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 .
Applicant's arguments and amendments to the claims filed on 10-16-2025 have been received and entered. Claims 2-3 have been amended. Claims 1-19 are pending.
Election/Restrictions
Applicant’s election without traverse of Group II (claims 2, 3, and 17) in the reply filed on 04-08-2024 is acknowledged.
Claims 1, 4-16, 18-19 are withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected subject matter, there being no allowable generic or linking claim. Election was made without traverse in the reply filed on 04-08-2024.
Claims 2, 3, and 17 are under consideration.
Priority
This application is a 371 of PCT/US2019/057929 filed on 10/24/2019 that claims priority from US provisional application 62/749,947 filed on 10/24/2018.
Information Disclosure Statement
The information disclosure statements (IDS) submitted on 04-10-2025, 09-24-2025, 11-21-2025 are in compliance with the provisions of 37 CPR 1.97. Accordingly, the information disclosure statements have been considered by the examiner.
Maintained in modified form - Claim Rejections - 35 USC § 103 - necessitated by amendments
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 2-3, 17 are rejected under 35 U.S.C. 103 as being unpatentable over Lu et al (Regen. Med. (2013) 8(4), 413–424, https://doi.org/10.2217/rme.13.36) (Applicant own work) in view of Amit et al (Pub. No.: US 2019/0284526 A1, Provisional application No .62/363,879 , filed on Jul. 19, 2016).
Claims interpretation:
The specification of the claimed invention teaches that the term "sphere" or "spheroid" means a three-dimensional spherical or substantially spherical cell agglomerate or aggregate (Page 12, lines 12-13). Since aggregate is clump of cells, cell clumps are interpreted as spheres.
According to the specification of the claim invention: "Hemogenic endothelial cells" refers to cells differentiated in vitro from PSCs that acquire hematopoietic potential and can give rise to multilineage hematopoietic stem and progenitor cells (line 10-11, page 15). Therefore, hemogenic endothelial cells (HECs) are interpreted as endothelial cells derived from PSCs that have the potential to become a hematopoietic stem and progenitor cells.
According to step (b) of claims 2-3, second spheres are interpreted as first spheres with PSCs being differentiated into HECs.
According to the specification of the claim invention: Scaffolds mimic the extracellular matrix of the native tissue, recapitulate the in vivo milieu and allow cells to influence their own microenvironments (line 34-35, page 11) …. the scaffold can be selected to mimic the in vivo niche to promote lineage specification such as NK cells, T lymphocytes, etc. (line 10-11, page 12). Therefore, “Scaffolds” in claims 2-3 are interpreted as Matrigel matrix which contains extracellular matrix or feeder-free culture systems.
Regarding to preamble of claims 2, 3, Lu et al teaches an efficient method to reproducibly generate large numbers of hemangioblasts (progenitor of hematopoietic cell lineages) from multiple hESC lines using an in vitro feeder and serum-free differentiation system. These cells can differentiate into multiple hematopoietic cells, including erythrocytes, megakaryocytes/platelets, macrophages, granulocytes, dendritic cells and natural killer cells (Page 414, left column).
Regarding to claims 2(a) and 3(a), Lu et al teach hESCs and iPSCs were grown on feeder-free culture, both hESCs and iPSCs were plated and grown on Matrigel in mTeSR1 medium (Page 414, right column, 2nd para.). Once collected, uniform cell clumps were created by gently pipetting the cell suspension to form clumps less than 100 µm in diameter (Page 414, right column, last para.).
However, Lu et al does not specifically teaches the use of carrier-free 3-dimensional (3D) sphere culturing under continuous agitation. Amit et al cure the deficiency.
Amit et al teach culture media for culturing pluripotent stem cells in a suspension culture devoid of substrate adherence (carrier-free) (Abstract). The culture is “devoid of substrate adherence” in which the pluripotent stem cells are capable of expanding without adherence to an external substrate such as components of extracellular matrix, a glass microcarrier or beads ([0276], page 15). The culture medium used by the method of this aspect of the present invention is serum-free, xeno-free , feeder-free (i.e., devoid of feeder cells) and protein carrier-free ([0283], page 15).
Amit et al teach “culturing according to this aspect of the present invention is effected using a controlled culturing system (preferably a computer-controlled culturing system ) in which culture parameters such as temperature, agitation , pH , and pO2 , is automatically performed using a suitable device” ([0310], page 17), and “for dynamic culturing of PSCs, the PSCs can be cultured in spinner flasks” ([0311], page 17), and “Culturing the cells with dynamic system (spinners) resulted in cells exceeding cell concentrations of 6 million cells per ml (Data not shown ). Cells cultured continuously with spinner flasks for more than a month (4 -5 passages)maintained the expression of pluripotency markers Oct4 and SSEA4” ([0434], page 17).
Therefore, it would have been prima facie obvious for a person of ordinary skill in the art before the effective filing date of the rejected claims to combine the teachings of prior art to modify the method of Lu et al by using carrier-free and feeder-free sphere culturing as taught by Amit et al as instantly claimed, with a reasonable expectation of success. Said modification amounting to combining prior art elements according to known methods to yield predictable results. One of ordinary skill in the art would be motivated to culture pluripotent stem cells in carrier-free and feeder-free sphere suspension culture because Amit et al provide advantages of hPSCs maintained all PSCs features after prolonged culture, including the developmental potential to differentiate into representative tissues of the three embryonic germ layers, unlimited and undifferentiated proliferative ability and maintenance of normal karyotype ([0087], page 5). Amit et al stated that their culture medium is capable of maintaining human pluripotent stem cells in a pluripotent state when cultured in a suspension culture devoid of substrate adherence ([0012], page 1), and their media with serum replacement is devoid of (completely free of) animal contaminants (xeno-free) ([0245]-[0246], page 12). One of ordinary skill in the art would have had a reasonable expectation of success in doing so because Amit et al provides proof of principle for culturing pluripotent stem cells in carrier-free and feeder-free sphere suspension culture, and Amit et al were successful in generating and maintaining human pluripotent stem cells in a pluripotent state when cultured in a suspension culture devoid of substrate adherence.
Regarding to claims 2(b) and 3(b), Lu et al teaches Hemangioblast differentiation: Hemangioblast differentiation occurs via a two-step process. The first process is the 3.5-day EB stage, and at day 3.5–4 EBs were trypsinized. Day 7 hemangioblasts were collected and analyzed (Page 415, bridging left – right column). Further, Lu et al teaches endothelial cell culture & differentiation from hemangioblasts (Page 415, right column, 3rd para.). Lu et al also teaches Figure 6: endothelial cell differentiation of hemangioblasts (hemogenic endothelial cells (HECs)) derived from human embryonic stem cells maintained on microcarriers (Page 420).
Amit et al teach “culturing according to this aspect of the present invention is effected using a controlled culturing system (preferably a computer-controlled culturing system ) in which culture parameters such as temperature, agitation , pH , and pO2 , is automatically performed using a suitable device” ([0310], page 17), and “Cells cultured continuously with spinner flasks for more than a month (4 -5 passages)maintained the expression of pluripotency markers Oct4 and SSEA4” ([0434], page 17). Amit et al also teach differentiation potential of PSCs in suspension 3D culture ([0082], page 5), and in suspension cultures , after at least the first passage in which the cells were transferred to spinner flasks
(75 rpm ) or shaker flasks, no splitting was needed ([0397], page 21).
Regarding to claims 2(c) and 2(d), Lu et al teaches endothelial cell culture & differentiation from hemangioblasts: Day 7 hemangioblasts were plated on fibronectin-coated dishes in endothelial cell growth medium. After 4 h, the media was changed and nonadherent cells were removed from culture. The media was changed daily. After 1 week, cells were dissociated with trypsin and collected. Cell number was determined and 125,000 cells were plated in 300 µl Matrigel in a four-well plate to determine angiogenesis functionality. (Page 415, right column, 3rd para.).
Regarding to claims 2(e), Lu et al teaches that hematopoietic colony-forming erythroid cells, colony-forming granulocytes, colony-forming macrophages and colony-forming multilineage cells were formed 10–12 days after plating these cells in serum-free methylcellulose media containing a spectrum of hematopoietic cytokines (Figure 5). These adherent cells took up acetylated low-density lipoprotein and formed capillary vascular-like structures that also took up acetylated low-density-lipoprotein after replating on the Matrigel matrix for less than 24 h (Figure 6), confirming their endothelial lineage (Page 420, right column, 2nd para). These cells can differentiate into multiple hematopoietic cells, including erythrocytes, megakaryocytes/platelets, macrophages, granulocytes, dendritic cells and natural killer cells (Page 414, left column).
Regarding to claims 3(c), Lu et al teaches hemogenic endothelial cells (HECs): Hemangioblasts can differentiate into multiple hematopoietic cells, including erythrocytes, megakaryocytes/platelets, macrophages, granulocytes, dendritic cells and natural killer cells (Page 414, left column). Amit et al teach the culture that is “devoid of substrate adherence” ([0276], page 15) for culturing somatic cells or stem cells such as hematopoietic stem cells. Thus, it would be obvious for one of ordinary skill in the art to culture the HECs in the second spheres in a scaffold-free third culture medium and differentiate into lymphoid lineage cells while permitting the lymphoid lineage cells to release from the second spheres.
Regarding to claim 17, Lu et al teaches the cells can differentiate into multiple hematopoietic cells, including erythrocytes, megakaryocytes/platelets, macrophages, granulocytes, dendritic cells and natural killer cells (Page 414, left column, 2nd para.).
Response to Arguments
Applicant's arguments filed 10-16-2025 have been fully considered but they are not persuasive.
1. Applicant argues that the person of ordinary skill in the art would not have been able to reach the key feature recited in the claims of "generating a plurality of first spheres comprising pluripotent stem cells (PSCs) in a first culture medium using carrier-free and feeder-free 3-dimensional (3D) sphere culturing under dynamic rotating conditions while monitoring sphere size, wherein the first spheres have an average size of about 60-150 micrometers" by merely combining Lu and Amit. The Action specifically characterizes Amit as teaching a culture medium for maintaining pluripotent stem cells in a pluripotent state, where the culture medium is "devoid of substrate adherence." The Action therefore alleges that it would have been obvious to modify Lu by using carrier-free and feeder-free sphere culturing as taught in Amit. The person of ordinary skill in the art would have lacked the motivation to combine Amit with Lu with a reasonable expectation of success due to the teachings of Amit. Further, by citing Amit, the Action appears to miss the point of the present claims, which are directed towards a method for production of lymphoid lineage cells by using 3D sphere culturing in dynamic conditions (the 1st and 2nd page of the remarks ).
Response to Arguments:
In response to applicant's arguments against the references individually, one cannot show nonobviousness by attacking references individually where the rejections are based on combinations of references. See In re Keller, 642 F.2d 413, 208 USPQ 871 (CCPA 1981); In re Merck & Co., 800 F.2d 1091, 231 USPQ 375 (Fed. Cir. 1986). In the instant case, Lu et al (applicant own work) teach production of lymphoid lineage cells: an efficient method to reproducibly generate large numbers of hemangioblasts (progenitor of hematopoietic cell lineages) from multiple hESC lines using an in vitro feeder and serum-free differentiation system. These cells can differentiate into multiple hematopoietic cells, including erythrocytes, megakaryocytes/platelets, macrophages, granulocytes, dendritic cells and natural killer cells (Page 414, left column). Lu et al teach hESCs and iPSCs were grown on feeder-free culture, both hESCs and iPSCs were plated and grown on Matrigel in mTeSR1 medium (Page 414, right column, 2nd para.). Once collected, uniform cell clumps were created by gently pipetting the cell suspension to form clumps less than 100 µm in diameter (Page 414, right column, last para.). Additionally, Amit et al teach culture media for culturing pluripotent stem cells in a suspension culture devoid of substrate adherence (carrier-free) (Abstract). The culture is “devoid of substrate adherence” in which the pluripotent stem cells are capable of expanding without adherence to an external substrate such as components of extracellular matrix, a glass microcarrier or beads ([0276], page 15). The culture medium used by the method of this aspect of the present invention is serum-free, xeno-free , feeder-free (i.e., devoid of feeder cells) and protein carrier-free ([0283], page 15). One of ordinary skill in the art would be motivated to culture pluripotent stem cells in carrier-free and feeder-free sphere suspension culture because Amit et al state that their culture medium is capable of maintaining human pluripotent stem cells in a pluripotent state when cultured in a suspension culture devoid of substrate adherence ([0012], page 1), and their media with serum replacement is devoid of (completely free of) animal contaminants (xeno-free) ([0245]-[0246], page 12).
2. Applicant argues that
Amit teaches repeatedly that it provides methods directed towards maintaining pluripotent stem cells (PSCs) in an undifferentiated state. See, e.g., Amit, paragraphs [0046], [0050], [0051 ], [0085]-[0088], etc. Accordingly, Amit teaches a specifically defined culture medium including Wnt3a, GSK3~i (GSK3~ inhibitor), phenylmethylsulfonyl fluoride (PMSF), and/or a Tosyl-L-lysyl-chloromethane hydrochloride (TLCK) intended to keep the PSCs in an undifferentiated state. See Amit, paragraph [0088]. Further, the PSC culture of Amit is effectively a single cell suspension by design, to prevent differentiation. See Amit, paragraph [0300].
This focus on maintaining PSCs in an undifferentiated state is incompatible with Lu. Lu teaches culture conditions for the formation of embryoid bodies under static conditions for the purpose of differentiation of the cells in these embryoid bodies into hemagioblasts that then may be further differentiated into hematopoietic and endothelial cells. See Lu, abstract. In other words, the goals of Lu and Amit are at odds; Lu seeks to provide culture conditions that cause the formation of embryoid body structures from the agglomeration of cells, which then facilitate differentiation of those cells, whereas Amit seeks to provide culture conditions that maintain the cells in an undifferentiated state and prevent agglomeration of cells as part of that process. Indeed, Amit is completely silent as to differentiation of its cells into hemangioblasts or the lymphoid lineage cells recited in the present claims. Instead, the most the person of ordinary skill in the art could have been motivated to do to combine Amit and Lu is to take the undifferentiated single cell PSC suspension culture of Amit and add it to the carrier system of Lu to form embryoid bodies for differentiation. The culture conditions of Amit are otherwise a poor partner for combination with Lu since they would prevent the hemangioblast differentiation that Lu is focused on achieving (the2nd and 3rd page of the remarks ).
Response to Arguments:
It appears that Applicant is arguing that the cited references do not expressly suggest the claimed invention. However, it is well established in case law that a reference must be considered not only for what it expressly teaches, but also for what it fairly suggests. In re Burkel, 201 USPQ 67 (CCPA 1979). Furthermore, in the determination of obviousness, the state of the art as well as the level of skill of those in the art are important factors to be considered. The teaching of the cited references must be viewed in light of these factors. It also appears that applicant is attempting to attack each reference individually. However, in a 103 rejection the references must be considered as a whole. Applicants selectively read the teachings of Amit et al to formulate the grounds for methods only directed towards maintaining pluripotent stem cells (PSCs) in an undifferentiated state. However, it is noted that Amit et al provide method to prepare pluripotent stem cell for use such as induction of differentiation into differentiated lineage: “FIGS. 18A -G depict images embryoid bodies (EBs) formation from PSCs which grew for at least 40 passages on the following culture media” ([0073], page 4), and “When cultured in suspension, after 20 passages in the tested medium hPSCs formed EBs similar to those created by ES cells grown on MEFs (FIGS. 18A-G ). Within these EBs, stem cells differentiated into cell types representative of the three embryonic germ layers ([0429], page 22). Additionally, Amit et al also teach that culture medium of some embodiments of the invention further comprises leukemia inhibitory factor (LIF) ([0211], page 11), and “ leukemia inhibitory factor (LIF)” refers to the pleiotropic cytokine which is involved in the induction of hematopoietic differentiation ([0213], page 11). Also, as applicant admitted, Lu et al concerned about culture conditions that cause the formation of embryoid body structures from the agglomeration of cells, which then facilitate differentiation of those cells, and Lu et al teach embryoid body (EB) formation and hemangioblast differentiation and their 3D system can be adapted to human induced pluripotent stem cells, which generate functional hemangioblasts with high efficiency (Abstract). Thus, a person of ordinary skill in the would have been motivated to combine and modify the teachings of Lu et al by culturing pluripotent stem cells in a suspension culture devoid of substrate adherence as taught by Amit et al because Amit et al were successful in generation of embryoid body (EB) formation in a suspension culture devoid of substrate adherence with the advantages of hPSCs being maintained all PSCs features after prolonged culture, including the developmental potential to differentiate into representative tissues of the three embryonic germ layers, unlimited and undifferentiated proliferative ability and maintenance of normal karyotype ([0087], page 5), and their media with serum replacement is devoid of (completely free of) animal contaminants (xeno-free) ([0245]-[0246], page 12). a person of ordinary skill in the would use hPSCs and culturing method of Amit et al for hemangioblast differentiation to generate lymphoid lineage cells as taught by Lu et al. Finally, there is no requirement for Huang et al. to teach that which is clearly taught by Lu et al. who clearly teach differentiation of hPSC cells into hemangioblasts or the lymphoid lineage cells as recited in the present claims.
3. Applicant argues that the person of ordinary skill would therefore not have combined Lu and Amit since the purposes of the cell culture systems described in these two references are incompatible. Further, the citation of Amit misses the point of the present claims. The present application discloses that PSCs are formed into well-controlled sphere sizes (e.g., 60-150 micrometers) in a carrier-free and feeder-free 3D sphere culture at the initiation of differentiation because it is "critical for homogenous specification of human PSCs toward mesoderm lineage with high efficiency and small variability." See Application, paragraph [0114]. No combination of Amit or Lu teaches this key feature of the present claims (the 3rd page of the remark).
Response to Arguments:
Lu et al teach PSCs are formed into well-controlled sphere sizes: hESCs and iPSCs were grown on feeder-free culture, both hESCs and iPSCs were plated and grown on Matrigel in mTeSR1 medium (Page 414, right column, 2nd para.). Once collected, uniform cell clumps were created by gently pipetting the cell suspension to form clumps less than 100 µm in diameter (Page 414, right column, last para.), and the hemangioblasts were homogenous in morphology (Page 420, left column). Additionally, Amit et al teach culturing pluripotent stem cells in a suspension culture devoid of substrate adherence (Abstract). Thus, an ordinary skill would have been motivated to combine Lu et al and Amit et al , and it is expected that the differentiation process of human PSCs toward mesoderm lineage would have the same results.
4. Applicant argues that
The carrier-free and feeder-free 3D sphere culture features of the present claims are a key advance over the art. Existing systems, such as those based on creating embryoid bodies using carriers, feeders, matrix, or other adhesion-based culture systems, are severely limited in their scalability. These systems of the art may be useful in creating small amounts of differentiated cells for research purposes, but cannot easily be scaled up for commercial purposes. A simple example is the system of Lu, which is limited by its reliance on a finite number of microcarriers. Similarly, other conventional systems may variously be limited by plate size or well number.
This is not the case for the present system. Since no carriers, feeders, or matrices are relied upon, the need for these additional factors do not provide any barriers to scalability. Instead, as the present application notes, 3D sphere culturing can be directly performed in far larger batches (e.g., ~1000x prior art batch sizes) in a spinner flask or stir-tank bioreactor. See Application, paragraph [0027].
The present methods have even further advantages over the prior art. Carriers, feeders, or matrices are additional components that add to the complexity of the culture system. Not only can such components significantly contribute to manufacturing costs, but clinical/therapeutic manufacture is regulated by the FDA to adhere to good manufacturing practices (GMP) guidelines. Methods including carriers, feeders, or matrices are inherently more difficult to perform in accordance with GMP guidelines, when compared to the present methods, simply because the additional components (e.g., carriers, feeders, or matrices) themselves need to be GMP compliant (the 3rd page of the remark).
Response to Arguments:
Before the effective filing date of instant invention, the process of culturing pluripotent stem cells in a suspension culture with no carriers, feeders, or matrices has been taught in the art : Amit et al teach culturing pluripotent stem cells in a suspension culture devoid of substrate adherence (Abstract), and the culture of the present invention is “devoid of substrate adherence” in which the pluripotent stem cells are capable of expanding without adherence to an external substrate such as components of extracellular matrix, a glass microcarrier or beads ([0276], page 15). Amit et al also teach mass production of hPSCs required for future industrial and clinical applications ([0088], page 5) with the use of bioreactors ([0398], page 21)
As per MPEP 716.02, Any differences between the claimed invention and the prior art may be expected to result in some differences in properties. The issue is whether the properties differ to such an extent that the difference is really unexpected. In re Merck & Co., 800 F.2d 1091, 231 USPQ 375 (Fed. Cir. 1986). In this case, the process of culturing pluripotent stem cells in a suspension culture with no carriers, feeders, or matrices has been taught in the prior art reference as described above, and there is no evidence of record that using the claimed method would lead to superior results over the cited prior art references.
Conclusion
No claim is allowed.
Applicant's amendment necessitated the new ground(s) of rejection presented in this Office action. Accordingly, THIS ACTION IS MADE FINAL. See MPEP § 706.07(a). Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action. In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any nonprovisional extension fee (37 CFR 1.17(a)) pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action. In no event, however, will the statutory period for reply expire later than SIX MONTHS from the mailing date of this final action.
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/KHOA NHAT TRAN/Examiner, Art Unit 1632
/PETER PARAS JR/Supervisory Patent Examiner, Art Unit 1632