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 .
Claim Status
1. The amendment filed 11/24/2025 has been entered. Claims 1 – 4, 6, 8, 9, 11 – 13, 15 – 28, 30, 31, 33, and new claims 47 – 52 are pending.
Election/Restrictions
2. Applicant’s election without traverse of Group I (claims 1 – 13 and 15 – 28) in the reply filed on 10/02/2024 is acknowledged. Because applicant did not distinctly and specifically point out the supposed errors in the restriction requirement, the election has been treated as an election without traverse (MPEP § 818.01(a)).
3. Claims 1 – 13, 15 – 28, 30, 31, and 33 remain pending in the current application, claims 30, 31, and 33 are withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected invention.
4. Claims 1 – 4, 6, 8, 9, 11 – 13, 15 – 28, and 47 – 52 are under consideration.
Priority
5. The present application is a continuation of PCT Application No. PCT/US2020/025955, filed March 31, 2020, which claims priority to U.S. patent application serial no. 62/827,383 filed April 1, 2019.
Information Disclosure Statement
6. The information disclosure statement (IDS) submitted on 07/30/2025 is acknowledged. The submission is in compliance with the provisions of 37 CFR 1.97. Accordingly, the information disclosure statement is being considered by the examiner.
Withdrawn Claim Rejections
7. The rejection of claims 1 – 4, 6, 8, 9, 11, and 12 under 35 U.S.C. 103 is withdrawn in view of Applicant’s amendments to claim 1 including where step (b) has been amended to remove Shh and add FGF8b; step (d) has been amended to require “inducing FOXN1 expression” with the BMP inhibitor Noggin; and step (e) has been amended to require BMP4 for inducing expression of FOXN1.
8. The rejection of claim 13 under 35 U.S.C. 103 is withdrawn in view of Applicant’s amendments to claim 1.
9. The rejection of claims 15 – 27 under 35 U.S.C. 103 is withdrawn in view of Applicant’s amendment to claim 15 to require retinoic acid and FGF8b in step (b) that was previously optional.
10. The rejection of claim 28 under 35 U.S.C. 103 is withdrawn in view of Applicant’s amendment to claim 15.
Claim Interpretation
11. For the purpose of applying prior art, step d of claim 1 is interpreted as culturing in the presence of Noggin to generate PP specification cells and step e of claim 1 is interpreted as culturing with BMP4 induces FOXN1 expression because Applicant’s specification discloses that BMP signaling is required for FOXN1 expression and cells exposed to BMP4 immediately after Noggin led to increases in FOXN1 confirming the need for Noggin exposure to develop sensitivity to BMP4 at page 14 para. 3.
Claim Objections/ Rejections Necessitated by Amendment
12. Claim 1 is objected to because of the following informalities: in line 6, “wherein differentiating DE cells into AFE” should read “wherein differentiating the DE cells into the AFE”. Appropriate correction is required.
13. Claim 1 is objected to because of the following informalities: in line 16, “thereby generating AFE cells” should read “thereby generating the AFE cells”. Appropriate correction is required.
14. Claim 1 is objected to because of the following informalities: in line 19, “wherein differentiating AFE cells into PE cells” should read “wherein differentiating the AFE cells into the PE cells”. Appropriate correction is required.
15. Claim 1 is objected to because of the following informalities: in line 28, “thereby generating PE cells” should read “thereby generating the PE cells”. Appropriate correction is required.
16. Claim 1 is objected to because of the following informalities: in line 30, “wherein differentiating PE cells into PP specification” should read “wherein differentiating the PE cells into the PP specification”. Appropriate correction is required.
17. Claim 1 is objected to because of the following informalities: in line 34, “thereby generating PP specification cells” should read “thereby generating the PP specification cells”. Appropriate correction is required.
18. Claim 1 is objected to because of the following informalities: in line 36, “into TECs or TEPs” should read “into the TECs or TEPs”. Appropriate correction is required.
19. Claim 1 is objected to because of the following informalities: in line 39, “thereby generating TECs or TEPs” should read “thereby generating the TECs or TEPs”. Appropriate correction is required.
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.
The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. Considering objective evidence present in the application indicating obviousness or nonobviousness.
This application currently names joint inventors. In considering patentability of the claims the examiner presumes that the subject matter of the various claims was commonly owned as of the effective filing date of the claimed invention(s) absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and effective filing dates of each claim that was not commonly owned as of the effective filing date of the later invention in order for the examiner to consider the applicability of 35 U.S.C. 102(b)(2)(C) for any potential 35 U.S.C. 102(a)(2) prior art against the later invention.
20. Claim(s) 1 – 4, 6, 8, 9, 11, 12, and 47 – 50 is/are rejected under 35 U.S.C. 103 as being unpatentable over Snoeck (WO2011139628-A1; previously cited), hereinafter Snoeck which is cited on the IDS filed 01/06/2023 in view of Parent (US20160010055A1; Filed 02/26/2014; Published 01/14/2016), hereinafter Parent which is cited on the IDS filed 10/01/2021 as evidenced by Sivakamasundari (Sivakamasundari, V., et. al. Gene Expression and Regulation in Mammalian Cells-Transcription Toward the Establishment of Novel Therapeutics. IntechOpen, 2018), hereinafter Sivakamasundari, and Wei (Wei, Qiaozhi, PloS one 6.11 (2011): e26795.), hereinafter Wei.
Regarding claim 1, Snoeck teaches an in vitro method of induction of definitive endoderm from pluripotent cells (step a), induction of definitive endoderm to form anterior foregut endoderm (step b), followed by further differentiation to pharyngeal endoderm (step c) and specification of specific derivatives (steps d and e) (page 8, lines 11 – 14; page 25, lines 19 – 22). Snoeck teaches anterior foregut cells are useful in generating cells or tissues for cell replacement therapy to treat conditions in which the thyroid is not functional (page 16, lines 1 – 11).
Regarding differentiating pluripotent stem cells into DE cells of step a of claim 1, and claims 2 – 3, Snoeck teaches generation of definitive endoderm from pluripotent stem cells comprising culturing cells for 5 days (claim 2) in BMP4 (0.5 – 10 ng/ml), bFGF (2.5 ng/mL) and activin A (100 ng/mL) in a serum-free medium (claim 3) (page 8, lines 16 – 28; page 17, lines 17 – 33; page 18, lines 1- 23; Figure 3B).
Regarding differentiating DE cells into AFE cells of step b (i) of claim 1, and claim 4, Snoeck teaches formation of anterior foregut endoderm from definitive endoderm is accomplished by adding Noggin and SB431542 (“Noggin” and “SB431542” of step b (i) of claim 1) at day 4 – 5 of culture and endoderm is cultured from days 5 to 7 (claim 4) (page 9, lines 5 – 28; Figure 3B; page 18, lines 2 – 17).
Regarding inducing expression of HOXA3 and TBX1 of step b (ii) and step c (i) and differentiating AFE cells into PE and generating PE cells of step c of claim 1, Snoeck teaches anterior foregut endoderm may be differentiated to pharyngeal endoderm (page 7, lines 27 – 29; page 8, lines 11 – 14). Snoeck teaches application of Noggin/SB-431542 yielded cells that expressed SOX2, TBX1, PAX9, and the pharyngeal endoderm marker FOXG1 (page 22, lines 28 – 33). Snoeck teaches PE expresses TBX1 and SOX2, while third PP cells express PAX1/9, HOXA3, and SIX1 (page 7, lines 10 – 14 and 27 – 29). Snoeck teaches FGF8b induces expression of TBX1 in Figure 1C-1 and 1C-2. Snoeck teaches retinoic acid induces expression of TBX1 and SOX2 in Figure 1C-1 and 1C-2. Therefore, Snoeck teaches generating PE cells and retinoic acid or FGF8b induces TBX1 expression. Snoeck does not teach culturing with both FGF8b and retinoic acid and where expression of HOXA3 is induced by retinoic acid.
Regarding inducing expression of PAX1 and PAX9 and Shh of step c (ii) of claim 1, Snoeck teaches culturing with SHH (“sonic hedgehog”) or with FGF8 and SHH in Figure 3b for inducing a parathyroid fate (page 4, lines 3 – 5; page 14, lines 22 – 28). Snoeck teaches Shh is upstream of Fgf8 in mouse pharyngeal pouch development (page 25, lines 11 – 16). Snoeck teaches Noggin and SB-431542 induced strong expression of TBX1 and PAX9 (page 21, lines 10 – 21; Figure 1c). Snoeck teaches third PP cells express PAX1/9, HOXA3, and SIX1 (page 7, lines 10 – 14 and 27 – 29) and Shh regulates expression of Pax1 and Pax9 as evidenced by Sivakamasundari (page 186, last para.; page 187, para. 3 – 4). Snoeck teaches Wnt3a, FGF10, KGF, BMP4, and EGF (WFKBE) induced PAX1 expression (Figure 2A and D; Figure 3A) and Noggin/SB431542 induced PAX9 expression (Figure 1C-1 and 1C-2, 1D, 1F, 1H, 1I).
Regarding step d of claim 1, Snoeck teaches culturing the Noggin/SB-431542 derived AFE with SHH and FGF8 resulted in pharyngeal pouch cells and PAX1/9, HOXA3, and SIX1 are pharyngeal pouch markers (page 25, lines 1 and 8 – 18; Figure 3b; page 6, lines 3 – 9; page 7, lines 10 – 14 and 27 – 29). Snoeck teaches culturing with Noggin produced cells positive for FGF8 that is a marker specific for pharyngeal pouch endoderm (page 22, lines 22 – 27). Therefore, Snoeck teaches generating distal PP specification cells with Noggin. Snoeck teaches the pharyngeal endoderm forms pharyngeal pouches (page 6, lines 16 – 20). Snoeck teaches thymic epithelial cells express FOXN1 (page 7, lines 29 – 30).
Regarding step e of claim 1, Snoeck does not teach “inducing FOXN1 expression in the PP specification cells” with BMP4. However, Snoeck teaches in vivo formation of TEPs from ES cells cultured in vitro with Noggin/SB431542 (page 23, lines 13 – 30; page 16, lines 12 – 14). Snoeck teaches thymic epithelial cells express FOXN1 (“FOXN1”) (page 7, lines 29 – 30).
Regarding claim 6, Snoeck teaches differentiation of anterior foregut endoderm to pharyngeal endoderm (page 8, lines 11 – 14). Snoeck teaches pharyngeal endoderm induction where at days 5 – 9 (“at about day 5”), Noggin and SB-431542 are added followed by addition of factors on days 7 – 19 (“for about 6 days to about 10 days”) (page 14, lines 2 – 13).
Regarding claim 8, Snoeck teaches FGF8b at 50 ng/mL in Figure 1C-1. Snoeck teaches SHH at concentrations of 10 ng/ml to 100 ng/ml (page 14, lines 29 – 32; page 15, lines 3 – 8).
Regarding claim 9, Snoeck teaches differentiation into pharyngeal pouch cells at day 11 to day 19 (Figure 3b; page 25, lines 8 – 18). Snoeck teaches exposure of Noggin/SB-431542 induced anterior foregut endoderm to WKFBE did not result in expression of terminal differentiation markers for thymus at day 13 or day 19 of culture (page 24, lines 29 – 31).
Regarding claim 47, Snoeck teaches expression of PAX9 and AIRE (page 4, lines 29; page 23, lines 24 – 27; page 21, lines 10 – 21). Snoeck does not teach FOXN1, PAX1, DLL4, ISL1, EYA1, SIX1, IL-7, KRT5 or KRT8. However, Snoeck teaches TECs express FOXN1 and third pharyngeal pouch cells express PAX1/9 and SIX1 (page 7, lines 28 – 30).
Regarding claim 49, Snoeck teaches 200 ng/ml of Noggin (page 18, lines 2 – 6; Figure 3a,b).
Regarding claim 50, Snoeck teaches Noggin is present in cultures at about 100 ng/ml in a most preferred embodiment (page 10, lines 20 – 21) and culturing with Noggin produced cells positive for FGF8 that is a marker specific for pharyngeal pouch endoderm (page 22, lines 22 – 27).
Snoeck does not teach culturing with both FGF8b and retinoic acid and where expression of HOXA3 is induced by retinoic acid of step b (ii) and step c (i) of claim 1, “inducing FOXN1 expression in the PP specification cells” with BMP4 of step e of claim 1 or the starting day of claim 11 or the concentration of BMP4 of claim 12 or FOXN1, PAX1, DLL4, ISL1, EYA1, SIX1, IL-7, KRT5 or KRT8 of claim 47 or the TEPs support T cell reconstitution in vivo of claim 48. However, Snoeck teaches that it appears critical that to obtain enriched or pure and functional populations of mature cells, developmental cues need to be sequentially applied to guide the differentiation of pluripotent cells in vitro (page 2, lines 5 – 8). Snoeck teaches anterior foregut cells are useful for generating cells for cell replacement therapy (Abstract; page 1, lines 18 – 23). Snoeck teaches the ability to generate populations of anterior foregut/pharyngeal endoderm cells from pluripotent cells would be useful in cell replacement therapy for tissues that develop from the pharyngeal endoderm that include the thymus (page 6, lines 16 – 25). Snoeck teaches thymic epithelial cells derived from pluripotent stem cells could improve humanized mouse models (page 16, lines 12 – 13).
Regarding culturing with both FGF8b and retinoic acid and where expression of HOXA3 is induced by retinoic acid of step b (ii) and step c (i) of claim 1, Parent teaches a method of inducing differentiation of pluripotent stem cells into TECs and TEPs in vitro comprising differentiating embryonic stem cells into DE, AFE, ventral pharyngeal endoderm (VPE), TEP, and finally TEC (Figure 1A; page 1, 0010; page 2, 0029; page 17, 0249 – 0250). Parent teaches conditions for differentiating DE to AFE and AFE to VPE where when retinoic acid is included in the media, HOXA3 expression is induced in Figure 1A and B (page 16, 0247 – 0248). Parent teaches condition 7 contains both retinoic acid (0.25 µM) and FGF8b (50 ng/ml) and this condition resulted in HOXA3 induction to levels similar to that found in fetal and adult thymus in Figure 1B page 14, 0224 – 0225; page 17, 0248). Parent teaches Hoxa3 is an early and essential regulator of thymus specification and of differentiation of TEPs into mature TECs and Hoxa3 expression is a readout for thymic specification in the method (page 16, 0246).
Regarding step e of claim 1, Parent teaches differentiating VPE to TEPs in media containing BMP4 where BMP4 induced expression of FOXN1 in Figure 1A and B (page 16, 0246 – 0248; page 17, 0248).
Regarding claim 11, Parent teaches the stage of producing TEP cells from VPE starts at about day 13 (where each of stages 1 and 2 can occur for a period of about 1 – 6 days and stage 3 can occur for a period of about 1 to 8 days; page 5, 0084; page 6, 0089, 0096) for a period of about 10 days (page 6, 0102). Parent teaches the TEP cells may be generated within about 15 days from the start of culturing of the pluripotent stem cells (page 7, 0108).
Regarding claim 12, Parent teaches 50 ng/ml BMP4 (page 14, 0225).
Regarding claim 47, Parent teaches the TEPs express FOXN1, PAX1, EYA1, DLL4, KRT5, and KRT8 (page 7, 0103; page 12, 0169; page 17, 0253; Figure 7E; page 18, 0253 – 0255; Figure 2C; Figure 1A). Parent does not teach the TECs express ISL1 or IL-7 of claim 47. However, Parent teaches the TEPs express one or more markers of TEP cells that are present in TEP cells in thymus of thymic tissue such as adult human thymus or fetal human thymus and at levels similar to levels expressed by TEP cells in adult or fetal thymus (page 11, 0169). Fetal and postnatal thymic epithelial cells express ISL1 and IL7 as evidenced by Wei (page 5, right col. para. 4 – 5; page 6, left col. para. 1 and 3; page 7, right col. para. 1).
Regarding claim 48, Parent teaches the TECs support the development of new T cells in vivo (page 18, 0258 – 0261; page 19, 0261 – 0266).
Parent teaches there is a need for methods for generating functional TEPs that can differentiate into functional TECs (page 1, 0005). Parent teaches the TEPs may be used in clinical applications for generating functional TECs in a subject having a genetic and/or developmental defect that results in reduced or undetectable thymus functions (page 14, 0213 – 0219).
It would have been obvious prior to the effective filing date of the invention as claimed for the person of ordinary skill in the art to combine the teachings of Snoeck regarding a method of induction of definitive endoderm from pluripotent cells, induction of definitive endoderm to form anterior foregut endoderm, followed by further differentiation to pharyngeal endoderm and pharyngeal pouch cells in vitro with the teachings of Parent regarding a method of generating TEPs in vitro from pluripotent stem cells to arrive at the claimed method where pluripotent stem cells are differentiated to DE cells; DE is incubated with retinoic acid and FGF8b to generate AFE and AFE is incubated with retinoic acid and FGF8b followed by Shh to produce PE cells; PE cells are differentiated into PP specification cells; and PP specification cells are differentiated into TECs or TEPs by incubating with BMP4 where the TEPs or TECs are FOXN1, PAX9, PAX1, DLL4, ISL1, EYA1, SIX1, IL-7, KRT5, KRT8, and AIRE positive. One would have been motivated to combine the teachings of Snoeck and Parent to produce therapeutic TEPs or TECs as Snoeck teaches the ability to generate populations of anterior foregut/pharyngeal endoderm cells from pluripotent cells would be useful in cell replacement therapy for tissues that develop from the pharyngeal endoderm that include the thymus and Parent teaches there is a need for methods for generating functional TEPs that can differentiate into functional TECs. One would have a reasonable expectation of success in combining the teachings as Snoeck teaches in vivo formation of TEPs from ES cells cultured in vitro with Noggin/SB431542 and Parent teaches the TEPs express markers of fetal or adult human thymus and at similar levels to human thymus and the TECs support the development of new T cells in vivo.
21. Claim(s) 13 is/are rejected under 35 U.S.C. 103 as being unpatentable over Snoeck (WO2011139628-A1; previously cited), hereinafter Snoeck which is cited on the IDS filed 01/06/2023 in view of Parent (US20160010055A1; Filed 02/26/2014; Published 01/14/2016), hereinafter Parent which is cited on the IDS filed 10/01/2021 as evidenced by Sivakamasundari (Sivakamasundari, V., et. al. Gene Expression and Regulation in Mammalian Cells-Transcription Toward the Establishment of Novel Therapeutics. IntechOpen, 2018), hereinafter Sivakamasundari and Wei (Wei, Qiaozhi, PloS one 6.11 (2011): e26795.), hereinafter Wei as applied to claims 1 – 4, 6, 8, 9, 11, 12, and 47 – 50 above, and further in view of Cha (US20160002604A1; Filed 10/11/2013; Published 05/23/2017; previously cited), hereinafter Cha, which is cited on the IDS filed 10/01/2021.
Snoeck in view of Parent make obvious the limitations of claim 1 as set forth above.
Snoeck and Parent do not teach the method further comprising contacting or incubating the TECs or TEPs with a survivin inhibitor. It is noted that the survivin inhibitor can optionally be YM155 and the term “optionally” does not require that the survivin inhibitor be YM155. However, Snoeck teaches undifferentiated stem cells generated teratomas in vivo (page, lines 13 – 17). Snoeck teaches it appears critical that to obtain enriched or pure and functional populations of mature cells, developmental cues need to be sequentially applied to guide the differentiation of pluripotent cells in vitro (page 2, lines 6 – 8).
Cha teaches in the development of a cell therapeutic agent using pluripotent stem cells, there is a potential danger of formation of teratoma from undifferentiated pluripotent stem cells (page 1, 0005). Cha teaches there is a demand for development of a technology capable of selectively removing undifferentiated cells having the potential danger of teratoma without affecting the differentiated cells (page 1, 0005). Cha teaches a method of preparing a cell sample including undifferentiated pluripotent stem cells and differentiated cells by differentiating the pluripotent stem cells and causing the selective death of the undifferentiated pluripotent stem cells by treating the resultant cell sample with YM-155 (page 1, 0013 – 0014).
It would have been obvious prior to the effective filing date of the invention as claimed for the person of ordinary skill in the art to combine the teachings of Snoeck regarding a method of induction of definitive endoderm from pluripotent cells, induction of definitive endoderm to form anterior foregut endoderm, followed by further differentiation to pharyngeal endoderm and pharyngeal pouch cells in vitro with the teachings of Parent regarding a method of generating TEPs in vitro from pluripotent stem cells with the teachings of Cha regarding treating a cell sample with YM-155 to arrive at the claimed method further comprising contacting or incubating the TECs or TEPs with YM155. One would have been motivated to combine the teachings of Snoeck, Parent, and Cha to prepare a cell population free of undifferentiated cells as Snoeck teaches it appears critical that to obtain enriched or pure and functional populations of mature cells, developmental cues need to be sequentially applied to guide the differentiation of pluripotent cells in vitro. One would have a reasonable expectation of success in combining the teachings as Snoeck teaches undifferentiated stem cells generated teratomas in vivo and Cha teaches YM-155 causes the selective death of undifferentiated pluripotent stem cells.
22. Claim(s) 15 – 27 and 51 – 52 is/are rejected under 35 U.S.C. 103 as being unpatentable over Snoeck (WO2011139628-A1; previously cited), hereinafter Snoeck which is cited on the IDS filed 01/06/2023 in view of Parent (US20160010055A1; Filed 02/26/2014; Published 01/14/2016), hereinafter Parent which is cited on the IDS filed 10/01/2021 as evidenced by Sivakamasundari (Sivakamasundari, V., et. al. Gene Expression and Regulation in Mammalian Cells-Transcription Toward the Establishment of Novel Therapeutics. IntechOpen, 2018), hereinafter Sivakamasundari and Wei (Wei, Qiaozhi, PloS one 6.11 (2011): e26795.), hereinafter Wei.
Regarding step a of claim 15, and claims 16 – 18, Snoeck teaches generation of definitive endoderm from human ES or iPS cells (claim 16) comprising culturing cells for 5 days (claim 17) in BMP4 (0.5 – 10 ng/ml), bFGF (2.5 ng/mL) and activin A (100 ng/mL) (claim 18) in a serum-free medium (page 8, lines 16 – 28; page 17, lines 17 – 33; page 18, lines 1- 23; Figure 3B).
Regarding step b (i) of claim 15, and claim 19, Snoeck teaches formation of anterior foregut endoderm from definitive endoderm is accomplished by adding Noggin and SB431542 to serum-free medium at day 4 – 5 of culture and endoderm is cultured from days 5 to 7 (claim 19) (page 9, lines 5 – 28; Figure 3B; page 5, lines 15 – 18).
Regarding step b (ii) and step c (i) and differentiating AFE cells into PE and generating PE cells of step c of claim 15, Snoeck teaches anterior foregut endoderm may be differentiated to pharyngeal endoderm (page 7, lines 27 – 29; page 8, lines 11 – 14). Snoeck teaches application of Noggin/SB-431542 yielded cells that expressed SOX2, TBX1, PAX9, and the pharyngeal endoderm marker FOXG1 (page 22, lines 28 – 33). Snoeck teaches PE expresses TBX1 and SOX2, while third PP cells express PAX1/9, HOXA3, and SIX1 (page 7, lines 10 – 14 and 27 – 29). Snoeck teaches FGF8b induces expression of TBX1 in Figure 1C-1 and 1C-2. Snoeck teaches retinoic acid induces expression of TBX1 and SOX2 in Figure 1C-1 and 1C-2. Therefore, Snoeck teaches generating PE cells and retinoic acid or FGF8b induces TBX1 expression. Snoeck does not teach culturing with both FGF8b and retinoic acid.
Regarding step c (ii) of claim 15, Snoeck teaches culturing with SHH (“sonic hedgehog”) or with FGF8 and SHH in Figure 3b for inducing a parathyroid fate (page 4, lines 3 – 5; page 14, lines 22 – 28). Snoeck teaches Shh is upstream of Fgf8 in mouse pharyngeal pouch development (page 25, lines 11 – 16). Snoeck teaches Noggin and SB-431542 induced strong expression of TBX1 and PAX9 (page 21, lines 10 – 21; Figure 1c).
Regarding step d of claim 15, Snoeck teaches culturing the Noggin/SB-431542 derived AFE with SHH and FGF8 resulted in pharyngeal pouch cells and PAX1/9, HOXA3, and SIX1 are pharyngeal pouch markers (page 25, lines 1 and 8 – 18; Figure 3b; page 6, lines 3 – 9; page 7, lines 10 – 14 and 27 – 29). Snoeck teaches culturing with Noggin produced cells positive for FGF8 that is a marker specific for pharyngeal pouch endoderm (page 22, lines 22 – 27). Therefore, Snoeck teaches generating distal PP specification cells with Noggin. Snoeck teaches the pharyngeal endoderm forms pharyngeal pouches (page 6, lines 16 – 20).
Regarding step e of claim 15, Snoeck does not teach “further differentiating the PP specification cells”” with BMP4. However, Snoeck teaches in vivo formation of TEPs from ES cells cultured in vitro with Noggin/SB431542 (page 23, lines 13 – 30; page 16, lines 12 – 14). Snoeck teaches thymic epithelial cells express FOXN1 (“FOXN1”) (page 7, lines 29 – 30).
Regarding claim 20, Snoeck teaches FGF8b at 50 ng/mL in Figure 1C-1 and retinoic acid at 0.1 µM in Figure 1C-1.
Regarding claim 21, Snoeck teaches differentiation of anterior foregut endoderm to pharyngeal endoderm (page 8, lines 11 – 14). Snoeck teaches pharyngeal endoderm induction where at days 5 – 9 (“at about day 5”), Noggin and SB-431542 are added followed by addition of factors on days 7 – 19 (“for about 6 days to about 10 days”) (page 14, lines 2 – 13).
Regarding claim 22, Snoeck teaches FGF8b at 50 ng/mL in Figure 1C-1 and retinoic acid at 0.1 µM in Figure 1C-1.
Regarding claim 23, Snoeck teaches FGF8 at concentrations of 10 ng/ml to 1 ug/ml and SHH at concentrations of 10 ng/ml to 100 ng/ml (page 14, lines 29 – 32; page 15, lines 3 – 8).
Regarding claim 24, Snoeck teaches at day 11 (“about day 12 – 18”), addition of FGF8+SHH formed GCM2 positive cells by day 19 (“for about 4 – 7 days”) (Figure 3b; page 6, lines 3 – 9).
Regarding claim 25, Snoeck teaches Noggin is present in cultures at a concentration of about 100 ng/mL (page 10, lines 20 – 21).
Regarding claim 51, Snoeck teaches expression of PAX9 and AIRE (page 4, lines 29; page 23, lines 24 – 27; page 21, lines 10 – 21). Snoeck does not teach FOXN1, PAX1, DLL4, ISL1, EYA1, SIX1, IL-7, KRT5 or KRT8. However, Snoeck teaches TECs express FOXN1 and third pharyngeal pouch cells express PAX1/9 and SIX1 (page 7, lines 28 – 30).
Snoeck does not teach culturing with both FGF8b and retinoic acid of step b (ii) and step c (i) of claim 15, or “further differentiating the PP specification cells”” with BMP4 of step e of claim 15 or the starting day of claim 26 or the concentration of BMP4 of claim 27 or FOXN1, PAX1, DLL4, ISL1, EYA1, SIX1, IL-7, KRT5 or KRT8 of claim 51 or the TEPs support T cell reconstitution in vivo of claim 52. However, Snoeck teaches that it appears critical that to obtain enriched or pure and functional populations of mature cells, developmental cues need to be sequentially applied to guide the differentiation of pluripotent cells in vitro (page 2, lines 5 – 8). Snoeck teaches anterior foregut cells are useful for generating cells for cell replacement therapy (Abstract; page 1, lines 18 – 23). Snoeck teaches the ability to generate populations of anterior foregut/pharyngeal endoderm cells from pluripotent cells would be useful in cell replacement therapy for tissues that develop from the pharyngeal endoderm that include the thymus (page 6, lines 16 – 25). Snoeck teaches thymic epithelial cells derived from pluripotent stem cells could improve humanized mouse models (page 16, lines 12 – 13).
Regarding culturing with both FGF8b and retinoic acid and of step b (ii) and step c (i) of claim 15, Parent teaches a method of inducing differentiation of pluripotent stem cells into TECs and TEPs in vitro comprising differentiating embryonic stem cells into DE, AFE, ventral pharyngeal endoderm (VPE), TEP, and finally TEC (Figure 1A; page 1, 0010; page 2, 0029; page 17, 0249 – 0250). Parent teaches conditions for differentiating DE to AFE and AFE to VPE using media comprising 0.25 µM retinoic acid and 50 ng/ml FGF8b in Figure 1A and B (page 14, 0225; page 16, 0247 – 0248). Parent teaches condition 7 contains both retinoic acid and FGF8b and this condition resulted in HOXA3 induction to levels similar to that found in fetal and adult thymus in Figure 1B (page 17, 0248). Parent teaches Hoxa3 is an early and essential regulator of thymus specification and of differentiation of TEPs into mature TECs and Hoxa3 expression is a readout for thymic specification in the method (page 16, 0246).
Regarding step e of claim 15, Parent teaches differentiating VPE to TEPs in media containing BMP4 where BMP4 induced expression of FOXN1 in Figure 1A and B (page 16, 0246 – 0248; page 17, 0248).
Regarding claim 26, Parent teaches the stage of producing TEP cells from VPE starts at about day 13 (where each of stages 1 and 2 can occur for a period of about 1 – 6 days and stage 3 can occur for a period of about 1 to 8 days; page 5, 0084; page 6, 0089, 0096) for a period of about 10 days (page 6, 0102). Parent teaches the TEP cells may be generated within about 15 days from the start of culturing of the pluripotent stem cells (page 7, 0108).
Regarding claim 27, Parent teaches 50 ng/ml BMP4 (page 14, 0225).
Regarding claim 51, Parent teaches the TEPs express FOXN1, PAX1, EYA1, DLL4, KRT5, and KRT8 (page 7, 0103; page 12, 0169; page 17, 0253; Figure 7E; page 18, 0253 – 0255; Figure 2C; Figure 1A). Parent does not teach the TECs express ISL1 or IL-7 of claim 47. However, Parent teaches the TEPs express one or more markers of TEP cells that are present in TEP cells in thymus of thymic tissue such as adult human thymus or fetal human thymus and at levels similar to levels expressed by TEP cells in adult or fetal thymus (page 11, 0169). Fetal and postnatal thymic epithelial cells express ISL1 and IL7 as evidenced by Wei (page 5, right col. para. 4 – 5; page 6, left col. para. 1 and 3; page 7, right col. para. 1).
Regarding claim 52, Parent teaches the TECs support the development of new T cells in vivo (page 18, 0258 – 0261; page 19, 0261 – 0266).
Parent teaches there is a need for methods for generating functional TEPs that can differentiate into functional TECs (page 1, 0005). Parent teaches the TEPs may be used in clinical applications for generating functional TECs in a subject having a genetic and/or developmental defect that results in reduced or undetectable thymus functions (page 14, 0213 – 0219).
It would have been obvious prior to the effective filing date of the invention as claimed for the person of ordinary skill in the art to combine the teachings of Snoeck regarding a method of induction of definitive endoderm from pluripotent cells, induction of definitive endoderm to form anterior foregut endoderm, followed by further differentiation to pharyngeal endoderm and pharyngeal pouch cells in vitro with the teachings of Parent regarding a method of generating TEPs in vitro from pluripotent stem cells to arrive at the claimed method where pluripotent stem cells are differentiated to DE cells; DE is incubated with retinoic acid and FGF8b to generate AFE and AFE is incubated with retinoic acid and FGF8b followed by Shh to produce PE cells; PE cells are differentiated into PP specification cells; and PP specification cells are differentiated into TECs or TEPs by incubating with BMP4 where the TEPs or TECs are FOXN1, PAX9, PAX1, DLL4, ISL1, EYA1, SIX1, IL-7, KRT5, KRT8, and AIRE positive. One would have been motivated to combine the teachings of Snoeck and Parent to produce therapeutic TEPs or TECs as Snoeck teaches the ability to generate populations of anterior foregut/pharyngeal endoderm cells from pluripotent cells would be useful in cell replacement therapy for tissues that develop from the pharyngeal endoderm that include the thymus and Parent teaches there is a need for methods for generating functional TEPs that can differentiate into functional TECs. One would have a reasonable expectation of success in combining the teachings as Snoeck teaches in vivo formation of TEPs from ES cells cultured in vitro with Noggin/SB431542 and Parent teaches the TEPs express markers of fetal or adult human thymus and at similar levels to human thymus and the TECs support the development of new T cells in vivo.
23. Claim(s) 28 is/are rejected under 35 U.S.C. 103 as being unpatentable over Snoeck (WO2011139628-A1; previously cited), hereinafter Snoeck which is cited on the IDS filed 01/06/2023 in view of Parent (US20160010055A1; Filed 02/26/2014; Published 01/14/2016), hereinafter Parent which is cited on the IDS filed 10/01/2021 as evidenced by Sivakamasundari (Sivakamasundari, V., et. al. Gene Expression and Regulation in Mammalian Cells-Transcription Toward the Establishment of Novel Therapeutics. IntechOpen, 2018), hereinafter Sivakamasundari and Wei (Wei, Qiaozhi, PloS one 6.11 (2011): e26795.), hereinafter Wei as applied to claims 15 – 27 and 51 – 52 above, and further in view of Cha (US20160002604A1; Filed 10/11/2013; Published 05/23/2017; previously cited), hereinafter Cha, which is cited on the IDS filed 10/01/2021.
Snoeck in view of Parent make obvious the limitations of claim 15 as set forth above.
Snoeck does not teach the method further comprising contacting or incubating the TECs or TEPs with a survivin inhibitor. It is noted that the survivin inhibitor can optionally be YM155 and the term “optionally” does not require that the survivin inhibitor be YM155. However, Snoeck teaches undifferentiated stem cells generated teratomas in vivo (page, lines 13 – 17). Snoeck teaches it appears critical that to obtain enriched or pure and functional populations of mature cells, developmental cues need to be sequentially applied to guide the differentiation of pluripotent cells in vitro (page 2, lines 6 – 8).
Cha teaches in the development of a cell therapeutic agent using pluripotent stem cells, there is a potential danger of formation of teratoma from undifferentiated pluripotent stem cells (page 1, 0005). Cha teaches there is a demand for development of a technology capable of selectively removing undifferentiated cells having the potential danger of teratoma without affecting the differentiated cells (page 1, 0005). Cha teaches a method of preparing a cell sample including undifferentiated pluripotent stem cells and differentiated cells by differentiating the pluripotent stem cells and causing the selective death of the undifferentiated pluripotent stem cells by treating the resultant cell sample with YM-155 (page 1, 0013 – 0014).
It would have been obvious prior to the effective filing date of the invention as claimed for the person of ordinary skill in the art to combine the teachings of Snoeck regarding a method of induction of definitive endoderm from pluripotent cells, induction of definitive endoderm to form anterior foregut endoderm, followed by further differentiation to pharyngeal endoderm and pharyngeal pouch cells in vitro with the teachings of Parent regarding a method of generating TEPs in vitro from pluripotent stem cells with the teachings of Cha regarding treating a cell sample with YM-155 to arrive at the claimed invention where comprising a step of contacting or incubating the TECs or TEPs at the end of the method with YM155. One would have been motivated to combine the teachings of Snoeck and Cha to prepare a cell population free of undifferentiated cells as Snoeck teaches it appears critical that to obtain enriched or pure and functional populations of mature cells, developmental cues need to be sequentially applied to guide the differentiation of pluripotent cells in vitro. One would have a reasonable expectation of success in combining the teachings as Snoeck teaches undifferentiated stem cells generated teratomas in vivo and Cha teaches YM-155 causes the selective death of undifferentiated pluripotent stem cells.
Applicant’s Arguments/ Response to Arguments
24. Applicant Argues: On page 11, paragraph 2, Applicant asserts that the Office has not med the burden of establishing a prima facie case of obviousness. On page 13, para. 1, Applicant asserts that Snoeck does not disclose or suggest (i) differentiating DE cells into AFE cells by contacting the cells with a BMP inhibitor and a TGFb inhibitor and then with RA and FGF8b, (ii) differentiating AFE into PE cells by contacting the cells with RA and FGF8b and then with FGF8b and Shh nor (iii) differentiating PE cells into TECs/TEPs by contacting the cells with noggin to obtain PP cells and then with BMP4 to obtain TECs/TEPs.
Response to Arguments: The previous rejection has been withdrawn in view of Applicant’s amendment to claim 1. In the new rejection, Snoeck is still cited as the primary reference because Snoeck teaches a method of inducing a ventral cell fate in anterior/pharyngeal endoderm or pharyngeal pouch fate that proceeds by induction of DE from pluripotent cells, induction of DE to form AFE, followed by further differentiation to pharyngeal endoderm and specification of specific derivatives (page 3, lines 31 – 32; page 8, lines 11 – 14). Snoeck teaches AFE cells express FOXA2 and SOX2, pharyngeal endoderm cells express TBX1, SOX2, and FOXG1, third pharyngeal pouch cells express PAX1/9, HOXA3, and SIX1, and thymic epithelial cells express FOXN1 (page 7, lines 25 – 30; page 22, lines 32 – 33; page 24, line 5). Snoeck teaches the combination of Noggin and SB-431542 to form AFE from DE where this combination (combination 17 in Figure 1C-1) significantly induces expression of FOXA2 and SOX2 (page 9, lines 26 – 28; Figure 1C-2; page 21, lines 10 – 21; page 22, lines 28 – 33). Snoeck also teaches the combination of Noggin and SB-431542 increases expression of the pharyngeal endoderm markers TBX1 and SOX2 and the third pharyngeal pouch cell marker PAX9 in Figure 1C-2.
Snoeck does not teach culturing with RA and FGF8b together but teaches culturing DE with each separately increases expression of FOXA2, SOX2, PAX9, and TBX1 in Figure 1C-2. Snoeck teaches RA can induce ventral anterior foregut from which the pharyngeal region arises and teaches RA in combination with several factors (WKFBE) that can specify lung fate (page 13, lines 13 – 27; page 24, lines 10 – 33; page 25, line 1 – 3; Figure 3A). Thus, Snoeck teaches Noggin/SB431542 differentiates DE to AFE, RA ventralizes the AFE, RA increases the expression of pharyngeal endoderm and pouch markers, but when RA is added along with WKFBE, the pharyngeal pouch marker PAX1 is decreased.
Snoeck teaches culturing AFE with WKFBE factors in the absence of RA and then adding FGF8, SHH, or the combination of FGF8 and SHH in Figure 3B where GCM2 expression is induced. Snoeck teaches GCM2 is a parathyroid-specific marker (page 25, line 13). Snoeck teaches the effects of SHH and FGF8 were not additive, mirroring in vivo epistasis studies showing that Shh is upstream of Fgf8 in mouse pharyngeal pouch development (page 25, lines 8 – 16). Snoeck references the teachings of Moore-Scott (Moore-Scott, Billie A., et. al. Developmental biology 278.2 (2005): 323-335.), which is cited on the IDS filed 10/01/2021, and Moore-Scott teaches that in E10.5 and E11.5 Shh-/- mouse mutants, Gcm2 expression was undetectable suggesting that Shh is required for the establishment of the dorsal parathyroid-specific domain in the third pouch and its proper patterning (page 332, left col. para. 2 – 3; Figure 9). Moore-Scott teaches Shh is required for dorsal-ventral regionalization of the third pouch endoderm (page 333, right col. para. 1). Moore-Scott teaches in Shh mutants, the third pouch-derived organ phenotype was in some ways reminiscent of the Hoxa3 knockout phenotype which fails to initiate formation of the thymus/parathyroid primordium and does not express Gcm2 (page 333, right col. last para.). Moore-Scott teaches Fgf8 expression is changed in Shh mutants (page 333, left col. last para.; page 334, left col. para. 1). Thus, Snoeck teaches that FGF8 and SHH are important for Gcm2 expression and pharyngeal pouch development.
Snoeck does not teach obtaining TECs/TEPs with BMP4 but teaches TECs derived from ES or iPS cells by a method described herein could also improve humanized mouse models and as TECs are the essential functional component of the thymus (page 16, lines 12 – 14 and 29 – 31). Snoeck teaches in vivo formation of TEPs expressing PAX9 and AIRE from ES cells cultured in vitro with Noggin/SB431542 (page 23, lines 13 – 30; page 16, lines 12 – 14). Parent teaches differentiating VPE to TEPs in media containing BMP4 where BMP4 induced expression of FOXN1 in Figure 1A and B (page 16, 0246 – 0248; page 17, 0248). Thus, the combination of Snoeck and Parent make obvious the claimed method.
Applicant Argues: On page 13, paragraph 2 – 3, Applicant asserts that Snoeck is silent on using RA in combination with FGF8b, switching from a combination of RA and FGF8b to a combination of Shh and FGF8b, and the successive use of a BMP inhibitor followed by the use of BMP4 to yield TEPs or TECs and Snoeck teaches away from the use of Shh in combination with FGFs because Snoeck describes the use of said combination of differentiating factors to obtain parathyroid fate in a cell – not a thymic fate.
Response to Arguments: As discussed above, Snoeck teaches RA and FGF8b individually increase expression of pharyngeal endoderm and pharyngeal pouch markers TBX1 and PAX9 but Snoeck does not teach the combination of RA and FGF8b. Parent teaches a method of inducing differentiation of pluripotent stem cells into TECs and TEPs in vitro comprising differentiating embryonic stem cells into DE, AFE, ventral pharyngeal endoderm (VPE), TEP, and TEC (Figure 1A; page 1, 0010; page 2, 0029; page 17, 0249 – 0250). Parent teaches conditions for differentiating DE to AFE and AFE to VPE where when retinoic acid is included in the media, HOXA3 expression is induced in Figure 1A and B (page 16, 0247 – 0248). Parent teaches condition 7 contains both retinoic acid (0.25 µM) and FGF8b (50 ng/ml) and this condition resulted in HOXA3 induction to levels similar to that found in fetal and adult thymus in Figure 1B page 14, 0224 – 0225; page 17, 0248). Parent teaches Hoxa3 is an early and essential regulator of thymus specification and of differentiation of TEPs into mature TECs and Hoxa3 expression is a readout for thymic specification in the method (page 16, 0246). As discussed above, Snoeck teaches culturing AFE in the absence of RA and then adding FGF8, SHH, or the combination of FGF8 and SHH in Figure 3B where GCM2 expression is induced. Snoeck teaches GCM2 is a parathyroid-specific marker (page 25, line 13). Snoeck teaches the effects of SHH and FGF8 were not additive, mirroring in vivo epistasis studies showing that Shh is upstream of Fgf8 in mouse pharyngeal pouch development (page 25, lines 8 – 16). Solely to rebut Applicant’s argument regarding teaching away because Snoeck describes obtaining parathyroid fate and not thymic fate, Gordon (Gordon, Julie, et al. Mechanisms of development 103.1-2 (2001): 141-143.) which is cited on the IDS filed 10/01/2021 teaches the thymus and parathyroids originate from a common primordium that develops from the third pharyngeal pouch in mice and humans (Abstract). Gordon teaches Gcm2 is expressed specifically in the developing second and third pharyngeal pouches at E9.5 and is further confined to a small domain of the third pouch endoderm by E10.5 (Abstract; page 141, left col. para. 1 – 2; page 142, right col. para. 2). Thus, Snoeck teaches the combination of SHH and FGF8 induce expression of Gcm2 which is a third pharyngeal pouch marker and the thymus develops from the third pharyngeal pouch. As discussed above, Snoeck does not teach the use of BMP4 to yield TEPs or TECs. However, Snoeck teaches the use of Noggin for specification of pharyngeal endoderm cells that when transplanted into a mouse develop into TECs. Parent teaches differentiating VPE to TEPs in media containing BMP4 where BMP4 induced expression of FOXN1 in Figure 1A and B (page 16, 0246 – 0248; page 17, 0248) and Snoeck teaches FOXN1 is a marker of TECs. Parent teaches the TECs support the development of new T cells in vivo (page 18, 0258 – 0261; page 19, 0261 – 0266) and the combination of Snoeck and Parent make obvious the limitations of new claim 47.
Applicant Argues: On page 13, last para. – page 15, para. 1 – 2, Applicant provides arguments regarding the teachings of Diman, Yamagishi, Patel, Bleul, and Vizcardo.
Response to Arguments: As the previous rejections using the teachings of Diman, Yamagishi, Patel, Bleul, and Vizcardo have been withdrawn, Applicant’s arguments are rendered moot.
Applicant Argues: On page 15, last para. – page 16, para. 1 – 2, Applicant asserts that differentiating stem cells into terminally differentiated cells is highly unpredictable and the references do not provide any reasonable expectation of success in differentiating stem cells into said TECs and/or TEPs because there is a high degree of uncertainty associated with each step and that the more complex the differentiation method the more unpredictable it becomes. Applicant asserts it is extremely difficult if not impossible to predict with any reasonable certainty that the combination of multiple individual steps from various differentiation protocols would lead to the claimed TECs or TEPs. Applicant asserts that the claimed method obtains cells that are purer and functional as demonstrated by their ability to reconstitute T cells in vivo and the claimed method results in superior technical effects as compared to the cells obtained by the methods described in the art. Applicant asserts that there is no motivation or suggestion in Snoeck that the protocol could or should be modified to yield greater results.
Response to Arguments: In the new rejection, the combination of Snoeck and Parent provide a reasonable expectation of success in differentiating pluripotent stem cells into TEPs/TECs because both methods use the steps of differentiating pluripotent stem cells to DE, DE to AFE, and AFE to pharyngeal endoderm, and Parent teaches further differentiation of the pharyngeal endoderm to TEPs/TECs that can reconstitute T cells in vivo. Regarding the claimed method yields “cells that are purer”, the claims do not require a step of purification, however, Snoeck teaches that it appears critical that to obtain enriched or pure and functional populations of mature cells, developmental cues need to be sequentially applied to guide the differentiation of pluripotent cells in vitro (page 2, lines 5 – 8). Parent teaches a substantially pure cell population can be recovered by isolation methods such as by using an affinity reagent (page 10, 0153 – 0156; page 11, 0157; page 12, 0179; page 14, 0214).
Applicant Argues: On page 17, para. 1, Applicant asserts that the TBX1 upregulating potential of FGF8 was a novel finding.
Response to Arguments: Snoeck teaches in Figure 1C-2 that FGF8b increases expression of TBX1.
Applicant Argues: On page 17, para. 2, Applicant asserts that when RA exposure was reduced and replaced with Shh this yields upregulation of PAX9, PAX1, and TBX1, which is another innovation of the claimed method.
Response to Arguments: Snoeck teaches in Figure 1C-2 that RA increases expression of TBX1 and PAX9, where PAX9 is a maker of PP cells. Snoeck teaches Shh without RA increases Gcm2, which as discussed above, is a marker for third PP cells and Shh regulates expression of Pax1 and Pax9 as evidenced by Sivakamasundari (page 186, last para.; page 187, para. 3 – 4). Claim 1 does not recite a concentration change of RA going from step b (ii) to step c (i), a step of removing RA going from step c (i) to step c (ii).
Applicant Argues: On page 17, para. 3, Applicant asserts that the use of Noggin and then BMP4 is the first report of using Noggin for in vitro thymic differentiation.
Response to Arguments: Snoeck teaches Noggin increases PAX9 expression in Figure 1C-2 and Noggin treated cells transplanted into mice generated PAX9 and AIRE positive TECs. However, Snoeck does not teach culturing PP cells with BMP4 to form TECs/TEPs. Parent teaches differentiating VPE to TEPs in media containing BMP4 where BMP4 induced expression of FOXN1 in Figure 1A and B (page 16, 0246 – 0248; page 17, 0248). Therefore, the combination of Snoeck and Parent make obvious the use of Noggin for generating PP cells and then treating PP cells with BMP4 to generate TEPs/TECs.
Applicant Argues: On page 20, para. 1, Applicant asserts that the Cha is completely silent about thymus in general, about TECs, TEPs in particular and notably about any method of differentiating stem cells into TECs and TEPs.
Response to Arguments: The combination of Snoeck and Parent make obvious the claimed method. The teachings of Cha are cited for reducing the amount of undifferentiated pluripotent stem cells that may cause teratomas when transplanted in vivo as Snoeck teaches undifferentiated stem cells generated teratomas in vivo (page, lines 13 – 17). Cha teaches in the development of a cell therapeutic agent using pluripotent stem cells, there is a potential danger of formation of teratoma from undifferentiated pluripotent stem cells (page 1, 0005). Cha teaches there is a demand for development of a technology capable of selectively removing undifferentiated cells having the potential danger of teratoma without affecting the differentiated cells (page 1, 0005). Cha teaches a method of preparing a cell sample including undifferentiated pluripotent stem cells and differentiated cells by differentiating the pluripotent stem cells and causing the selective death of the undifferentiated pluripotent stem cells by treating the resultant cell sample with YM-155 (page 1, 0013 – 0014). One would have been motivated to combine the teachings of Snoeck and Cha to prepare a cell population free of undifferentiated cells as Snoeck teaches it appears critical that to obtain enriched or pure and functional populations of mature cells, developmental cues need to be sequentially applied to guide the differentiation of pluripotent cells in vitro. One would have a reasonable expectation of success in combining the teachings as Snoeck teaches undifferentiated stem cells generated teratomas in vivo and Cha teaches YM-155 causes the selective death of undifferentiated pluripotent stem cells.
Applicant Argues: On page 20, para. 3 – 5, Applicant asserts that at least for the same reasons detailed above regarding the rejection of claims 1 – 4, 6, 8, 9, and 11 – 13, Snoeck, Diman, Yamagishi, Patel, Bleul, and Vizcardo do not disclose or suggest the presently claimed method.
Response to Arguments: Applicant is directed to the Responses above.
Conclusion
No claims 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.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to ZANNA M BEHARRY whose telephone number is (571)270-0411. The examiner can normally be reached Monday - Friday 8:45 am - 5:45 pm.
Examiner interviews are available via telephone, in-person, and video conferencing using a USPTO supplied web-based collaboration tool. To schedule an interview, applicant is encouraged to use the USPTO Automated Interview Request (AIR) at http://www.uspto.gov/interviewpractice.
If attempts to reach the examiner by telephone are unsuccessful, the examiner’s supervisor, Peter Paras can be reached at (571)272-4517. The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300.
Information regarding the status of published or unpublished applications may be obtained from Patent Center. Unpublished application information in Patent Center is available to registered users. To file and manage patent submissions in Patent Center, visit: https://patentcenter.uspto.gov. Visit https://www.uspto.gov/patents/apply/patent-center for more information about Patent Center and https://www.uspto.gov/patents/docx for information about filing in DOCX format. For additional questions, contact the Electronic Business Center (EBC) at 866-217-9197 (toll-free). If you would like assistance from a USPTO Customer Service Representative, call 800-786-9199 (IN USA OR CANADA) or 571-272-1000.
/Z.M.B./Examiner, Art Unit 1632
/MARCIA S NOBLE/Primary Examiner, Art Unit 1632