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 .
Election/Restrictions
Applicant's election with traverse of Group I, claims 70-84 in the reply filed on 08/21/2025 is acknowledged. The traversal is on the ground(s) that Group II is drawn to the product made by the method of Group I and Group II is drawn to a kit comprising the elements necessary to carry out the method of Group I. This is not found persuasive because the cells of Invention II can be made by a different method and the claims of Group II read on a product of nature because the construct in the engineered iPSC is not required to be maintained. The kit of Group III has other uses that are distinct from the elected method of differnetiating iPSCs into CD7+ NK/T progenitors. Searching Group I does not require a search of the limitations of Group III. The requirement is still deemed proper and is therefore made FINAL.
Claims 85-89 are withdrawn from further consideration pursuant to 37 CFR 1.142(b), as being drawn to a nonelected invention, there being no allowable generic or linking claim. Applicant timely traversed the restriction (election) requirement in the reply filed on 08/21/2025.
Claim Rejections - 35 USC § 112
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 70,72-84 remain rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, because the specification, while being enabling for the claimed method of obtaining a population of CD7+ T/NK progenitor cells wherein the culturing the population of cells in the presence of a Notch ligand for between 9 and 17 days comprises co-culturing the population of differentiating cells in the presence of a stromal cell feeder layer, does not reasonably provide enablement for carrying out this step without stromal cells. 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/or use the invention commensurate in scope with these claims.
Enablement is considered in view of the Wands factors (MPEP 2164.01(a)). The court in Wands states: "Enablement is not precluded by the necessity for some experimentation such as routine screening. However, experimentation needed to practice the invention must not be undue experimentation. The key word is 'undue,' not 'experimentation.' " (Wands, 8 USPQ2d 1404). Clearly, enablement of a claimed invention cannot be predicated on the basis of quantity of experimentation required to make or use the invention. "Whether undue experimentation is needed is not a single, simple factual determination, but rather is a conclusion reached by weighing many factual considerations." (Wands, 8 USPQ2d 1404). The factors to be considered in determining whether undue experimentation is required include: (1) the quantity of experimentation necessary, (2) the amount or direction or guidance presented, (3) the presence or absence of working examples, (4) the nature of the invention, (5) the state of the prior art, (6) the relative skill of those in the art, (7) the predictability or unpredictability of the art, and (8) the breadth of the claims. While all of these factors are considered, a sufficient amount for a prima facie case are discussed below.
The nature of the invention relates to increasing efficiency of directing differentiation of pPSCs to become CD7+ T/NK progenitors by early/premature induction of Notch signaling along with a culture protocol that guides pPSCs to become CD7+ T/NK progenitors. The invention further matures the progenitors into either T or NK cells by co-culture with different densities of OP9 stromal cells that secrete a Notch ligand.
The claims are drawn to generation of T/NK progenitors and recites a step of culturing the cells in the presence of a Notch ligand for between 9 and 17 days.
Oh (2019, Mol. Cells, 42:200-209, IDS), for example, reviews the directed differentiation of pluripotent stem cells and supports that a variety of cell types, including neurons, pancreatic b-cells, can be reliably obtained including neurons, pancreatic-cells, skeletal and cardiac muscle and hepatocytes by varying culture conditions and forced expression of different sets of genes. Ebrahimi (2020, Stem Cell Research and Therapy, 11:483, pages 1-13, IDS) reviews the directed differentiation of pluripotent stem cells into erythroid cells and teaches,
“This developmental procedure is controlled by cell-cell/cell-matrix interactions along with several cytokines and growth factors including IL-3, IL-6, erythropoietin (EPO) (the main erythropoietic stimulating hormone), EPO-receptor, members of the transforming growth factor-β (TGF-β), activin A, activin receptor-II, Flt3 ligand (Flt3-L), vascular endothelial growth factor (VEGF), stem cell factor (SCF), thrombopoietin (TPO), and granulocyte colony-stimulating factor (G-CSF)” (Ebrahimi, 2).
With regard to cardiac differentiation, Fujita (2019, Stem Cells, 37:992-1002; IDS) states, “complex genetic and epigenetic circuits coordinately regulate cardiac differentiation and maturation from hPSCs “(see page 992). Gene expression is not the only factor in controlling differentiation as Podkalicka (2020, Biomolecules, 10:1614, pages 1-30; IDS) teaches the importance of oxygen levels in differentiation as early embryogenesis occurs in relatively hypoxic conditions (see page 3 and Section 5.1, page 10).
The specification teaches directing differentiation of pluripotent stem cells to a population of cells comprising a specific CD7+ T/NK progenitor cells. This involves tight regulation of gene expression and culture conditions including media components and oxygen levels. The specification refers to this culture process as an iT protocol. This protocol is diagrammed as follows:
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Herrerra (frontiers in immunology, 2017,8:755, pages 1-10) compared stromal cell layers for the differentiation of NK cells and found OP9 to be superior and that M2-10B4 provides good support for cells in long-term culture and OP9 is superior to M2-10B4 cells for the generation of NK cells, exemplifying the importance of the stromal cells in this process. Roth (J Mol Med (2007) 85:1047–1056) teaches that stromal cells secrete factors and carry cell surface proteins that are important in NK cell differentiation. The in vivo environment for NK/T cell differentiation comprises stromal cells. Stromal cells provide many signals, not just the notch ligand (see Abel, 2018, Front Immunol., 9:1869, pages 1-23). Addition of notch ligand to the environment created by stromal cells, can lead to T cell development, as supported by the Specification. However, there is nothing of record to support that Notch ligand alone will lead cells to become CD7+ NK/T precursors without the presence of stromal cells. See also Miller (1994, Blood, 83:9,1-8).
Thus, it would have required undue experimentation to carry out the method as claimed without the presence of stromal cells.
Applicant’s Remarks and the Mostoslavsky declaration dated 05/18/2026 have been fully considered but are not persuasive. The declaration points to evidence published in Heinze (2022, Appendix B dated 05/18/2026). The Heinz publication and the declaration support that CD7+ cells can be obtained in culture with a solid surface with immobilized Notch ligand when culture is carried out for 40 days. This does not support obtaining CD7+ T/NK progenitor cells in such culture conditions for 9-17 days or in culture with conditioned media (generic or OP9 conditioned) as encompassed by the claims. The Heinze reference teaches at page 2625, para 2, that the T cell lineage begins to emerge at day 20. Thus, this brings into question whether 40 days on a substrate with immobilized Notch ligand will result in a population of T-cells or T/NK progenitors, as claimed. At page 2622, last paragraph, Heinze states that the protocol using OP9 feeders was adapted with coated plates optimized for the differentiation of T lymphocytes and references the experimental procedures. The experimental procedures and supplemental Experimental procedures don’t appear to address what the optimizations were. The only feeder-free conditions referenced are with regard to T-cell maturation while the claims are to deriving T/NK progenitors, cells that have potential to differentiate into either T cells or NK cells. Thus, the teachings of Heinze support that additional experimentation to transition from OP9-supported culture to feeder-free was needed. Neither Heinze nor the Mostoslovsky declaration support the claimed conditioned media conditions.
The Supplemental Experimental Procedures found by the Examiner to be relevant are below. Applicant is invited to redirect the Examiner to other teachings that support their remarks. It is noted, however, that changes to the protocol to adapt it to feeder-free conditions need support in the specification.
Differentiation into hematopoietic progenitors and T/NK cells Progenitor identity was checked by flow cytometry for CD34, CD45, and CD235a. Higher percentages of CD34/45 double positive cells negative for CD235a were considered optimal. T cell differentiation was accomplished using the OP9-DLL4:MHCII feeder layer as described above. Co-cultures were initiated in 10cm plates in T cell differentiation media containing αMEM, 20%FBS, glutamax, aMTG, ascorbic acid, 5ng/ml IL-7, 5ng/ml FLT3L, and 50ng/ml SCF. Cultures were passaged onto new feeder cells every 7 days. After the first 7 days, SCF was removed and 10υM dexamethasone was added. For passaging, the OP9-DLL4:MHCII feeder layer was rinsed with existing media several times, and then the entire monolayer was broken up using the 10ml serological pipette. The resulting suspension was passed through a 40υM cell strainer, centrifuged, and resuspended in new T cell media over a new feeder layer. Progress of T cell differentiations were followed by weekly flow cytometry checking CD7, CD4, CD8, and CD5 with additional markers as needed (including CD3, CD1a, CD56, and others). For stimulation assays, day 40 bulk iPSC-derived T cells were passaged onto new 10cm dishes of OP9-DLL4:MHCII feeder cells in OP9 media supplemented with 5ng/ml IL-7, 5ng/ml FLT3L, 10υM dexamethasone, 5ng/ml IL-2 and either 25ul/ml ImmunoCult™ Human CD3/CD28 T Cell Activator (StemCell), or PMA/ionomycin (25ng/ml and 250ng/ml respectively). Cells were fed once during the week of stimulation and analyzed by flow cytometry after 7 days. For the feeder-free T cell maturation, day 12 CD34+ progenitor cells were enriched using the EasySep™ Human CD34 Positive Selection Kit II (StemCell), and plated at 25 x 103 cells/well in 24w non tissue-culture treated plates coated with StemSpan™ Lymphoid Differentiation Coating Material (StemCell) in StemSpan™ Lymphoid Progenitor Expansion media (StemCell). Seven days later, the cells were transferred to newly coated wells in expansion media. On day 14 of T cell culture (day 26 overall), the resulting cells were transferred to newly coated wells in StemSpan™ T Cell Progenitor Maturation media (StemCell). Cells were fed twice weekly until day 40 overall, when they were either analyzed by flow cytometry or stimulated with ImmunoCult™ Human CD3/CD28 T Cell Activator (StemCell) at 12.5ul/ml in the presence of 10ng/ml IL-15. Stimulated cells were analyzed by flow cytometry one week later for activation markers CD69 and CD25. For the analysis in Figure 2 E, pre-stimulation cells were stained with cell trace violet (Invitrogen) per the manufacturer’s protocol and cellular proliferation measured by flow cytometry one week later in the live CD7+/CD5+ population. For all flow cytometry experiments, data was analyzed in Flow Jo (BD Biosciences).
New Matter
Claims 70,72-84 are rejected under 35 U.S.C. 112, first paragraph, as containing subject matter which was not described in the specification in such a way as to reasonably convey to one skilled in the relevant art that the inventor(s), at the time the application was filed, had possession of the claimed invention. 37 CFR 1.118 (a) states that “No amendment shall introduce new matter into the disclosure of an application after the filing date of the application”.
Claim 70 is drawn to a method where the population of cells is cultured from 9 to 18 days or until the cells express CD7. The specification provides no implicit or explicit support for the context of the breadth of culture until the cells express CD7. The specification has only provided support for obtaining progenitors when the cells are cultured in claim 70, step d, for 9 to 17 days and not indefinitely or not for any extended period where CD7 is expressed. Applicants are reminded that it is their burden to show where the specification supports any amendments to the claims. See 37 CFR 1.121 (b)(2)(iii), the MPEP 714.02, 3rd paragraph, last sentence and also the MPEP 2163.07, last sentence.
MPEP 2163.06 notes “If new matter is added to the claims, the examiner should reject the claims under 35 U.S.C. 112, first paragraph - written description requirement. In re Rasmussen, 650 F.2d 1212, 211 USPQ 323 (CCPA 1981).” MPEP 2163.02 teaches that “Whenever the issue arises, the fundamental factual inquiry is whether a claim defines an invention that is clearly conveyed to those skilled in the art at the time the application was filed...If a claim is amended to include subject matter, limitations, or terminology not present in the application as filed, involving a departure from, addition to, or deletion from the disclosure of the application as filed, the examiner should conclude that the claimed subject matter is not described in that application. MPEP 2163.06 further notes “When an amendment is filed in reply to an objection or rejection based on 35 U.S.C. 112, first paragraph, a study of the entire application is often necessary to determine whether or not “new matter” is involved. Applicant should therefore specifically point out the support for any amendments made to the disclosure [or point to case law supporting incorporation of such a limitation as in the instant case]” (emphasis added).
Conclusion
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VALARIE E. BERTOGLIO, Ph.D.
Examiner
Art Unit 1632
/VALARIE E BERTOGLIO/Primary Examiner, Art Unit 1632
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