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 .
DETAILED ACTION
Amendments
In the reply filed 12/08/2025, Applicant has amended claims 1, 4, 7-9, 16, 18, 20 and 22, and newly canceled claims 17, 24, 25, 29-32 and 34-36.
Claim Status
Claims 1-2, 4, 6-9, 11, 14, 16, 18, 20 and 22 are pending and are considered on the merits.
Withdrawn Claim Objections
The prior objection to claims 7, 8 and 20 because of missing conjunction is withdrawn in light of Applicant’s amendment to the claims.
New Claim Objections
Claims 1 and 4 are objected to because of the following informalities:
Claim 1 recites the phrase “thereby induing…” in the newly added limitation, which contains a typographic error. It should be changed to “thereby inducing”.
Claim 4 (c) recites “KDR or KDRlo”. It seems the term “KDR” misses an annotation directed to the expression level (e.g., KDR+, KDR- or KDRhigh).
Appropriate correction is required.
Withdrawn Claim Rejections - 35 USC § 112
The prior rejection of claims 4, 7-9, 16, 18 and 22 under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite is withdrawn in light of Applicant’s amendment to the claims.
New Claim Rejections - 35 USC § 112(d)
The following is a quotation of 35 U.S.C. 112(d):
(d) REFERENCE IN DEPENDENT FORMS.—Subject to subsection (e), a claim in dependent form shall contain a reference to a claim previously set forth and then specify a further limitation of the subject matter claimed. A claim in dependent form shall be construed to incorporate by reference all the limitations of the claim to which it refers.
The following is a quotation of pre-AIA 35 U.S.C. 112, fourth paragraph:
Subject to the following paragraph [i.e., the fifth paragraph of pre-AIA 35 U.S.C. 112], a claim in dependent form shall contain a reference to a claim previously set forth and then specify a further limitation of the subject matter claimed. A claim in dependent form shall be construed to incorporate by reference all the limitations of the claim to which it refers.
Claim 4 is rejected under 35 U.S.C. 112(d) or pre-AIA 35 U.S.C. 112, 4th paragraph, as being of improper dependent form for failing to further limit the subject matter of the claim upon which it depends, or for failing to include all the limitations of the claim upon which it depends.
Claim 4 (c) recites the limitation “KDR or KDRlo”. The term “KDR” is being examined as “KDR-” as being supported by specification (e.g., p. 2, para 4). Since base claim 1 has recited the new limitation of “KDR- or KDRlo endothelial cells”, the KDR embodiment in claim 4 (c) is axiomatically included in the scope of claim 1.
Applicant may cancel the claim, amend the claim to place the claim in proper dependent form, rewrite the claim in independent form, or present a sufficient showing that the dependent claim complies with the statutory requirements.
Withdrawn Claim Rejections - 35 USC § 103
The prior rejection of claims 1-2, 4, 6-9, 11, 14, 16, 18, 20 and 22 under 35 U.S.C. 103 as being unpatentable over Masuda et al., in view of Prasain et al. is withdrawn in light of Applicant’s amendment to claim 1 to recite new limitation of “KDR- or KDRlo endothelial cells”.
New Claim Rejections - 35 USC § 103
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
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.
Claims 1-2, 4, 6-9, 11, 14, 16, 18, 20 and 22 are rejected under 35 U.S.C. 103 as being unpatentable over Masuda et al., (Regenerative Therapy. 2018; 9: 1-9. Cited in IDS 09/06/2022) in view of Prasain et al., (Nature Biotechnology. 2014;32(11): 1151-1157. Cited in IDS 09/06/2022) and Sahara et al., (Cell Research. 2014; 24:820-841).
With respect to claim 1, Masuda teaches a method of preparing iPSC-derived CD31+ endothelial cells using three-dimensional suspension culture (see title, abstract), thus teaches the preamble a method of generating endothelial cells from pluripotent stem cells.
In regard to step (a) culturing the PSCs in a 3-D suspension culture and the last wherein clause, Masuda teaches the iPSCs are cultured in a 3-D suspension culture in a bioreactor (see e.g., abstract and p. 3, left col, para 3.1, also see Fig 1). Masuda teaches “small colonies of hiPS cells were seeded into 30-mL culture vessels” and “cell aggregates on day 7 were dissociated and subjected to MACS” (p. 3, left col, para 3.1), thus teaches step (a) and the last wherein clause the PSCs form cellular aggregates in the suspension.
In regard to step (b), Masuda teaches inducing iPSCs to undergo endothelial differentiation by (i) culturing the PSCs with growth factors and small molecules including BMP-4, Activin A and bFGF (also known as FGF-2) during day 2 to day 5 and (ii) replacing with medium containing VEGF and bFGF during day 5 to day 7 and day 7 to day 10 (see scheme in Fig 1). Thus, Masuda teaches the PSCs are cultured in a first endothelial differentiation medium and a second endothelial differentiation medium comprising the growth factors and small molecules recited in instant claim 1.
However, Masuda does not specifically teach the exact components or the exact culture duration in the first and the second endothelial differentiation medium in claim 1.
Prasain teaches a method of differentiating human PSCs into cells similar to cord-blood endothelial colony-forming cells (CB-ECFC-like cells), which display a stable endothelial phenotype with high clonal proliferative potential and the capacity to form human vessels in mice and to repair the ischemic mouse retina and limb (see e.g., abstract). Prasain tests multiple protocols (see p. 1151, right col, last two paragraphs, also see supplementary Figs 1, 2, and 3) and develops a protocol having a combination of growth factors/culture duration to enhance generation of stable CB-ECFC-like cells with clonal proliferative potential and in vivo vessel-forming capacity (p. 1152, right col, see Fig 1a). Prasain teaches the protocol comprises “[a]fter 2 d (-D2) of culture in mTeSR1 media, cultures were directed toward the mesodermal lineage with addition of activin A (10 ng/ml) in the presence of FGF-2, VEGF165 and BMP4 (10 ng/ml) for 24 h. The following day, activin-A containing media was removed and replaced with 8 ml of Stemline II complete media containing FGF-2, VEGF165 and BMP4 to promote endothelial cell emergence and expansion” (see Online Methods, p. 1, left col, para “Directed differentiation of hESCs and hiPSCs into the ECFC lineage”). Thus, Prasain teaches culturing the PSCs for about 24 hours in a first endothelial differentiation medium comprising Activin-A, BMP-4, FGF-2 and VEGF and then replacing with a second endothelial differentiation medium comprising BMP-4, FGF-2 and VEGF in claim 1 (b) (i) and (ii).
Therefore, it would have been obvious for one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of generating endothelial cells from PSCs comprising culturing the PSCs in a first and a second endothelial differentiation medium containing BMP-4, Activin A, bFGF and VEGF disclosed by Masuda, by substituting with culturing the PSCs for about 24 hours in a first endothelial differentiation medium comprising Activin-A, BMP-4, FGF-2 and VEGF and then replacing with a second endothelial differentiation medium comprising BMP-4, FGF-2 and VEGF as taught by Prasain with a reasonable expectation of success. Since Masuda has used a protocol using all the recited growth factors and small molecules for inducing PSCs to undergo endothelial differentiation, and since Prasain compares multiple protocols and develops a protocol with the recited growth factors/small molecules in the media and the recited culture duration to enhance generation of stable endothelial cells with clonal proliferative potential and vessel-forming capacity (p. 1152, right col, see Fig 1a), one of ordinary skill in the art would have had a reason to substitute with Prasain’s first and second medium and culture duration in the method of Masuda in order to enhance the generation of stable endothelial cells with high proliferative potential and vessel forming capacity for forming pre-vascularized network structures in 3-D functional tissues (see Prasain p. 1152, right col and Masuda, abstract).
In regard to the new limitation thereby inducing the PSCs to differentiate into KDR- or KDRlo endothelial cells, as a first matter, it must be noted that this thereby clause does not recite an active step in the claimed method, but only the results of the inducing step comprising culturing the PSCs in the first and the second endothelial differentiation medium for the defined duration as suggested by Masuda in view of Prasain. MPEP 2111.04 I states a whereby clause “in a method claim is not given weight when it simply expresses the intended result of a process step positively recited.” Therefore, this new limitation does not provide any patentable weight in determining patentability of the claimed method.
Nevertheless, in order to provide compact prosecution, the limitation is examined as follows. The instant specification provides definition of the term "lo" … to mean a significant decrease in the expression of said marker… as compared to a reference level (p. 12, para 3). Thus, this limitation of KDRlo endothelial cells is reasonably interpreted as late-passage endothelial cells that have decreased percentage of KDR+ cells as compared to those in the intermediate stage.
As stated supra, both Masuda and Prasain teach inducing the PSCs to differentiate into endothelial cells. In regard to the dynamic expression of KDR in the endothelial cells during differentiation, Prasain indicates the KDR-expressing cells are progressively decreased after long-term in vitro culture, evidenced by there being about 77% KDR+ cells at day 6 of hPSC differentiation (see Fig 3b inset for the flow cytometry plot), while in late-passage (Passage 14) hPSC-ECFCs, KDR was expressed in 50-60% of cells (Fig. 4c) (p. 1156, left col, lines 1-2). This pattern is supported by the teaching of prior art Sahara, who teaches during human PSC differentiation into endothelial cells, endothelial cells exhibited weaker expression of KDR from Day 6 (99% KDR+) to Day 14 (78% KDR+) when cultured with VEGF-A (see Figure 3E for flow cytometry plots. It is noted that the method of Masuda in view of Prasain comprises culturing with VEGF), presumably indicating EC maturation (p. 826, left col, para 1). Sahara also teaches after 4 days, VEGF-A treatment promoted endothelial differentiation of KDR+ precursors and decreased KDR expression (p. 828, left col, para 1, see Fig 4A row 3 from 100% sorted KDR+ cells at Day 4 to 56% KDR+ at Day 8).
Therefore, one of ordinary skill in the art would have immediately expected that the method of generating endothelial cells from PSCs as suggested by Masuda in view of Prasain would have resulted in inducing the PSCs to differentiate into late-passage endothelial cells (i.e., KDRlo endothelial cells) that have decreased percentage of KDR+ cells as compared to the intermediate stage as suggested by Prasain and Sahara.
With respect to claim 2 directed to the PSCs being human PSCs, and claim 4 directed to the endothelial cells being human endothelial cells, Masuda teaches the PSCs are human induced pluripotent stem cells (hiPS cells) (see p. 2, left col, para 2.3), and Prasain teaches the PSCs are human ESCs and human iPSCs (see Online Methods, p. 1, left col, para “Directed differentiation of hESCs and hiPSCs into the ECFC lineage”), thus both teach the PSCs are human PSCs and the endothelial cells are human endothelial cells.
With respect to claim 6 and claim 7 directed to the endothelial cells being human endothelial cells which express CD31, as stated supra, both Masuda and Prasain teach the endothelial cells are human endothelial cells. Furthermore, both Masuda and Prasain teach the human endothelial cells express CD31 (see e.g., Masuda, abstract and Prasain, p. 1152, right col).
With respect to claim 8 directed to the concentration of each of Activin-A, BMP-4, FGF-2 and VEGF in the first endothelial differentiation medium, as stated supra, Prasain teaches the concentration of activin A (10 ng/ml) and FGF-2, VEGF165 and BMP4 (10 ng/ml) in the first endothelial differentiation medium (see Online Methods, p. 1, left col, para “Directed differentiation of hESCs and hiPSCs into the ECFC lineage”), within the recited range of 1 ng/ml to 100 ng/ml. Accordingly, one of ordinary skill in the art would have chosen the taught concentrations of the growth factors/small molecules since Prasain has reduced to practice the concentrations that successfully enhances generation of stable endothelial cells (Prasain p. 1152, right col).
With respect to claim 9 directed to the endothelial cells being CD31+ and NRP-1lo for three to 12 passages, as stated supra, this limitation of NRP-1lo endothelial cells is reasonably interpreted as late-passage endothelial cells that have decreased percentage of NRP-1+ cells as compared to those in the intermediate stage. Prasain teaches NRP-1 expression was progressively downregulated in late-passage hPSC-ECFCs (p. 1155, right col, see Fig 4A in which the NRP-1+ cells in the CD31+ cells decrease from 91% at Passage 4 to 53% at Passage 18), thus teaches the endothelial cells being CD31+ and NRP-1lo for three to 12 passages. Accordingly, one of ordinary skill in the art would have immediately expected that the method of generating endothelial cells from PSCs as suggested by Masuda in view of Prasain would have resulted in the endothelial cells being CD31+ and NRP-1lo for three to 12 passages.
With respect to claim 11 directed to the endothelial cells being functional without co-culture, Masuda teaches the hiPS cell-derived endothelial cells form a sprouted branch-like structure on the Matrigel (see e.g., Fig. 2D), and Prasain teaches hiPSC-ECFCs form complete capillary networks on Matrigel (see e.g., Fig 1f), thus both teach the endothelial cells are functional without co-culture.
With respect to claim 14 directed to the 3-D suspension culture being carried out in a stirred-tank bioreactor, Masuda teaches the 3-D suspension culture is carried out in a bioreactor with a magnetic stirrer (p. 2, left col, para 2.3).
With respect to claim 16 directed to the 3-D suspension culture being serum-free, Masuda teaches small colonies of hiPS cells were seeded into culture vessels in mTeSR1 containing Y27632 and cultured until day 2 (p. 2, left col, para 2.3), thus teaches the 3-D suspension culture being serum-free.
With respect to claim 18 directed to the 3-D suspension culture using a culture vessel that is feeder-cell free, as stated supra, Masuda teaches the 3-D suspension culture is carried out in a bioreactor with a magnetic stirrer, and small colonies of hiPS cells were seeded into culture vessels in mTeSR1 containing Y27632 and cultured until day 2 (p. 2, left col, para 2.3). It is noted that mTeSR1 is a feeder-free, serum-free medium. Thus, Masuda teaches the 3-D suspension culture uses a culture vessel that is feeder-cell free.
With respect to claim 20 directed to the PSCs being expanded prior to the 3-D suspension culture, Masuda teaches the human iPS cells are maintained in Primate ES Cell Medium supplemented with bFGF and are passaged as small clumps every 3 days (p. 2, left col, para 2.2), thus teaches the PSCs are expanded prior to the 3-D suspension culture.
With respect to claim 22 directed to further comprising a step of isolating endothelial cells, as stated supra, Masuda teaches the iPSC-derived endothelial cells are isolated by enzymatic dissociation and MACS (p. 2, left col, para 2.3).
Hence, the claimed invention as a whole was prima facie obvious to a person of ordinary skill before the effective filing date of the claimed invention in the absence of evidence to the contrary.
Response to Traversal:
Applicant’s arguments filed on 12/08/2025 are acknowledged.
Applicant argues that neither motivation to combine nor reasonable expectation of success has been established. Specifically, Applicant argues that (1) Masuda is silent as to the importance of the timing or the combinations/timings used. Prasain describes a 2D culture system and describes a method which is feeder-free. While the cell line used in Masuda requires co-culture with feeder cells, and acknowledges that the results and methods cannot be generalized between cell lines. Thus, there is no motivation to combine (Remarks, p. 11); and (2) Masuda’s method differs to that of the present invention with respect to molecules and timing. The method of Masuda produces predominantly a population of endothelial cells that is predominantly KDR+, whereas the endothelial cells of the present invention are KDR- or KDRlo. Therefore, a skilled person would have no reasonable expectation of success to produce KDR- or KDRlo endothelial cells (Remarks, p. 11-12).
Applicant’s arguments have been fully considered but they are not persuasive.
In response to Applicant’s first argument, as a first matter, Masuda contemplates to improve the method repeatedly, such as “We consider the development of a culturing method that enables long-term amplification of iPS cell-derived CD31+ cells as a future subject” (p. 8, right col, para 3), “In the future, standardization of differentiation induction method using plural strains and development of proliferation method of iPS cell-derived endothelial cells are required” (p. 8, right col, para 4) and “it is suggested that improving proliferative ability is a key factor for preparing a large number of hiPS cell-derived endothelial cells in the future” (p. 8, right col, para 5). One of ordinary skill in the art would have understood that the method required for improvement as suggested by Masuda would have included both reagents and timing of incubation. Therefore, Masuda acknowledges the importance of the timing or the combinations/timings used in the method.
Secondly, although Masuda teaches a 3-D suspension culture in the first stage of the culture, the KDR+ cells are re-cultured onto tissue culture dishes in the later stage of differentiation (e.g., p. 2, left col, para 2.3), which is similar to Prasain. Furthermore, one of ordinary skill in the art would have understood, as agreed by Applicant, that the important determinant for differentiation is the combinations of agents and timings used in the method. Thus, one of ordinary skill in the art would have had a reason to combine Prasain’s 2-D culture in Masuda’s 2-D and 3-D method.
Thirdly, regarding feeder cells, both Masuda and Prasain use a feeder-free culture condition for differentiating PSCs into endothelial cells. Applicant may have confused Masuda’s using feeder cells in maintaining PSCs before differentiation.
Fourthly, regarding Masuda acknowledging that the results and methods cannot be generalized between cell lines, as discussed above, Masuda contemplates to improve the method repeatedly (see above). Thus, one of ordinary skill in the art would have had a reason to combine Prasain in the method of Masuda.
In response to Applicant’s second argument, as a first matter, Applicant is reminded, 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, Masuda teaches a method of generating endothelial cells from PSCs comprising the steps recited in the instant invention. The only difference between Masuda and the instant invention is that Masuda does not use the defined combination of molecules and duration. Prasain is cited to teach the defined combination of molecules and the defined duration as claimed in the instant invention.
Secondly, Applicant’s argument that the method of Masuda produces predominantly a population of endothelial cells that is predominantly KDR+, is not supported by Masuda. The citation of Masuda in the Remarks (page 12) teaches that “70%-80% of KDR+ cells became positive for CD31” (Masuda, p. 8, left col.), which is not equivalent to the endothelial cells (i.e. CD31 positive cells) being predominantly KDR+. This is supported by both Prasain and the newly cited art Sahara, who teaches that “after 4 days, VEGF-A treatment promoted endothelial differentiation of KDR+ precursors and decreased KDR expression” (Sahara, p. 828, left col, para 1, see Fig 4A row 3 from 100% sorted KDR+ cells at Day 4 to 56% KDR+ at Day 8). Thus, both Prasain and Sahara teach that the KDR+ cells are differentiated into endothelial cells, in which the KDR expression is in turn decreased (i.e., KDRlo endothelial cells). Therefore, a skilled person would have had a reasonable expectation of success to produce KDR- or KDRlo endothelial cells by the method of Masuda in view of Prasain and Sahara.
Conclusion
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 extension fee 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 date of this final action.
No claims are allowed.
Examiner Contact Information
Any inquiry concerning this communication or earlier communications from the examiner should be directed to Jianjian Zhu whose telephone number is (571)272-0956. The examiner can normally be reached M - F 8:30AM - 4PM (EST).
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If attempts to reach the examiner by telephone are unsuccessful, the examiner’s supervisor, James Douglas (Doug) Schultz can be reached on (571) 272-0763. The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300.
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/JIANJIAN ZHU/Examiner, Art Unit 1631
/JAMES D SCHULTZ/Supervisory Patent Examiner, Art Unit 1631