CD56-positive, Perforin-positive and EOMES-positive, would have necessarily occurred.
Based on Murphy, the produced NK cells would have been expected to be ‘highly pure’ for the invention rendered obvious by the references. Specifically, Murphy discloses that the NK cells produced by their method are CD3-negative (paragraph [0009]). Moreover, Murphy teaches that the NK cell population produced by their method comprises no less than 99% CD3-CD56+ cells (paragraph [0500]) – thus NK cells that are not CD3-negative, i.e., CD3-positive NK cells, would be 1% or less. Furthermore, Spanholtz `118 teaches that the acquisition of cytotoxic and IFN-gamma producing NK cell functions by IL-12 was correlated with induced expression of the perforin gene (page 5, lines 14-17). Since IL-12 is included in the third medium of the method rendered obvious by Murphy in view of Roeven March 2014 and Spanholtz `118, then there would have been a reasonable expectation that the produced NK cells are Perforin-positive.
Further still, there would have been a reasonable expectation that at least 10% CD56 positive cells are capable of secreting IFN-gamma upon stimulation with K562 target cells because the studies disclosed in Spanholtz `118 found that IL-12 modulates the differentiation towards an IFN-gamma producing NK cell, and that IL-12 induced ex vivo generated NK cells had an enhanced cytotoxic activity against K562 cells. Since IL-12 is included in the third medium of the method rendered by the references, then the claimed IFN-gamma secretion upon stimulation with K562 target cells would have been expected.
Therefore, Murphy in view of Roeven March 2014 and Spanholtz `118 renders obvious instant claim 1.
Regarding instant claim 5, Murphy discloses that in certain embodiments, the stem cell mobilizing compound is StemRegenin-1 (SR-1) (paragraph [0397]), which is an aryl hydrocarbon receptor inhibitor (paragraph [0022]), i.e. aryl hydrocarbon receptor antagonist. SR-1 was used in Examples 1 and 2 as a component of Stage 1 (referring to the step of culturing in a first medium of Murphy) and Stage 2 (referring to the step of culturing in a second medium of Murphy) media of the three-stage method of Murphy (paragraphs [0621]-[0622]; paragraph [0630]). Examples 1 and 2 of Murphy teach culturing CD34+ cells (Example 2 is according to Example 1; see paragraphs [0630] and [0620]). Thus, instant claim 5 (elected species SR1) is rendered obvious.
Regarding instant claim 8, see Figure 1A of Murphy, which shows the fold expansion for various culturing conditions, including ‘NK cell expansion’ for previous NK expansion media resulting in about 1000 fold expansion as compared to Murphy’s three-stage method using SR1 (an aryl hydrogen receptor antagonist) at 1 µM which resulted in 10,000 fold expansion (paragraph [0278]). The greater fold expansion using SR1 is directed to a difference falling in the range of ‘at least about two-fold higher’ of instant claim 8. In including SR1 as the aryl hydrogen receptor antagonist of the method rendered obvious by Murphy in view of Roeven March 2014 and Spanholtz `118, then the same difference in mean overall expansion as compared to when no aryl hydrocarbon receptor antagonist is used, would have been expected. Therefore, instant claim 8 is rendered obvious.
Response to Arguments
Applicant’s arguments, filed May 27, 2026, with respect to the objection to claims 1, 5-8, and 16, and the rejection under 35 U.S.C. 103 of claims 1, 5-8, and 16 as being unpatentable over Murphy in view of Roeven March 2014 and Spanholtz `118, have been fully considered and are persuasive. In particular, the objection has been overcome by the amendment to claim 1. The rejection under 35 U.S.C. 103 has been overcome by the amendment to claim 1 and the canceling of claims 6, 7, and 16. Therefore, this objection and this rejection have been withdrawn.
However, upon further consideration, a new ground(s) of rejection is made in view of the previously cited references Murphy, Roeven March 2014, and Spanholtz `118. The amendments to the claims necessitated the new grounds of rejection under 35 U.S.C. 103, as well as requiring a new claim objection.
Applicant asserts that it could not have been foreseen that the presently claimed method would result in such highly functional and pure NK-cells according to the present invention. In response to the allegation in the last Office Action that Murphy would disclose a population of CD3-negative NK-cells, Applicant submits that while Murphy mentions in paragraph [0009] that NK-cells are produced that are CD56-positive and CD3-negative and in paragraph [0500] that the NK cell population produced by their method comprises no less than 99% CD3-negative and CD56-postiive NK-cells, Applicant asserts that Figure 1B shows that the obtained population of NK-cells by Murphy are at most around 90% CD56-positive and CD3-negative. However, Murphy does not provide details regarding the three-stage method for obtaining the results shown in Figure 1B. Murphy only discusses Figure 1 in paragraph [0278]. It is unclear the exact contents of the media used for obtaining the data shown in Figure 1. The cell purity shown in Figure 1B does not mean that other combinations of media according to Murphy would not result in as high as 99% CD3-CD56+ cells as disclosed in paragraph [0500] of Murphy. Therefore, Applicant’s arguments are unpersuasive with respect to the new grounds of rejection under 35 U.S.C. 103 over Murphy in view of Roeven March 2014 and Spanholtz `118.
Conclusion
No claims are allowed.
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/SUSAN E. FERNANDEZ/Examiner, Art Unit 1651
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