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
The instant application is a national stage entry of PCT application PCT/US2022/018991, filed 08/29/2023 under 35 USC 371. Acknowledgement is made of the applicant’s claim for benefit to prior-filed U.S. provisional patent applications 63/157, 112, which was filed 03/05/2021.
Election/Restrictions
Applicant's election with traverse of group I, claims 1-2, 9, 12, 18-19, 27, 32-33, 37, 52, 55-56, 62-63, and 73, in the reply filed on 06/17/2026 is acknowledged. The traversal is on the grounds that there would be no undue search and examination burden for examining groups II and III (Remarks, p8); in addition, the cited reference Jacobs et al. do not disclose or suggest any methods of producing a population of CD56+ NK cells as claimed, let alone features such as the specifically claimed inhibiting and differentiating steps, stromal-free methods of NK cell differentiation, or the population of CD56+ NK cells comprises at least 60% CD56+ NK cells (Remarks, p9).
Applicant’s arguments are fully considered but they are not persuasive. Instant application is a national stage filing of an international application. Burden is not a consideration in a finding of lack of inventive unity; rather, according to M.P.E.P. §1850, the only consideration is whether the inventions share a special technical feature. In instant case, as stated in the office action mailed 2/20/2026, the shared technical feature is a population of CD56+ NK cells, this technical feature is not a special technical feature as it does not make a contribution over the prior art in view of Jacobs et al.. Jacobs et al. teach the reconstitution of NK cells in patients after bone marrow transplantation (BMT) was analyzed. In contrast to normal NK cells, the presence of two distinct NK cell subpopulations, namely, CD16+ CD56+dim and CD16- CD56+bright cells could be detected (p3239, left column). Herein the CD56+ NK cells in Jacob et al.’s teaching read on the technical feature. Thus groups I-III lack unity of invention.
The requirement is still deemed proper and is therefore made FINAL. Accordingly, claims 1-2, 9, 12, 18-19, 27, 32-33, 37, 52, 55-56, 62-63, 73 and 132 (new added claim which depends from claim 1) have been considered on the merits. Claims 118, 119, 122 and 130 are withdrawn from consideration pursuant 37 CFR 1.142(b).
Claim Objections
Claim 1 is objected to because of the following informalities:
Claim 1 recites multiple abbreviations including “NK” (in line 1), “G9a” and “GLP “. Abbreviations should be spelled out at the first encounter in the claims.
Appropriate correction is required.
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 52, 55 and 56 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.
Claim 52 recites the limitation “the resultant population of CD34+hemogenic endothelium” in lines 1-2. There is insufficient antecedent basis for this limitation in the claim.
Claim 55 recites the limitation "the pluripotent stem cells " in line 2. There is insufficient antecedent basis for this limitation in the claim.
Claim 55 recites the limitation " the resultant population of CD34+ hemogenic endothelium" in line 2. There is insufficient antecedent basis for this limitation in the claim.
Claims 52 and 55 recite “the resultant population of CD34+ hemogenic endothelium” renders instant claims indefinite. It is not clear the phrase “the resultant population of CD34+ hemogenic endothelium” means a CD34+ hemogenic endothelium derived from the pluripotent stem cells, or a CD34+ hemogenic endothelium derived from the pluripotent stem cells and inhibited by a histone methyltransferase.
Claim 56 depends from claim 55, and thus inherit the deficiency and are rejected on the same basis.
Claim Interpretation
As stated above, claims 52 and 55 are indefinite. In the interest of compact prosecution, the claims are interpreted as the population of CD34+ hemogenic endothelium derived from the pluripotent stem cells.
Claim 2 recites “aggregation media”. The specification provides the definition of “aggregation media”: the term "aggregation media" refers to a series of cell culture media that are used to differentiate a population of pluripotent stem cells, or a derivative cell population thereof such as embryoid bodies, into a population of CD34+ hemogenic endothelium (parag 00462). The claim is interpreted in light of the definition in the Specification.
Claim Rejections - 35 USC § 103
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
Claims 1, 2, 9, 12, 18, 19, 27, 33, 37, 52, 62, 63, 73 and 132 are rejected under 35 U.S.C. 103 as being unpatentable over Valamehr et al. (US 2018/0072992 A1, cited in IDS) in view of Vo et al. (US 2019/0225940 A1, as cited in IDS).
Valamehr et al. teach culture platforms, cell media, and methods of differentiating pluripotent cells into hematopoietic cells (Abstract).
Regarding claim 1, it is noted that the preamble “producing a population of CD56+ NK cells” is an intended use. The purpose or intended use of the invention, rather than any distinct definition of any of the claimed invention’s limitations, then the preamble is not considered a limitation and is of no significance to claim construction. See MPEP 2111.02. Valamehr et al. teach methods and compositions for the generation of hematopoietic cell lineages through definitive hemogenic endothelium (HE) and definitive hematopoietic stem cells (HSC) derived from pluripotent stem cells (parag 0008). Valamehr et al. teach differentiation towards definitive hemogenic endothelium resulting in the derivation of hematopoietic stem cells and differentiated progeny such as T, B, NKT and NK cells (parag 0011). Valamehr et al. teach in Example 17, to demonstrate the capacity of hiPSC-derived HE cells in giving rise to iNK cell progenitors in the stromal-free differentiation platform, Day 10 CD34+ THE sorted cells were cultured in iNK-A2 media in cultures containing DLL4 protein or control protein. Plate bound DLL4 supports the differentiation of CD56+CD7+CD161+ NK cell progenitors but not CDllb+ myeloid cells (parag 0345). DLL4 is one of Notch activators (see parag 0171). Therefore Valamehr et al. teach a method comprising b) differentiating the population of CD34+ hemogenic endothelium in Natural Killer (NK)- cell-differentiation media in the presence of a Notch ligand (herein DLL4) for a sufficient time to promote differentiation into a population of CD56+ NK cells.
Valamehr et al. do not teach step (a), i.e., (i) inhibiting a histone methyltransferase in a population of CD34+ hemogenic endothelium. However, this was disclosed by Vo et al. at the time of instant invention.
Vo et al. teach production methods and compositions of engineered immune cells, such as B or T lymphocytes, from limited lineage myeloid progenitor cells, or from pluripotent stem cells, or from multilineage hematopoietic progenitor cells comprising the addition of various cell differentiation transcription factors and inhibiting epigenetic histone methylations in said cells (Abstract).
Regarding claim 1, Vo et al. teach that inhibition of specific histone modifying enzymes targeting H3K9 and H3K27 promotes lymphoid potential of hematopoietic progenitors derived from pluri potent stem cells. The histone modifying enzymes are histone lysine methyltransferases (parag 0311).
It would have been prima facie obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify Valamehr et al.’s methods of differentiating hiPSC-derived hemogenic endothelium cells to NK cells, and inhibit a histone methyltransferase in the CD34+ hemogenic endothelium cells as taught by Vo et al.. The skilled artisan would have been motivated to inhibit a histone methyltransferase in a population of CD34+ hemogenic endothelium during the differentiation of hiPSC-derived HE cells to NK cells since Vo et al. teach inhibition of histone methyltransferases targeting H3K9 and H3K27 promotes lymphoid potential of hematopoietic progenitors derived from pluripotent stem cells (see parag 0311). There would be a reasonable expectation of success of inhibiting a histone methyltransferase in a population of CD34+ hemogenic endothelium since Vo et al. teach the histone methyltransferase, i.e., H3K27 histone methyltransferase is EZHl (see parag 0319).
Regarding claim 2, Valamehr et al. teach in Example 2, hiPSCs are cultured in iHSC-A medium (see parag 0176), the culture was subsequently switched to iHSC-B (see parag 0177), 48hrs post the initiation of differentiation, followed by a switch to iHSC-C (see parag 0178) on day 4-5 post initiation of differentiation (parag 0316, also figure 1). Cells are analyzed for CD34 surface expression. Using FACS Aria or MACS enrichment, CD34 positive cells were collected (parag 0317). This teaching reads on the step of differentiating iPSCs to CD34+ hemogenic endothelium as recited in in instant claim.
Regarding claim 9, Valamehr et al. teach in some embodiments, one or more of the Notch ligands can be introduced as soluble peptide, or immobilized on a solid material. The solid material may include, but not limited to, polystyrene plates, or beads. The beads for Notch ligands immobilization may be agarose beads, magnetic beads, and latex beads. In one embodiment, the Notch ligand peptide is conjugated/immobilized to beads. In another embodiment, the Notch ligand peptide is conjugated/immobilized to the surface of a polystyrene plate (parag 0171). This teaching reads on the Notch ligand is attached to a cell culture dish (which is a polystyrene plate). In addition, Valamehr et al. teach in Example 17, the Plate bound DLL4 is used (see parag 0345), and DLL4 is one of Notch activators (see parag 0171).
Regarding claim 12, Valamehr et al. teach Example 17, stromal-free differentiation cultures for the generation of T and NK cells. Day 10 CD34+ THE sorted cells were cultured in iNK-A2 media in cultures containing DLL4 protein or control protein. Then the hiPSC-derived CD34+ cells were differentiated towards the NK cell lineage for 20 days and then placed in suspension culture for further
maturation (parag 0345). The method des not comprise co-culturing with a stromal cell expressing a Notch ligand or does not comprise co-culturing with OP9-DLL1 cells or OP9-DLL4 cells, as recited in instant claim. Moreover, the Notch ligand used in the method is DLL4.
Regarding claim 18, Valamehr et al. teach specific to NK cells, CD34 positive cells were treated with differentiation medium including IL15, iNK-A1 medium for approximately 10 days (FIG. 2) and switched to iNK-B1 medium (FIG. 2) for an additional 10-20 days (see CD56 positive population in FIG. 10) (parag 0322). Therefore it takes about 20-30 days to differentiate to CD56+ NK cells, which overlaps with the limitation of “4 weeks” as recited in instant claim. In the case where the claimed ranges "overlap or lie inside ranges disclosed by the prior art" a prima facie case of obviousness exists. See MPEP 2144.05(I).
Regarding claim 19, Valamehr et al. teach in Example 13, sorted CD34+ HE cells were further differentiated towards the T and NK lymphoid lineages. Specific to NK cells, upon sorting, the HE cells were cultured as aggregates for 16 hours on low attachment tissue culture plates in iNK-A serum-free differentiation media containing BMP4, SCF, IL3, IL15, Flt3L and IL7 (FIG. 14)(parag 0339). This teaching reads on the NK-cell-differentiation media is serum-free, and comprises SCF, FLT3 and IL7, as recited in instant claim.
Regarding claim 27, following the discussion above, Valamehr et al.. teach in Example 13, specific to NK cells, upon sorting, the HE cells were cultured as aggregates for 16 hours on low attachment tissue culture plates in iNK-A serum-free differentiation media containing BMP4, SCF, IL3, IL15, Flt3L and IL7 (parag 0339). After 16hrs the aggregated cells were transferred to adherent cultures containing DLL4-expressing stromal cells in iNK-B differentiation media containing SCF, IL3, IL15, Flt3L and IL7. After 5 days the iNK-B medium was maintained to complete NK cell differentiation. After approximately 10-15 days of culture (post HE isolation) the culture was assessed for the generation of NK cell progenitors followed by mature NK subsets following an additional 10-15 days of culture (parag 0339). Valamehr et al. teach the iNK-A1 medium (p20, table 6) and iNK-B1 culture medium (p20, table 7), which comprising 0.1-25 ng/ml of IL15, therefore the IL15 (0.1-25 ng/ml) is used for at least 15-20 days of culture (post HE isolation), reads on “the NK-cell-differentiation media further comprises 10 ng/mL interleukin-15 (IL-15) starting after at least the first 2 weeks of differentiating in the NK-cell-differentiation media” as recited in instant claim.
Regarding claim 33, following the discussion of claim 2, Valamehr et al. teach in Example 1, hiPSC generation. Somatic cells including fibroblast and blood cells were induced to reprogram towards a pluripotent state using various factor combinations including OCT4/SOX2/LargeT, OCT4/SOX2 or OCT4/SOX2/NANOG/LargeT in the presence of reprogramming medium containing ROCK, MEK,GSK3 pathway and TGF~ receptor inhibitors (parag 0314, Example 1). This teaching reads on the population of pluripotent stem cells comprises induced pluripotent stem cells (iPS cells), and the induced pluripotent stem cells are produced by introducing into mature cells (i.e., somatic cells) OCT4 and SOX2, as recited in instant claim.
Regarding claim 37, following the discussion above, Valamehr et al. teach Example 2, hematopoietic differentiation: To initiate differentiation towards hematopoietic cell lineage, naive hiPSCs were seeded as a mono layer in the maintenance medium (parag 0316). On day 9 (this time point can be extended, optimally by day 14), the cells were dissociated into single cells and analyzed for CD34 and CD45 surface expression (parag 0317). This teaching reads on the hiPSCs is differentiated in to population of CD34+ hemofenic endothelium using 2D adherent culture, and the sufficient time is at least 8 days (i.e., 9 days or 14 days).
Regarding claim 52, Valamehr et al. teach CD34+ cells can be isolated by FACS (parag 334). Valamehr et al. also teach one or more cell populations, cell lines, or clonal cells of (i) pluripotent stem cell-derived CD34+ HE cells (iCD34), wherein the iCD34 cells have capacity to differentiate into multipotent progenitor cells, and wherein the iCD34 cells are CD34+CD43-; (ii) pluripotent stem cell-derived definitive hemogenic endothelium (iHE), wherein the iHE cell line or clonal cells are CD34+; (iii) pluripotent stem cell-derived definitive HSCs (iHSC), wherein the iHSC is CD34+CD45+, and is suitable for long-term engraftment; (iv) pluripotent stem cell-derived multipotent progenitor cells (iMPP), wherein the iMPP cells are CD34+CD45+ (parag 0298). This teaching reads on the cell population of iCD34 and iHE are CD34+ but CD45 negative or low.
Regarding claim 62, Valamehr et al. do not teach he histone methyltransferase catalyzes the addition of methyl group to the histone 3 lysine residue 9 (H3K9) and/or histone 3 lysine residue 27 (H3K27). However, Vo et al. teach the histone methyltransferase catalysis the addition of methyl group to the hi stone H3 lysine residue 9 (H3K9) and/or histone H3 lysine residue 27 (H3K27) (parag 0065).
Regarding claim 63, following the discussion above, Vo et al. teach the histone methyltransferase small molecule inhibitor include but are not limited to AMI-1, A-366, BIX-01294, BIX01338, BRD4770, chaetocin, UNC0224, UNC0631, UNC0638, UNC0642, UNC0646, EPZ5676, EPZ005687, GSK343, EPZ-6438, 3-deazaneplanocin A (DZNeP) HCl, UNC1999, MM-102, SGC 0946, Entacapone, EPZ015666, UNC0379, Ell, MI-2 (Menin-MLL Inhibitor), MI-3 (Menin-MLL Inhibitor), PFI-2, GSK126, EPZ004777, BRD4770, and EPZ-6438 (parag 0072).
It would have been prima facie obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify Valamehr et al.’s methods of differentiating hiPSC-derived HE cells to NK cells, and inhibit histone methyltransferase H3K9 and/or H3K27 by small molecule inhibitor such as BIX-01294 as taught by Vo et al.. The skilled artisan would have been motivated to inhibit histone methyltransferase H3K9 and/or H3K27 by small molecule inhibitor such as BIX-01294 in a population of CD34+ hemogenic endothelium during differentiating hiPSC-derived HE cells to NK cells since Vo et al. teach inhibition of specific histone modifying enzymes targeting H3K9 and H3K27 promotes lymphoid potential of hematopoietic progenitors derived from pluripotent stem cells (parag 0311). There would be a reasonable expectation of success of inhibiting histone methyltransferase H3K9 and/or H3K27 by small molecule inhibitor such as BIX-01294 in a population of CD34+ hemogenic endothelium since Vo et al. teach the available histone methyltransferase small molecule inhibitors (see parag 0072).
Regarding claim 73, Vo et al. teach in vitro screen for epigenetic modifiers that restrict definitive lymphoid potential (parag 0112) in figure 1, which includes MBD protein MBD2 and histone methyltransferases such as SUV39H1 and SMYD2.
It would have been prima facie obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify Valamehr et al.’s methods of differentiating hiPSC-derived HE cells to NK cells, and inhibit an epigenetic regulator such as MBD protein or histone methyltransferases such as SUV39H1 and SMYD2as taught by Vo et al.. The skilled artisan would have been motivated to inhibit an epigenetic regulator such as MBD protein or histone methyltransferases in a population of CD34+ hemogenic endothelium during differentiating hiPSC-derived HE cells to NK cells since Vo et al. teach epigenetic regulation maintains cell identity during development (parag 0243), therefore regulating epigenetic factors can regulate cell fate (i.e., promotion of the differentiation of hiPSC-derived HE cells to NK cells). There would be a reasonable expectation of success of inhibiting an epigenetic regulator such as MBD protein or histone methyltransferases in a population of CD34+ hemogenic endothelium since Vo et al. teach the epigenetic regulators such as MBD protein or histone methyltransferases (see figure 1), as well as the inhibitors such as shRNAs target to the epigenetic regulators (see i.e., figure 1).
Regarding claim 132, Valamehr et al. teach figure 22B, the CD56+ NK cells comprise more than 60% CD56+ NK cells.
Claims 1, 2, 9, 12, 18, 19, 27, 32, 33, 37, 52, 62, 63, 73 and 132 are rejected under 35 U.S.C. 103 as being unpatentable over Valamehr et al. (US 2018/0072992 A1, cited in IDS) in view of Vo et al. (US 2019/0225940 A1, as cited in IDS), as applied to 1, 2, 9, 12, 18, 19, 27, 33, 37, 52, 62, 63, 73 and 132 above, further in view of Naume et al. (J Immunol Methods. 1991 Jan 24;136(1):1-9).
The teaching of Valamehr et al. in view of Vo et al. is set forth above.
Regarding claim 32, Valamehr et al. and Vo et al. do not teach a step of CD56+ NK cell enrichment. However, this was disclosed by Naume et al. at the time of instant invention.
Naume et al. teach the immunomagnetic isolation of human natural killer (NK) and lymphokine activated killer (LAK) cells (Abstract).
Regarding claim 32, Naume et al. teach antibodies against CD56 and sheep anti-mouse IgG-coated magnetic monodisperse particles were used for the positive isolation of CD56 + cells from unstimulated mononuclear cells (PBMC). A highly enriched population of CD56 + cells (~< 3% contaminating cells) was obtained with this method (Abstract).
It would have been prima facie obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify Valamehr et al.’s methods of differentiating hiPSC-derived HE cells to NK cells, and use magnetic beads for immunomagnetic isolation of CD56+ NK cells as taught by Naume et al.. The skilled artisan would have been motivated to use magnetic beads for immunomagnetic isolation of CD56+ NK cells since Naume et al. teach this method can yield a highly enriched population of CD56 + cells (Abstract). There would be a reasonable expectation of success of using magnetic beads for immunomagnetic isolation of CD56+ NK cells since Naume et al. teach the method (see i.e., p2-3).
Claims 1, 2, 9, 12, 18, 19, 27, 33, 37, 52, 55, 56, 62, 63, 73 and 132 are rejected under 35 U.S.C. 103 as being unpatentable over Valamehr et al. (US 2018/0072992 A1, cited in IDS) in view of Vo et al. (US 2019/0225940 A1, as cited in IDS), as applied to 1, 2, 9, 12, 18, 19, 27, 33, 37, 52, 62, 63, 73 and 132 above, further in view of Glienke et al. (Front Pharmacol. 2015 Feb 12;6:21).
The teaching of Valamehr et al. in view of Vo et al. is set forth above.
Regarding claims 55 and 56, Valamehr et al. and Vo et al. do not teach genetically modifying the cells such as the resultant population of CD56+ NK cells, such as expressing a chimeric antigen receptor (CAR). However, this was disclosed by Glienke et al. at the time of instant invention.
Glienke et al. summarize the data on CAR expressing NK cells focusing on the possible advantage using these short-lived efector cells and discuss the necessity of suicide switches (Abstract).
Regarding claims 55 and 56, Glienke et al. teach GMP-conform manufacturing of chimeric antigen receptor expressing natural killer cells with/without suicide genes (figure 1). The CAR cell engineering by retro-/lentiviral transduction is a genetically modifying of the CD56+ NK cells, which expressing a chimeric antigen receptor on NK cells.
It would have been prima facie obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify Valamehr et al.’s methods of differentiating hiPSC-derived HE cells to NK cells, and genetically modifying the resultant population of CD56+ NK cells such as expressing a chimeric antigen receptor (CAR) in said CD56+ NK cells as taught by Glienke et al.. The skilled artisan would have been motivated to genetically modify the resultant population of CD56+ NK cells since Glienke et al. teach CAR-modified NK cells can represent a complementary therapeutic
option to CAR-expressing T cells, and CAR-modified primary human NK cells redirected against CD19, CD20, CD244, and HER2 as well as CAR-expressing NK-92 cells targeting a broader range of cancer antigens (p2, left column). There would be a reasonable expectation of success of genetically modifying the resultant population of CD56+ NK cells since Glienke et al. teach the genetic modification method, i.e., using retro- or lentivirus-based vectors (p2, right column).
Conclusion
No claims are allowed.
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/Q.G./Examiner, Art Unit 1633
/FEREYDOUN G SAJJADI/Supervisory Patent Examiner, Art Unit 1699
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