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 .
Continued Examination Under 37 CFR 1.114
A request for continued examination under 37 CFR 1.114, including the fee set forth in 37 CFR 1.17(e), was filed in this application after final rejection. Since this application is eligible for continued examination under 37 CFR 1.114, and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114. Applicant's submission filed on 10-2-25 has been entered.
Claims 1-5, 24, 25 have been canceled. Claims 6-23, 26, 27 are pending.
Applicant's arguments filed 10-2-24 have been fully considered but they are not persuasive.
The text of those sections of Title 35, U.S. Code not included in this action can be found in a prior Office action.
Election/Restrictions
Applicants elected Group VIII, claims 6-18, differentiating a clonal multipotent cell into an adipocyte that expresses UCP1 using a TGFβ superfamily protein and a PPARγ agonist in the reply filed on 4-2-24 without traverse.
Claim 6 as amended is drawn to differentiating pluripotent cells into brown adipocytes that express UCP1, C19orf80, or ADIPOQ but not COX7A1.
Claims 6-23, 26, 27 remain under consideration as they relate to differentiating multipotent cells into adipocytes that express UCP1 but not COX7A1.
Claim 6 and its dependents are not under consideration as they relate to differentiating pluripotent cells into adipocytes that express C19orf80 or ADIPOQ but not COX7A1.
Claim 10 and its dependents are not under consideration as they relate to differentiating pluripotent cells into adipocytes that express FABP4, C19orf80, ADIPOQ, NTNG1, or THRSP but not COX7A1.
Specification
The title will have to be changed to more closely reflect the claimed subject matter, i.e. ---METHOD OF GENERATING BROWN ADIPOCYTES FROM PLURPOTENT CELLS---.
Claim Objections
The steps of “a) differentiating a human pluripotent cell line in the presence of DMEM/F12, B27 supplement, and basic Fibroblast Growth Factor (bFGF) thereby forming an embryoid body; and b) producing clonal human progenitor cell lines from the embryoid body” in claim 6 does not make sense because it starts with differentiating pluripotent cells into embryoid bodies but results more broadly by obtaining any clonal “progenitor” cell line which encompasses totipotent, pluripotent, or multipotent cells. The phrase also does not make sense because “clonal” infers an active step of “cloning” the cells which is missing from the claim.
The phrase “thereby forming” in step a) of claim 6 does not necessarily indicate the “differentiating” has the function of producing an embryoid body. It simply says the active step of “differentiating” must be performed using DMEM/F12, B27 supplement, and bFGF. If the final product at the end of step a) is an embryoid body, then just say ---differentiating human pluripotent cells into embryoid bodies…---.
Step b) of claim 6 is actually a cloning step and should be set forth as such, i.e. ---cloning cells from the embryoid body---. But it is unclear what kind of “progenitor cells” are being cloned here, so the concept of “producing [any] clonal human progenitor cell lines from the embryoid body” in step b) does not make sense.
It is unclear what kind of “progenitor cells” express HOXA5 & IL13RA but not COX7A1 in step c), so the concept of “isolating [any kind of] clonal human progenitor cell line that expresses HOXA5 & IL13RA but not COX7A1” from a cloned embryoid body in step c) does not make sense.
The phrase “thereby producing” in step d) of claim 6 does not necessarily indicate the “differentiating” has the function of producing “differentiated progeny”. It also fails to set forth into what the “progeny” have differentiated. It simply says the active step of “differentiating” must be performed using TGFβ or PPARγ agonist. If the final product at the end of step d) is a specific kind of progenitor cell, then just say ---differentiating the clonal [multipotent?] cell that expresses HOXA5 and IL13RA2 but not COX7A1 into __[?]__ cells…---.
The phrase “one or more of the genes UCP1, C19orf80, or ADIPOQ” in claim 6 is improper Markush language. The list is of proteins, but the set up of the phrase says they are genes. Proper Markush language would be ---express a gene selected from the group consisting of a UCP1 gene, a C19orf80 gene, or an ADIPOQ gene--- or ---express a protein selected from the group consisting of UCP1, C19orf80, or ADIPOQ---. It would be simpler to say ---a cell line that expresses UCP1, C19orf80, or ADIPOQ but not COX7A1--- which is open claim language and encompasses cells that express one or more of UCP1, C19orf80, or ADIPOQ but that do not express COX7A1.
It is unclear whether the clonal progenitors in claim 9 refer to the ones being produced in step b) of claim 6, isolated in step c), or differentiated in step d).
Claim 10 is unclear for reasons set forth above in claim 6.
Claims 14-19 are unclear for reasons set forth in claim 9.
Claim 19 is unclear because it is a sentence fragment and grammatically incorrect. It should be ---further comprising---.
Claim Rejections - 35 USC § 112
Enablement
Claims 6-23, 26, 27 remain rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, because the specification, while being enabling for a method of culturing human clonal pluripotent stem cells in media conditions comprising rosiglitazone such that brown fat cells expressing UCP1 are obtained, does not reasonably provide enablement for the claims as written. The specification does not enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and/or use the invention commensurate in scope with these claims.
A) The specification does not enable culturing human pluripotent cells in the presence of DMEM/F12, B27 supplement, and bFGF such that an embryoid body (EB) is obtained, cloning cells of the EB, isolating any kind of “progenitor cell” from the EB, isolating any kind of “progenitor cell” that expresses HOXA5 & IL13RA2 but not COX7A, or differentiating any “progenitor cell” that expresses HOXA5 & IL13RA2 into any “progenitor” that expresses UCP1, C19orf80, or ADIPOQ but not COX7A1 as required in claims 6 and 10.
Claims and scope
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It is unclear what well-known progenitors are encompassed by “clonal progenitors” that are produced from EBs as required in step b) or “clonal progenitors” that express HOXA5 and IL13RA2 but not COX7A1 obtained from the EBs in step c). The claims do not require a step of cloning, so it is unclear how the intermediary cells are “clonal”. The “progenitor cell” in step b) and c) also encompasses any totipotent, pluripotent, or multipotent cell which does not make sense. The specification does not enable obtaining intermediary cells that are totipotent as encompassed by step b) or c) because totipotent cells are less differentiated than pluripotent cells. The specification does not enable obtaining intermediary cells that are HOXA5+ IL13RA2+ COX7A1- pluripotent cells because the concept is not described in the specification or the art.
The specification does not enable culturing pluripotent cells in any culture medium comprising DMEM/F12, B27 supplement, and bFGF such that HOXA5+ IL13RA2+ COX7A1- multipotent cells are obtained because bFGF is used to maintain pluripotency. This is shown by Haghighi (Cell Comm. & Signaling, 2018, Vol. 16, pg 1-14) (see title, abstract, materials & methods, results) and West (Regenerative Med., 2008, Vol. 3, pp, 287-308) who cultured human pluripotent cells in bFGF to maintain pluripotency (pg 300, col. 2, 1st full paragraph).
West cloned pluripotent cells (pg 301, “Isolation & expansion of clonal cell lines”) and cultured them in a variety of conditions (pg 289 col. 1, lines 3-7) including mesenchymal conditions (Supplementary Table A1, cited on pg 288 col, 2, para 1, lines 7-17; see lines 59, 89, 193, 196, 220, 303 of Table A1; see also pg 302, col. 1, “Mesencult basal medium” plus “supplement”).
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Supplementary Table A2 shows expression patterns of HOXA5, IL13RA2, and COX7A1 (lines 330, 4045, 372, respectively)
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But it is unclear from the massive number of cells whether any of the cells had the expression pattern claimed. The cells lines across the top of Supp. Table A2 of West include those described by applicants as being part of the invention on pg 31, lines 3-5 (E3, E72, E75, C4ELS5.1, C4ELSR2, and NP110SM). C4ELS5.1 made by West is in column Q of Table A2, is described by applicants on pg 32, line 22; pg 41, line 26, and described in 2010/0184033 (12/504630), 8685386 (13/384289), 2013/0115673 (13683241), 2014/0178994 (14/172765), 2014/0234964 (14/131429) which express TAC1, EBF2, SCARA5, EYA4, and TBX1, but not HOXA10, ZIC2, or MKX).
West cultured human pluripotent clonal cell line C4ELS5.1 in bFGF, but the cells did not express HOXA5, IL13RA2, or COX7A1 (Supp Table A2; column Q, lines 330, 4045, and 372 respectively). Thus the ability to culture pluripotent cells in any culture media comprising bFGF and obtain any HOXA5+, IL13RA2+, and COX7A1- progenitor was unpredictable.
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Pg 31, lines 26-31; pg 42, line 29, discuss the marker pattern of cell line NP110SM in quiescence.
“When RNA was extracted from these NP110SM cells at passage 10 and induced into quiescence for 5 days a condition sometimes referred to as "control" or "Ctrl" herein, the cells displayed the following gene expression markers: DLK1, HOXA5, SLC7A14, NTNG1, HEPH, PGM5, IL13RA2, SLC1A3, and SBSN but unlike fetal or adult-derived BAT progenitors do not express COX7A1, or one or more markers chosen from POSTN, KRT34, MKX, HAND2, TBX15, HOXAIO, PLXDC2, DHRS9, NNAT, and HOXDll.”
However, NP110SM cells are HOXA5+ IL13RA2+ COX7A1- as required in step b), but they are clonal pluripotent cells.
Applicants do not teach how to overcome the teachings of West and culture NP110SM cells or any other clonal pluripotent cells “into quiescence” in any culture media comprising DMEM/F12, B27 supplement, and bFGF such that any HOXA5+, IL13RA2+, and COX7A1- pluripotent cells are obtained.
The specification does not teach how to obtain pluripotent cells that are HOXA5+, IL13RA2+, and COX7A1- as broadly encompassed by “progenitor” because C4ELS5.1 and all of the other clones described by West were cultured in bFGF and cloned, but they did not have the marker pattern claimed.
The specification does not teach how to obtain totipotent cells that are HOXA5+, IL13RA2+, and COX7A1- as broadly encompassed by “progenitor” because the specification and the art do not teach de-differentiating the pluripotent cells into totipotent cells.
The description of Fig. 15 on pg 11 says NP110SM cells were cultured in “reduced growth factors to induce quiescence” without describing the specific culture media comprising bFGF, the “growth factors” that are “reduced”, or the amount of “growth factors” used to obtain HOXA5+, IL13RA2+, and COX7A1- pluripotent cells. The remainder of the paragraph goes on to describe differentiating the “quiescent” NP110SM cells using HyStem beads BMP4 as the TGFβ protein and rosiglitazone as the PPARγ agonist.
The specification does not teach how to obtain NP110SM pluripotent cells in “quiescence” that are HOXA5+, IL13RA2+, and COX7A1- as broadly encompassed by step b) because while NP110SM had the marker pattern claimed, the specification did not teach how to recapitulate obtaining pluripotent cells having the same structure as NP110SM, or the specific culture conditions required to induce “quiescence” of NP110SM cells to make them HOXA5+, IL13RA2+, COX7A1-.
Perhaps the culture medium in step a) is supposed to be mesenchymal stem cell medium because adipose tissue is derived from mesenchymal cells, but the specification does not teach culturing NP110SM or any other clonal pluripotent cell in mesenchymal stem cell medium such that HOXA5+, IL13RA2+, COX7A1- progenitors are obtained.
It’s possible the cells produced in steps b) and c) are mesenchymal stem cells, but the function of progenitors isolated in step b) and c) is missing from the claims.
The specification and the art at the time of filing teach that the pluripotent cells in step a) are cloned. This step is missing from the claim.
Applicants and the art do not describe “cloning” progenitors that are HOXA5+, IL13RA2+, and COX7A1- as inferred by step b) and c).
Applicants do not disclose the specific starting clonal cells or the specific differentiation medium to overcome the unpredictability of West and arrive at HOXA5+, IL13RA2+, and COX7A1- multipotent cell.
The specification does not teach the specific mesenchymal differentiation medium and supplements required to overcome the unpredictability of West and turn E3, E72, E75, C4ELS5.1, C4ELSR2, NP110SM, C4ELS5.1, or any other clonal pluripotent cell into HOXA5+, IL13RA2+, and COX7A1- multipotent cells as encompassed by step b).
It is unclear whether the HOXA5+, IL13RA2+, and COX7A1- progenitors in step b) are well-known mesenchymal stem cells, well-known preadipocyte progenitors, or if they are some newly identified progenitor. If they are newly identified progenitors, applicants do not disclose whether they are MSCs, adipocyte progenitors, brown adipocyte-specific progenitors, white adipocyte-specific progenitors, beige adipocyte -specific progenitors.
Given the lack of guidance in the specification taken with the art at the time of filing, it would have required those of skill undue experimentation to determine how to culture human pluripotent cells in the presence of DMEM/F12, B27 supplement, and bFGF such that any HOXA5+ IL13RA2+ COX7A-progenitor is obtained as required in steps a), b) and c) of claims 6 and 10 other than culturing human pluripotent cells in bFGF and cloning them as described by West.
B) The specification does not enable contacting any HOXA5+, IL13RA2+, and COX7A1- progenitor with any TGFβ superfamily protein or PPARγ agonist such that UCP1+ COX7A1- brown adipocytes are obtained as required in step c) of claims 6 and 10 other than culturing well-known progenitors with BMP4 or BMP7 and rosiglitazone.
The starting material of step c) encompasses any totipotent, pluripotent, or multipotent HOXA5+, IL13RA2+, and COX7A1- cell.
The TGFβ superfamily encompasses TGFβ1, TGFβ2, TGFβ3, activin, inhibins, BMP2-14, growth differentiation factors (GDF), glial-derived neurotrophic factors (GDNFs).
The PPARγ agonists encompass:
Thiazolidinediones (TZDs):
Pioglitazone (Actos)
Rosiglitazone (Avandia)
Troglitazone (removed from market due to liver toxicity)
Dual PPAR alpha/gamma agonists:
Lobeglitazone
Muraglitazar
Other PPAR gamma agonists:
Elafibranor
Saroglitazar
Elabd (Stem Cells, 2009, Vol. 27, pg 2753-2760) differentiated human brown preadipocytes into brown adipocytes expressing UCP1 (pg 2754, col. 1, last 2 lines). Elabd also taught: “in contrast to primary and clonal preadipocytes of WAT from various species, no primary or clonal precursor cells of human brown adipocytes have been so far obtained that could be used as tools to develop therapeutic drugs and to gain further insights into the molecular mechanisms of brown adipogenesis [29]” (pg 2754, col. 1, about 20 lines from top). (Cannon, “Cultures of adipose precursor cells from brown adipose tissue and of clonal brown-adipocyte-like cell lines”, Methods Mol. Biol., 2001, Vol. 155, pg 213-222) is reference 29).
Schulz (PNAS, Jan. 4, 2011, Vol. 108, No. 1, pg 143-148) differentiated multipotent mouse (SI, pg and human (SI, pg 2, col. 1, line 5) cells into brown adipocytes using the PPARγ agonist rosiglitazone in combination with BMP7 (pg 147, col. 1, “BMP7 acts….”; SI, pg 7, Fig. S9B). UCP1 expression was obtained (Fig. S9H).
Scott (Stem Cells and Development, Oct. 2011, Vol. 20, No. 10, pg 1793-1804) differentiated clonal multipotent mouse adipocytes (3T3-L1) into brown adipocytes expressing UCP1 using rosiglitazone (pg 1794, Table 1; pg 1798, “Use of PPARγ agonists”). The multipotent adipocyte cell line 3T3-L1 is clonal is structurally and functionally equivalent to a “clonal embryonic progenitor cell derived from human pluripotent stem cells” in claim 6 and a “pluripotent stem-cell derived clonal progenitor cell line” in claim 10. Without evidence to the contrary the 3T3-L1 cell line expresses DLK, SBSN and IL13RA2 but not COX7A1 as in claim 10 because it is encompassed by the claim and because it is capable of making brown adipocytes.
Boyden (20120321671) differentiated progenitors into brown adipocytes expressing UCP1 using rosiglitazone and BMP4 or BMP7 (para 40, 47).
The description of Fig. 15 on pg 11 says NP110SM cells were cultured in “reduced growth factors to induce quiescence” followed by differentiating the “quiescent” NP110SM cells using HyStem beads BMP4 as the TGFβ protein and rosiglitazone as the PPARγ agonist.
Pg 43, line 19 says NP110SM does not express COX7A1 in either the undifferentiated or differentiated states” but does not teach brown adipocytes obtained from NP110SM are UCP1+ COX7A1-.
The specification mentions other TGFβ proteins and PPARγ agonists without clearly correlating the amounts required to obtain UCP1+ COX7A1- brown adipocytes from NP110SM or any other pluripotent cell as broadly claimed.
Despite calling the UCP1+ COX7A1- cells in step c) “brown adipocytes”, it is unclear whether the UCP1+ COX7A1- cells in step c) are brown, beige, or white adipose because the specification is so confusing. Applicants argued brown/beige/white adipocytes do not all share the same marker profile (pg 26, 1st full para, of the response filed 6-7-17).
Pg 25, lines 4-15,
“As used in the present invention, all cells of the present invention expressing UCP1 and one or more of ADIPOQ or C19orf80 are designated as "brown" fat cells. While small molecule drugs such as the thiazolidinedione class of compounds (rosiglitazone, also known as Avandia) have shown usefulness as antidiabetic agents, such compounds can often have serious side effects. Therefore, the concept of brown fat cell transplantation as a therapeutic regimen has emerged. Reports suggest that the loss of brown or beige fat cells may correlate with obesity, cardiovascular disease, hypertension, and type II diabetes and restoration of these cells by transplantation can reverse obesity and type II diabetes in nonhuman animal studies. There remains, however, a need for a method for the manufacture brown fat cellular components that express UCP1 and certain adipokines expressed by brown fat tissue such as adiponectin and C19orf80 on an industrial scale suitable for transplantation in humans for the treatment of these large and growing health problems.”
Pg 25, lines 21-28,
“The present invention teaches methods and compositions for the manufacture of specific cellular components of BAT tissue, including: 1) UCP1-expressing brown adipocytes that express low to undetectable levels of the adipokines adiponectin and betatrophin; 2) adiponectin +, betatrophin + adipocytes that express low or no levels of UCP1; 3) UCP1-expressing brown adipocytes that express ADIPOQ and C19orf80 at levels comparable to fBAT cells, and 4) vascular endothelial cells expressing ITLN1 or ITLN2; and combinations of these three cell types with collagen and hyaluronic acid-based hydrogels with or without added cells from autologous adipose-derived SVF.”
Therefore, the specification does not enable making UCP1+ COX7A1- cells that are “brown adipocytes” because the markers are associated with brown, beige, or white adipose as well as cells that are “browning”.
Pg 50, line 33, teaches differentiating human pluripotent cells “in preparation [step 1] for the generation of candidate cultures [step 2] which function as stock cultures from which progenitor cells with the gene expression profile of the NP110SM line can be isolated [step 3] (pg 51, lines 1-3).
In step 1, hES cells are cultured in a specific neural differentiation medium (“NP (-)”) until EBs are formed (pg 51, lines 5-11). Every 48 hours, the medium was replaced with fresh medium supplemented with Noggin and bFGF. On day 21, the medium was replaced with fresh medium supplemented only with bFGF.
In step 2 (pg 51, line 17), “Generation of Stock Candidate Cultures”, the EBs obtained on day 22 are dissociated and cultured in “NP (-)” medium supplemented with bFGF (“NP (+)”). Medium was changed 24 hrs later, then 3x/week until confluence and then expanded and plated at clonal dilution (pg 51, line 31). This appears to be a starting dilution that is not clonal, per se, because the cells are 500-7000 cells/50ml in a 15cm dish; these are then further diluted in step 3.
In step 3 (pg 52, line 4), the “Stock Candidate Cultures” are further diluted in “NP (+)” supplemented with bFGF to obtain clonal cell populations. Applicants do not disclose any of the multipotent cells obtained from pluripotent cells express expressing DLK1, SBSN, HOXA5, SLC7A14, HEPH, PGM5, SLC1A3 and IL13RA2, but not COX7A1, as required in claim 10.
In step 4 (pg 53, line 10), the multipotent cells are differentiated using BMP4 medium and tested for “potential for differentiation into cellular components of brown adipose tissue". Samples expressing FABP4 and CD36 “are considered to be differentiation into [sic] adipocytic lineages” and those expressing FABP4 and BETATROPHIN (C19orf80, LOC55908) are “candidates for progenitors of brown adipose tissue cells.” Applicants do not disclose the cells obtained may express UCP1, C19orf80, or ADIPOQ as required in claim 6 or FABP4, C19orf80, ADIPOQ, UCP1, PCK1, NNAT, CEBPA, CIDEA, and THRSP as required in claim 10
In step 5 (pg 54, line 1), “Progenitor cells” are differentiated using a TGFβ super family member. The “progenitor cells may be any progenitor cell disclosed infra.” In particular, pg 54, line 11, states the cell line can be chosen from C4ELSR2, C4ELS5.1….
Step 5 does not teach which step cell lines C4ELSR2, C4ELS5.1… came from. In particular, pg 31-42 discuss the marker patterns for these cell lines, none of which express FABP4, CD36 or C19orf80.
Pg 56, line 27, describes “Progeny of progenitor cell lines” such as a “human embryonic progenitor cell line (hEP)”. However, the structure and/or function of cells is not disclosed. In particular, it is unclear how such cells correlate to cells of step 1, 2, or 3.
Pg 58, line 7, describes using HyStem-C to cryopreserve and culture any of a number of “progenitor" cells. In over 3000 experiments cells cultured in HyStem-4D with BMP4 exhibited increased MYH11 expression, FABP4 and TIMP4 expression (pg 58, line 28). The structures of HyStem-C or HyStem-4D are not disclosed or known in the art.
Pg 59, line 10, teaches “diverse clonal embryonic progenitor cell lines show correspondingly diverse differentiation responses to growth factors of the TGFβ superfamily.”
Pg 59, line 12, teaches “some cell lines strongly express markers of adipocytes such as FABP4 and CD36. Because the clonal progenitor cell lines capable of adipocyte differentiation represent mesenchymal anlagen [sic] of diverse anatomical origin, the corresponding adipocytes may represent fat-forming cells with diverse phenotypes.
Pg 59, lines 17-22, teaches cell lines E72, E75, and E163 expressed C19orf80, Hoxa10, Hoxd11 but not Hoxc9, Hoxc10 or Hoxc11.
Pg 59, lines 22- pg 60, line 5, and discusses E72.
Pg 60, lines 6-20, teaches “The cell lines E72, E75, and E163, or cells with a similar pattern of gene expression, are capable of differentiating into C19orf80-expressing adipocytes when exposed to adipogenic differentiation conditions such as differentiation in HyStem-C (BioTime, Inc. Alameda, CA) as described with chondrogenic medium supplemented with 10 ng/mL BMP4 for 14-21 days, but without TGFβ3. Bone marrow-derived mesenchymal stem cells (MSCs), differentiated in HyStem-C (BioTime, Inc. Alameda, CA) beads in the presence of 50 ng/mL of BMP2, or 10 ng/mL of BMP4, or 100 ng/mL of BMP7 for 14 days caused the differentiation of the cells into adipocytes as evidenced by their expression of FABP4, whereas in the undifferentiated state, the MSCs did not express detectable FABP4.
Example 1 (pg 89) teaches “only a subset of diverse clonal hEP cell lines [] gave rise to FABP4+ adipoyctes in the presence of the “HyStem BMP4/BMP7” “confluence adipo” and “HyStem Osteo” conditions tested. Line 16 teaches a subset of cells expressing FABP4 expressed C19orf80. Example 1 does not correlate using any “clonal embryonic progenitor cell derived from human pluripotent stem cells” (claim 6) or "pluripotent stem-cell derived clonal progenitor cell line” expressing “HOXA5 and IL13RA2,” (claim 10) for making cells expressing ADIPOQ, C19orf80 or UCP1 (claim 6) or FABP4, C19orf80, ADIPOQ, UCP1, NTNG1, or THRSP (claim 10).
Given the lack of guidance in the specification taken with the art at the time of filing, it would have required those of skill undue experimentation to determine how to contact any HOXA5+, IL13RA2+, and COX7A1- progenitor with any TGFβ superfamily protein and a PPARγ agonist such that UCP1+ COX7A1- brown adipocytes are obtained as required in step c) of claims 6 and 10 other than culturing well-known progenitors with BMP4 or BMP7 and rosiglitazone.
Response to arguments
Applicants point to case law which is not persuasive because the fact pattern in those cases is not the same as in this case.
Applicants point pg 51, lines 1-15, which teach culturing pluripotent cells in DMEM/F12, B27 supplement, and bFGF. Applicants’ argument is not persuasive. The specification does not disclose cloning any progenitor cells from an embryoid body as claimed. The specification does not teach what kind of progenitor cells are HOXA5+ IL13RA2+ COX7A1- as claimed. The specification does not teach the structure or potency of NP110SM. The specification does not teach how to put NP110SM or any other clonal pluripotent cell into “quiescence” as described on pg 31, line 19, 22, & 26; pg 32, line 27-28; pg 33, line 6, 9 to obtain HOXA5+ IL13RA2+ COX7A1- cells. The specification does not teach how to obtain HOXA5+ IL13RA2+ COX7A1- NP110SM cells or any other clonal pluripotent cells on pg 32-37 are HOXA5+ IL13RA2+ COX7A1- (in either their normal or quiescent state) as broadly encompassed by step b) or c).
Applicants argue the amendment overcomes the rejection (pg 8-9 of the response). Applicants’ argument is not persuasive for reasons set forth above.
Written Description
Claims 6-23, 26, 27 remain rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, as failing to comply with the written description requirement. The claim(s) contains subject matter which was not described in the specification in such a way as to reasonably convey to one skilled in the relevant art that the inventor or a joint inventor, or for applications subject to pre-AIA 35 U.S.C. 112, the inventor(s), at the time the application was filed, had possession of the claimed invention.
A) The specification lacks written description for culturing human pluripotent cells in the presence of DMEM/F12, B27 supplement, and bFGF such that any HOXA5+ IL13RA2+ COX7A-progenitor is obtained as required in steps a) and b) of claims 6 and 10 other than culturing human pluripotent cells in bFGF and cloning them as described by West.
The claims, their scope, the state of the art, the teachings in the specification are discussed above.
It is unclear what well-known progenitors are encompassed by “clonal progenitors” that are produced from EBs as required in step b) or “clonal progenitors” that express HOXA5 and IL13RA2 but not COX7A1 obtained from the EBs in step c). The claims do not require a step of cloning, so it is unclear how the intermediary cells are “clonal”. The “progenitor cell” in step b) and c) also encompasses any totipotent, pluripotent, or multipotent cell which does not make sense. The specification does not enable obtaining intermediary cells that are totipotent as encompassed by step b) or c) because totipotent cells are less differentiated than pluripotent cells. The specification does not enable obtaining intermediary cells that are HOXA5+ IL13RA2+ COX7A1- pluripotent cells because the concept is not described in the specification or the art.
The specification does not enable culturing pluripotent cells in any culture medium comprising DMEM/F12, B27 supplement, and bFGF such that HOXA5+ IL13RA2+ COX7A1- multipotent cells are obtained because bFGF is used to maintain pluripotency. This is shown by Haghighi (Cell Comm. & Signaling, 2018, Vol. 16, pg 1-14) (see title, abstract, materials & methods, results) and West (Regenerative Med., 2008, Vol. 3, pp, 287-308) who cultured human pluripotent cells in bFGF to maintain pluripotency (pg 300, col. 2, 1st full paragraph).
West cloned pluripotent cells (pg 301, “Isolation & expansion of clonal cell lines”) and cultured them in a variety of conditions (pg 289 col. 1, lines 3-7) including mesenchymal conditions (Supplementary Table A1, cited on pg 288 col, 2, para 1, lines 7-17; see lines 59, 89, 193, 196, 220, 303 of Table A1; see also pg 302, col. 1, “Mesencult basal medium” plus “supplement”).
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Supplementary Table A2 shows expression patterns of HOXA5, IL13RA2, and COX7A1 (lines 330, 4045, 372, respectively)
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But it is unclear from the massive number of cells whether any of the cells had the expression pattern claimed. The cells lines across the top of Supp. Table A2 of West include those described by applicants as being part of the invention on pg 31, lines 3-5 (E3, E72, E75, C4ELS5.1, C4ELSR2, and NP110SM). C4ELS5.1 made by West is in column Q of Table A2, is described by applicants on pg 32, line 22; pg 41, line 26, and described in 2010/0184033 (12/504630), 8685386 (13/384289), 2013/0115673 (13683241), 2014/0178994 (14/172765), 2014/0234964 (14/131429) which express TAC1, EBF2, SCARA5, EYA4, and TBX1, but not HOXA10, ZIC2, or MKX).
West cultured human pluripotent clonal cell line C4ELS5.1 in bFGF, but the cells did not express HOXA5, IL13RA2, or COX7A1 (Supp Table A2; column Q, lines 330, 4045, and 372 respectively). Thus the ability to culture pluripotent cells in any culture media comprising bFGF and obtain any HOXA5+, IL13RA2+, and COX7A1- progenitor was unpredictable.
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Pg 31, lines 26-31; pg 42, line 29, discuss the marker pattern of cell line NP110SM in quiescence.
“When RNA was extracted from these NP110SM cells at passage 10 and induced into quiescence for 5 days a condition sometimes referred to as "control" or "Ctrl" herein, the cells displayed the following gene expression markers: DLK1, HOXA5, SLC7A14, NTNG1, HEPH, PGM5, IL13RA2, SLC1A3, and SBSN but unlike fetal or adult-derived BAT progenitors do not express COX7A1, or one or more markers chosen from POSTN, KRT34, MKX, HAND2, TBX15, HOXAIO, PLXDC2, DHRS9, NNAT, and HOXDll.”
However, NP110SM cells are HOXA5+ IL13RA2+ COX7A1- as required in step b), but they are clonal pluripotent cells.
Applicants do not teach how to overcome the teachings of West and culture NP110SM cells or any other clonal pluripotent cells “into quiescence” in any culture media comprising DMEM/F12, B27 supplement, and bFGF such that any HOXA5+, IL13RA2+, and COX7A1- pluripotent cells are obtained.
The specification does not teach how to obtain pluripotent cells that are HOXA5+, IL13RA2+, and COX7A1- as broadly encompassed by “progenitor” because C4ELS5.1 and all of the other clones described by West were cultured in bFGF and cloned, but they did not have the marker pattern claimed.
The specification does not teach how to obtain totipotent cells that are HOXA5+, IL13RA2+, and COX7A1- as broadly encompassed by “progenitor” because the specification and the art do not teach de-differentiating the pluripotent cells into totipotent cells.
The description of Fig. 15 on pg 11 says NP110SM cells were cultured in “reduced growth factors to induce quiescence” without describing the specific culture media comprising bFGF, the “growth factors” that are “reduced”, or the amount of “growth factors” used to obtain HOXA5+, IL13RA2+, and COX7A1- pluripotent cells. The remainder of the paragraph goes on to describe differentiating the “quiescent” NP110SM cells using HyStem beads BMP4 as the TGFβ protein and rosiglitazone as the PPARγ agonist.
The specification does not teach how to obtain NP110SM pluripotent cells in “quiescence” that are HOXA5+, IL13RA2+, and COX7A1- as broadly encompassed by step b) because while NP110SM had the marker pattern claimed, the specification did not teach how to recapitulate obtaining pluripotent cells having the same structure as NP110SM, or the specific culture conditions required to induce “quiescence” of NP110SM cells to make them HOXA5+, IL13RA2+, COX7A1-.
Perhaps the culture medium in step a) is supposed to be mesenchymal stem cell medium because adipose tissue is derived from mesenchymal cells, but the specification does not teach culturing NP110SM or any other clonal pluripotent cell in mesenchymal stem cell medium such that HOXA5+, IL13RA2+, COX7A1- progenitors are obtained.
It’s possible the cells produced in steps b) and c) are mesenchymal stem cells, but the function of progenitors isolated in step b) and c) is missing from the claims.
The specification and the art at the time of filing teach that the pluripotent cells in step a) are cloned. This step is missing from the claim.
Applicants and the art do not describe “cloning” progenitors that are HOXA5+, IL13RA2+, and COX7A1- as inferred by step b) and c).
Applicants do not disclose the specific starting clonal cells or the specific differentiation medium to overcome the unpredictability of West and arrive at HOXA5+, IL13RA2+, and COX7A1- multipotent cell.
The specification does not teach the specific mesenchymal differentiation medium and supplements required to overcome the unpredictability of West and turn E3, E72, E75, C4ELS5.1, C4ELSR2, NP110SM, C4ELS5.1, or any other clonal pluripotent cell into HOXA5+, IL13RA2+, and COX7A1- multipotent cells as encompassed by step b).
It is unclear whether the HOXA5+, IL13RA2+, and COX7A1- progenitors in step b) are well-known mesenchymal stem cells, well-known preadipocyte progenitors, or if they are some newly identified progenitor. If they are newly identified progenitors, applicants do not disclose whether they are MSCs, adipocyte progenitors, brown adipocyte-specific progenitors, white adipocyte-specific progenitors, beige adipocyte -specific progenitors.
Accordingly, the specification lacks written description for culturing human pluripotent cells in the presence of DMEM/F12, B27 supplement, and bFGF such that any HOXA5+ IL13RA2+ COX7A-progenitor is obtained as required in steps a) and b) of claims 6 and 10 other than culturing human pluripotent cells in bFGF and cloning them as described by West.
B) The specification lacks written description for contacting any HOXA5+, IL13RA2+, and COX7A1- progenitor with any TGFβ superfamily protein and a PPARγ agonist such that UCP1+ COX7A1- brown adipocytes are obtained as required in step c) of claims 6 and 10 other than culturing well-known progenitors with BMP4 or BMP7 and rosiglitazone.
The starting material of step c) encompasses any totipotent, pluripotent, or multipotent HOXA5+, IL13RA2+, and COX7A1- cell.
The TGFβ superfamily encompasses TGFβ1, TGFβ2, TGFβ3, activin, inhibins, BMP2-14, growth differentiation factors (GDF), glial-derived neurotrophic factors (GDNFs).
The PPARγ agonists encompass:
Thiazolidinediones (TZDs):
Pioglitazone (Actos)
Rosiglitazone (Avandia)
Troglitazone (removed from market due to liver toxicity)
Dual PPAR alpha/gamma agonists:
Lobeglitazone
Muraglitazar
Other PPAR gamma agonists:
Elafibranor
Saroglitazar
The teachings in the art and specification are described above.
The specification lacks written description for making UCP1+ COX7A1- cells that are “brown adipocytes” because the markers are associated with brown, beige, or white adipose as well as cells that are “browning”.
Accordingly, the specification lacks written description for contacting any HOXA5+, IL13RA2+, and COX7A1- progenitor with any TGFβ superfamily protein and a PPARγ agonist such that UCP1+ COX7A1- brown adipocytes are obtained as required in step c) of claims 6 and 10 other than culturing well-known progenitors with BMP4 or BMP7 and rosiglitazone.
Response to arguments
Applicants point pg 51-53, which teach culturing pluripotent cells in DMEM/F12, B27 supplement, and bFGF. Applicants’ argument is not persuasive. The specification does not disclose cloning any progenitor cells from an embryoid body as claimed. The specification does not teach what kind of progenitor cells are HOXA5+ IL13RA2+ COX7A1- as claimed. The specification does not teach the structure or potency of NP110SM. The specification does not teach how to put NP110SM or any other clonal pluripotent cell into “quiescence” as described on pg 31, line 19, 22, & 26; pg 32, line 27-28; pg 33, line 6, 9 to obtain HOXA5+ IL13RA2+ COX7A1- cells. The specification does not teach how to obtain HOXA5+ IL13RA2+ COX7A1- NP110SM cells or any other clonal pluripotent cells on pg 32-37 are HOXA5+ IL13RA2+ COX7A1- (in either their normal or quiescent state) as broadly encompassed by step b) or c).
Applicants argue the amendment overcomes the rejection (pg 9-10 of the response). Applicants’ argument is not persuasive for reasons set forth above.
Indefiniteness
Claims 6-23, 26, 27 remain rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
A) Step a) of claims 6 and 10 is indefinite because it requires “differentiating” pluripotent cells in the presence of bFGF. Haghighi (Cell Comm. & Signaling, 2018, Vol. 16, pg 1-14) (see title, abstract, materials & methods, results) and West (Regenerative Med., 2008, Vol. 3, pp, 287-308) cultured human pluripotent cells in bFGF to maintain pluripotency (pg 300, col. 2, 1st full paragraph). The art at the time of filing teaches bFGF is limited to maintain pluripotent cells; therefore, the concept doesn’t make sense, thereby making step a) indefinite.
Step a) is also indefinite because it requires contacting pluripotent cells with bFGF “thereby producing an embryoid body; b) isolating a clonal human progenitor cell line expressing HOXA5 and IL13RA2, and does not express COX7A1”; however, the “progenitor cell line” in step b) encompasses totipotent, pluripotent, or multipotent cells, which is broader than “pluripotent” cells and does not correlate to “differentiating” pluripotent cells.
Step b) does not make sense because obtaining “clonal” cells infers an active step of “cloning” the cells which is missing from the claim. “Producing clonal” cell lines does not result in “clonal” cells without an active step of cloning the cells after “differentiating”.
The phrase “thereby forming” in step a) does not necessarily indicate the “differentiating” has the function of producing multipotent cells that express HOXA5 and IL13RA2 but not COX7A1. The phrase “thereby producing” simply says the active step of “differentiating” must be performed.
It is unclear whether the final product at the end of step b) is multipotent cells, i.e. ---differentiating human pluripotent cells into multipotent cells in the presence of basic fibroblast growth factor (bFGF)---. It is unclear whether the final product at the end of step a) is pluripotent cells, i.e. ---maintaining human pluripotent cells into multipotent cells in the presence of DMEM/F12, B27 supplement and basic fibroblast growth factor (bFGF)---. It is unclear whether the final product at the end of step b) is “quiescent” (discussed throughout pg 31-37 of the specification and elsewhere), in which case it is unclear how to guide applicants at this time.
Given all of these issues, steps a)-c) do not make sense, the metes and bounds of the active steps encompassed by the step cannot be determined, the structure/function of the final “progenitors” cannot be determined, and those of skill would not be able to determine whether they were infringing on the metes and bounds of step a)-c).
B) While step c) of claims 6 and 10 requires obtaining HOXA5+ IL13RA2+ COX7A1- progenitors, step b) is indefinite because it is encompasses obtaining totipotent HOXA5+ IL13RA2+ COX7A1- cells which is broader than “differentiating” pluripotent cells as required in step a). Therefore, step c) does not make sense, thereby making step c) indefinite.
Step c) is also indefinite because it requires “isolating a clonal human progenitor cell line expressing HOXA5 and IL13RA2, and does not express COX7A1”; however, this does not make sense because obtaining “clonal” cells at the end of the step infers an active step of “cloning” the cells which is missing from the claim. “Isolating” progenitors does not result in “clonal” cells without an active step of cloning the cells which is missing from the claim.
Response to arguments
Applicants point to case law which is not persuasive because the fact pattern in those cases is not the same as in this case.
Applicants point pg 51, lines 1-15, which teach culturing pluripotent cells in DMEM/F12, B27 supplement, and bFGF. Applicants’ argument is not persuasive. The specification does not disclose cloning any progenitor cells from an embryoid body as claimed. The specification does not teach what kind of progenitor cells are HOXA5+ IL13RA2+ COX7A1- as claimed. The specification does not teach the structure or potency of NP110SM. The specification does not teach how to put NP110SM or any other clonal pluripotent cell into “quiescence” as described on pg 31, line 19, 22, & 26; pg 32, line 27-28; pg 33, line 6, 9 to obtain HOXA5+ IL13RA2+ COX7A1- cells. The specification does not teach how to obtain HOXA5+ IL13RA2+ COX7A1- NP110SM cells or any other clonal pluripotent cells on pg 32-37 are HOXA5+ IL13RA2+ COX7A1- (in either their normal or quiescent state) as broadly encompassed by step b) or c).
Applicants argue the amendment overcomes the rejection (pg 8-9 of the response). Applicants’ argument is not persuasive for reasons set forth above.
Claim Rejections - 35 USC § 103
A) Claims 6-15, 19, 26, 27 remain rejected under 35 U.S.C. 103 as being unpatentable over West (Regenerative Med., 2008, Vol. 3, pp, 287-308) in view of Schulz (PNAS, Jan. 4, 2011, Vol. 108, No. 1, pg 143-148) or Boyden (20120321671) taken with Scott (Stem Cells and Development, Oct. 2011, Vol. 20, No. 10, pg 1793-1804).
West cultured human pluripotent cells in DMEM/F12, B27 supplement, and bFGF to maintain pluripotency (pg 300, col. 2, 1st full paragraph) then cloned the pluripotent cells (pg 301, “Isolation & expansion of clonal cell lines”) and cultured them in a variety of conditions (pg 289 col. 1, lines 3-7) including into embryoid bodies then into mesenchymal cell lineages (Supplementary Table A1, cited on pg 288 col, 2, para 1, lines 7-17; see lines 59, 89, 193, 196, 220, 303 of Table A1; see also pg 302, col. 1, “Mesencult basal medium” plus “supplement”).
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Supplementary Table A2 shows expression patterns of HOXA5, IL13RA2, and COX7A1 (lines 330, 4045, 372, respectively)
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.
Two adipogenesis protocols in applicants’ disclosure cite West as the means for performing steps a) and b), i.e. 1) pg 82, “Adipogenesis Protocol 1” “Procedures”, “Clonal embryonic preadipocyte maintenance and passage”; and 2) pg 82, “Adipogenesis Protocol 2”. And the cells lines across the top of Supp. Table A2 of West include those described by applicants as being part of the invention on pg 31, lines 3-5 (E3, E72, E75, C4ELS5.1, C4ELSR2, NP110SM, and C4ELS5.1). For example, C4ELS5.1 is in column Q of Table A2 of West and is described by applicants on pg 32, line 22; pg 41, line 26, and described in 2010/0184033 (12/504630), 8685386 (13/384289), 2013/0115673 (13683241), 2014/0178994 (14/172765), 2014/0234964 (14/131429). Therefore, the process of West inherently MUST result in HOXA5+, IL13RA2+, and COX7A1- clonal progenitors as required in steps a) and b) of claims 6 and 10 because the starting materials and reagents are the same as those used by applicants and claimed.
West did not teach contacting clonal pluripotent cells with a TGFβ superfamily protein and a PPARγ agonist such that UCP1+ COX7A1- brown adipocytes are obtained as required in step d) of claims 6 and 10.
However, Schulz differentiated multipotent mouse (SI, pg and human (SI, pg 2, col. 1, line 5) cells into brown adipocytes using the PPARγ agonist rosiglitazone in combination with BMP7 (pg 147, col. 1, “BMP7 acts….”; SI, pg 7, Fig. S9B). UCP1 expression was obtained (Fig. S9H).
Boyden differentiated progenitors into brown adipocytes expressing UCP1 using i) rosiglitazone, and ii) BMP4 or BMP7 (para 40, 47). Boyden taught that it was well known to use PPARγ agonists, e.g. rosiglitazone, for differentiating progenitors into brown adipose in paragraphs 42, 43, 48, 49, 50, 55, 77, 91, 96. Boyden discusses using BMP7 in context of differentiating progenitors into brown adipose in paragraph 52. The progenitors are human (claim 2; para 100-101, et al.) as required in claim 6.
Thus, it would have been obvious to those of ordinary skill in the art at the time of filing to culture pluripotent cells in bFGF, clone them, and isolate HOXA5+ IL13RA2+ COX7A1- progenitors as described by West followed by contacting them with BMP4 or BMP7 and rosiglitazone as described by Schulz and Boyden. Those of ordinary skill in the art at the time of filing would have been motivated to do so to obtain adipocytes for obtaining brown adipocytes in culture.
Those of skill would have had a reasonable expectation of differentiating any of a number of progenitors using a PPARβ agonist and BMP as required in step c) because Scott (Stem Cells and Development, Oct. 2011, Vol. 20, No. 10, pg 1793-1804) differentiated a variety of progenitors (including clonal progenitor 3T3-L1) (pg 1794-1797) using PPARγ agonists, e.g. rosiglitazone, and BMPs (pg 1798, “Use of PPARγ agonists”, “BMPs”) into adipocytes (throughout “Results”). Scott taught “rosiglitazone both speeds and increases the degree of differentiation of adipoprogenitors” - sentence bridging col. 1-2 of pg 1798.
Therefore, the combined teachings of West and Schulz or Boyden taken with Scott meet the limitations of claims 6 and 10.
BMP7 (Schulz and Boyden) and BMP4 (Boyden) are BMPs as required in claims 7.
Schulz and Boyden used rosiglitazone (see above) as required in claim 8.
Boyden taught culturing the cells in a matrix comprising hyaluronic acid and collagen (praa 61) which is “embedding” the cells in a “mixture of hyaluronic acid and collagen-based matrix” as required in claim 9.
BMP7 (Schulz and Boyden) and BMP4 (Boyden) are BMPs as required in claims 11.
Boyden taught BMP4 (para 40, 47) as required in claim 12.
Schulz and Boyden used rosiglitazone (see above) as required in claim 13.
Boyden taught culturing the cells in a matrix comprising hyaluronic acid and collagen (praa 61) which is “contacting” or “embedding” the cells with or in a “mixture of hyaluronic acid and collagen-based matrix” as required in claims 14 and 15.
Boyden taught using a β3-adrenergic agonist (para 4, 8, 52, 55, 77, 78; claim 32) as required in claim 19.
Claims 26 and 27 have been included because Schulz taught using 1 μM rosiglitazone (pg 1 “Cell Culture” of Supporting Information of Schultz):
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Response to arguments
Applicants argue there was no motivation provided. Applicants’ argument is not persuasive because a motivational statement was provided.
Applicants argue the amendment overcomes the rejection (pg 8-9 of the response). Applicants’ argument is not persuasive for reasons set forth above.
Applicants argue there was no reasonable expectation of success. Applicants’ argument is not persuasive because it is unfounded. Differentiating progenitors into brown adipocytes using rosiglitazone and BMP7in culture definitely had a high expectation of success as shown above.
B) Claims 16-18, 21-23 remain rejected under 35 U.S.C. 103 as being unpatentable over West (Regenerative Med., 2008, Vol. 3, pp, 287-308) in view of Schulz (PNAS, Jan. 4, 2011, Vol. 108, No. 1, pg 143-148) or Boyden (20120321671) taken with Scott (Stem Cells and Development, Oct. 2011, Vol. 20, No. 10, pg 1793-1804) as applied to claims 6-15, 19, 26, 27 above and further in view of Hassan (Stem Cell Res. & Therap., March 21, 2013, Vol. 4, No. 32, pg 1-11) and Espandar (Archives of Ophthalmology, Feb. 2012, Vol. 130, No. 2, pg 202-208) as