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 .
Remarks
The amendments and remarks filed on 11/13/2025 have been entered and considered. The text of those sections of Title 35, U.S. Code not included in this action can be found in a prior office action. The rejections and/or objections presented herein are the only rejections and/or objections currently outstanding. Any previously presented objections or rejections that are not presented in this Office Action are withdrawn. Claims 1-6, 9-12, 21-24, and 27-28 are pending; Claims 7-8, 13-20, and 25-26 are cancelled; Claims 1, 12, and 21 are amended; and Claims 1-6, 9-12, 21-24, and 27-28 are under examination.
Withdrawal of Objections
The objection to the specification is withdrawn due to the amendment to the specification filed on 11/13/2025.
The objection to Claims 1 and 25 is withdrawn due to the amendment to the claims filed on 11/13/2025.
Withdrawal of Rejections
The rejection of claim 12 under 35 U.S.C. 112(b) is withdrawn due to the amendment to the claim filed on 11/13/2025.
The rejection of Claim 12 under 35 U.S.C. 102/103 as being anticipated by or, obvious over Kotaro et al. is withdrawn due to the amendment to the claim filed on 11/13/2025.
The rejection of Claims 1-4, 10-12, 21-24, and 28 under 35 U.S.C. 103 over Stubbers et al. is withdrawn due to the amendment to the claims.
The rejection of Claims 1-6, 10-12, 21-24, and 28 under 35 U.S.C. 103 as being unpatentable over Stubbers et al. in view of Izadpanah et al. is withdrawn due to the amendment to the claims.
The rejection of Claims 1-4, 9-12, 21-24, and 27-28 under 35 U.S.C. 103 over Stubbers et al. in view of Veremeyev et al. is withdrawn due to the amendment to the claims.
The rejection of Claims 7-8 and 25-26 under 35 U.S.C. 103 over Stubbers et al. in view of Lockhart et al. and Hesse et al. is withdrawn due to the cancellation of the claims.
Claim Objections
Claim 12 is objected to due to the recitation of “cell” and “cells” in the preamble or the steps of the claim. These terms are being used interchangeably. To be consistent, applicant is requested to review the claim and elect either the plural or the singular form of the term/cell to be used throughout the claim. Appropriate correction is required.
Claim Rejections - 35 USC § 103
Claims 1-4, 10-12, 21-24, and 28 are rejected under 35 U.S.C. 103 as being unpatentable over Stubbers et al. (WO 2009/073724, 2009, cited in IDS) in view of Lockhart et al. (J. Stem Cell Res Ther, 2015, 5:12, pages 1-8, cited in IDS) and Hesse et al. (US Patent NO: 5853976, 1998, cited in IDS).
Stubbers et al. teach a method for dispersing an adipose tissue and preparing a stromal vascular fraction (SVF) from the adipose tissue, comprising steps: treating an adipose tissue with an enzyme solution containing a collagenase and a dispase, and collecting/recovering the stromal vascular fraction/SVF (i.e. SVF cells), wherein the enzyme solution comprises collagenase (60,000 U) and dispase (120 U) (Example 4: para 102 and para 103/last 2 lines; paras 0048/lines 7-8, 0049, and 0072), and wherein the dispase is a neutral protease (para 0072/second line from bottom).
Regarding the limitation that the collagenase and neutral protease are derived from Clostridium histolyticum in Claims 1, 12, and 21, Stubbers et al. are silent about sources where collagenase and neutral protease are derived from. However, Stubbers et al. further teach that there are non-limiting examples of enzymes suitable for treating adipose tissues, including protease, collagenase, dispase (i.e. neutral protease) and/or a mixture thereof (para 0072, lines 7-9), which indicates that a mixture of collagenase and protease/neutral protease from different sources (e.g. dispase being replaced by other sourced neutral protease) is suitable for treating adipose tissue in the method of Stubbers et al. Stubbers et al. further teach that collagenase can be specifically collagenases I and II (para 0072, line 3 from bottom).
Lockhart et al. teach isolating SVF cells from adipose tissue is commonly achieved by using tissue dissociation enzymes (TDE) to breakdown the adipose tissue (page 1: left col/para 2/lines 1-4). Lockhart et al. further teach that collagenases from the bacterium C. histolyticum (including types I and II collagenases of C. histolyticum) are primary constitute of the tissue dissociation enzymes (TDE) (page 1: right col/para 2-3). Lockhart et al. further teach that neutral protease (ChNP) from C. histolyticum is the second major component in the TDE for breaking down the adipose tissue (page 2, left col, para 2, lines 1-3).
Hesse et al. teach that C. histolyticum produces types I and II collagenases, which are used for digesting tissues for isolating cells from the tissues (col 1, lines 8-19). Hesse et al. further teach that C. histolyticum also produces a neutral protease and the neutral protease of C. histolyticum is used for digesting a tissue to release cells from the tissue, wherein the tissue may be a fat tissue, i.e. adipose tissue (claims 10-11 and 13, col 1/lines 6-8 and 18-21). Hesse et al. further demonstrate that a combination of the neutral protease of C. histolyticum with the types I and II collagenases of C. histolyticum effectively digest tissues and releasing cells from the tissue (example 4, col. 9/lines 2-4).
It would have been obvious to include types I and II collagenases from Clostridium histolyticum as the collagenases I and II (i.e. types I and II collagenases) in the enzyme solution and further to include a neutral protease (ChNP) of C. histolyticum as the replacement of dispase in the enzyme solution in the method taught by Stubbers et al. for treating and dispersing adipose tissue and for preparing the SVF cells. One of ordinary skill in the art would have been motivated to do so, because types I and II collagenases as well as neutral protease from Clostridium histolyticum are commonly used in the art for breaking down adipose tissue for releasing cells from tissue and preparing SVF cells, as supported by Lockhart et al. and Hesse et al. In addition, it is well known in the art to use the collagenases and neutral protease from Clostridium histolyticum as the major components of tissue dissociation enzymes for breaking down adipose tissue for releasing cells, as supported by Lockhart et al. and Hesse et al. Furthermore, it is well known in the art that a combination of neutral protease of C. histolyticum with types I and II collagenases of C. histolyticum are effective at dissociating tissues and releasing cells from the tissues, as supported by Lockhart et al. and Hesse et al. Moreover, Stubbers et al. specifically teach using types I and II collagenases (i.e. collagenases I and II) in the enzyme solution for treating adipose tissue in their method, and teach that either dispase or other protease/neutral protease is suitable for treating/dissociating adipose tissue. Given the neutral protease of C. histolyticum is an art-recognized equivalent of dispase for the same purpose. Substitution of one known element for another and the results of the substitution would have been predictable. One of ordinary skill in the art has a reasonable expectation of success at modifying the method of Stubbers et al. by applying an enzyme solution comprising collagenases and neutral protease of C. histolyticum for treating adipose tissue and preparing SVF cells, because collagenases and neutral protease of C. histolyticum are effective at dissociating adipose tissue and releasing cells from the dissociating adipose tissue, thus allowing the SVF cells to be collected, as supported by Lockhart et al. and Hesse et al.
Regarding the ratios of neutral protease activity vs. collagenase activity recited in claims 1-3 and 21-23, Stubbers et al. teach a ratio of 120 U neutral protease activity vs. 60,000 U collagenase activity, which is equivalent to a ratio of 20 U vs. 10,000 U, i.e. not less than 20 U of neutral protease activity with respect to 10,000 U of collagenase activity, meeting the claimed ratios in the claims 1-3 and 21-23. It is noted that Stubbers et al. do not specifically teach the activities of collagenase and neutral protease are determined respectively by assays of using commercial EnzChek collagenase assay kit and the labeled Gly-Phe-Tyr peptide substrate, as defined in the specification (pages 18-19 and 20-21). However, methods for assaying activities of neutral protease and collagenase are well established in the art. It is an obvious design choice to assay the activities of collagenase and neutral protease by using the methods as defined in the specification of the instant application for preparing the enzyme solution having collagenase and neutral protease in the method suggested by Stubbers et al., Lockhart et al. and Hesse et al.
Regarding claims 4 and 24, the claimed ratio range from 34,000 : 9 to 34,000 : 45 is equivalent to a range from 10,000 : 2.65 to 10,000 : 13.24. The ratio 10,000 : 20 taught by Stubbers et al. does not exactly match the claimed ratio range; and the ratio of Stubbers et al. has a slightly higher neutral protease activity, compared to the claimed ratio range. However, it is considered that the ratio of Stubbers et al. can be readily modified by routine optimization based on specific digestion/treatment conditions and specific neutral protease and collagenase enzymes used in the treatment for achieving a desired result for dissociating adipose tissues and releasing cells. Furthermore, it is well settled that routine optimization is not patentable, even though it results in significant improvement over the prior art (see MPEP 2144.05).
Regarding Claims 10 and 28, the enzyme solution suggested by Stubbers et al. Lockhart et al. and Hesse et al. comprises collagenases and neutral protease from C. histolyticum, which is free of clostripain or thermolysin.
Regarding Claim 11, Stubbers et al. teach human adipose tissues and teach their method is particularly suitable for isolation of cells from human adipose tissues (paras 0018/lines 2-3 from bottom, and 0068/lines 5-6).
Regarding Claim 12, Stubbers et al. teach collecting or recovering SVF cells, and then culturing/proliferating or enriching the SVF cells in a flask (para 00104/lines 1-6, para 103/last 2 lines). Furthermore, Stubbers et al. teach that adipose tissue is a rich source of endothelial cells and endothelial progenitor cells, which may have utility in treatment of various medical conditions (page 2/para 0005/lines 1-3); that endothelial cells and endothelial progenitor cells contribute to neovascularization for supplying oxygenated blood to ischemic tissue; and isolation of reparative cell populations including endothelial cells and endothelial progenitor cells has potential therapeutic applications (para 0003, lines 2-8). In view of the teachings of Stubbers et al., it would have been obvious to collect/recover a SVF having endothelial cells and endothelial progenitor cells, and specifically enrich and proliferate endothelial cells and endothelial progenitor cells in the collected SVF in the method suggested by Stubbers et al., Lockhart et al. and Hesse et al. for obtaining reparative cell populations comprising endothelial cells and endothelial progenitor cells to be used for medical research or for potential therapeutic applications.
Therefore, the invention as a whole would have been prima facie obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention.
Claims 1-6, 10-12, 21-24, and 28 are rejected under 35 U.S.C. 103 as being unpatentable over Stubbers et al. (WO 2009/073724, 2009, cited in IDS) in view of Lockhart et al. (J. Stem Cell Res Ther, 2015, 5:12, pages 1-8, cited in IDS) and Hesse et al. (US Patent NO: 5853976, 1998, cited in IDS), as applied to Claims 1-4, 10-12, 21-24, and 28, further in view of Izadpanah et al. (US 20130190729, 2013, of record), as evidenced by Etchison (Sports Health, 2011, 3(6):499, of record).
The teachings of Stubbers et al. as modified by Lockhart et al. and Hesse et al. are described above.
Regarding claims 5 and 6, Stubbers et al. are silent about a specific weight of the adipose tissue treated by collagenase and neutral protease, and do not teach a collagenase content of not less than 500 U per gram of adipose tissue and a neutral protease content of not less than 0.05 U per gram of adipose tissue. However, Stubbers et al. use an enzyme solution comprising collagenase (60,000 U) and neutral protease (120 U) for treating/dispersing the adipose tissue for preparing a stromal vascular fraction.
It would have been obvious to apply collagenase and neutral protease of C. histolyticum respectively at contents of not less than 500 U or not less than 0.05 U per 1 gram of adipose tissue in the enzymatic treatment step in the method suggested by Stubbers et al. and other cited prior art for preparing a stromal vascular fraction, because it is well known in the art that the collagenase and neutral protease contents in the claimed ranges can effectively treat/digest the adipose tissue when an enzyme solution containing collagenase (60,000 U) and neutral protease (120 U) is applied to the adipose tissue for preparing a stromal vascular fraction. In support, Izadpanah et al. teach a highly similar method for treating/dispersing an adipose tissue and preparing a stromal vascular fraction (SVF), comprising steps: providing lipoaspirate (i.e. adipose tissue) at a volume of approximately 100 ml, treating an adipose tissue with an enzyme solution containing a collagenase (60,000 U) and a neutral protease/dispase (120 U); collecting the SVF (SVF cells); and culturing the SVF cells (Example 7: para 0108/page 11/lines 1-4 and 8-9 and page 12/lines 1-3, para 0109/last 5 lines; claim 1). It is noted that the ~ 100 ml of adipose tissue taught by Izadpanah et al. has a weight of ~ 91.96 g, because adipose tissue has a density of 0.9196 g/mL, as evidenced by Etchison, who teaches the density of adipose tissue is 0.9196 kg/L (i.e. 0.9196 g/mL) (page 499, left col, para 1, line 6). As such, about 91.96 g of adipose tissue is treated with collagenase (60,000 U) and neutral protease (120 U) in the method of Izadpanah et al., which can be converted to a collagenase content of ~ 652.5 U per gram of adipose tissue and a neutral protease content of ~ 1.31 U per gram of adipose tissue, which respectively read on the claimed ranges. Furthermore, Examiner notes that the specific ratio of Stubbers et al. can be readily optimized for reaching a desirable effect on tissue treatment based on specific treatment conditions and specific neutral protease and collagenase enzymes used in the treatment for achieving a desired result for dissociating adipose tissues and releasing cells. It is well settled that routine optimization is not patentable, even though it results in significant improvement over the prior art (see MPEP 2144.05).
Therefore, the invention as a whole would have been prima facie obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention.
Claims 1-4, 9-12, 21-24, and 27-28 are rejected under 35 U.S.C. 103 as being unpatentable over Stubbers et al. (WO 2009/073724, 2009, cited in IDS) ) in view of Lockhart et al. (J. Stem Cell Res Ther, 2015, 5:12, pages 1-8, cited in IDS) and Hesse et al. (US Patent NO: 5853976, 1998, cited in IDS), as applied to Claims 1-4, 10-12, 21-24, and 28, further in view of Dendo et al. (Transplantation, 2015, 99: 1349–1355).
The teachings of Stubbers et al. as modified by Lockhart et al. and Hesse et al. are described above.
Regarding claims 9 and 27, Stubbers et al. do not teach that the enzyme solution further comprises clostripain. However, Stubbers et al. further teach that there are non-limiting examples of enzymes suitable for treating adipose tissues in their method, which include protease, collagenase, neutral protease/dispase and/or a mixture thereof (para 0072, lines 7-9), indicating that a mixture of collagenase, neutral protease, and an additional protease are suitable for treating adipose tissue in the method of Stubbers et al.
Dendo et al. teach that a combination of clostripain (CP), neutral protease (ChnP), collagenase subtypes ColG and ColH (i.e. collagenases I and II, types I and II collagenases) from C. histolyticum generates a strong synergistic effect on treating/dispersing animal tissues for cell isolation, i.e. isolating islet cells, and effectively increasing cell isolation efficiency (abstract, page 1349/right col/lines 6-8, page 1350/left col/paras 4/lines 1-4 and last para). Dendo et al. further teach that clostripain is a main protease in C. histolyticum and it has tryptic-like activity, but the collagenases and neutral protease do not have the tryptic-like activity; and accordingly the clostripain may exhibit biological synergy when being combined with the collagenases and neutral protease given they all are produced by the same bacterium C. histolyticum (page 1350/left col/para 3).
It would have been obvious to combine clostripain with the collagenases and neutral protease (ChNP) of C. histolyticum in the enzyme solution in the method of Stubbers et al. for generating a synergetic effect for preparing stromal vascular fraction (SVF cells), as taught by Dendo et al. One of ordinary skill in the art would have motivated to do so, because Stubbers et al. teach including an additional protease in the enzyme solution (in addition to collagenase and neutral protease) for dissociating adipose tissue and releasing cells therein in their method. Furthermore, it is well known in the art that an enzyme mixture comprising clostripain, collagenases, neutral protease (ChNP) from the same bacterium C. histolyticum generates a strong synergistic effect at dissociating animal tissues and effectively increasing cell isolation efficiency, as supported by Dendo et al. One of ordinary skill in the art has a reasonable expectation of success at modifying the method suggested by Stubbers et al. and other cited prior art by further including clostripain in the enzyme solution of Stubbers et al. for treating the adipose tissue, because Dendo et al. teach that clostripain of C. histolyticum has tryptic-like protease activity, which is not possessed by the collagenases and neutral protease of C. histolyticum, thus compensating the digesting activity of the collagenases and neutral protease.
Therefore, the invention as a whole would have been prima facie obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention.
Response to Arguments
Applicant's arguments about the objection to claims and specification as well as the rejection of claim 12 under 35 U.S. C. 112(b) in the response filed 11/13/2025 (pages 9-10) have been fully considered but they are moot because the objections and rejection has been withdrawn as indicated above.
Applicant's arguments about the 102/103 rejection of Claim 12 as being anticipated by or, obvious over Kotaro et al. in the response filed on 11/13/2025 (page 10) have been fully considered but they are moot, because the rejection has been withdrawn as indicated above.
Applicant's arguments about the 103 rejections of Claims 1-6, 9-12, 21-24, and/or 27-28 over Stubbers et al. either alone or in view of Izadpanah et al. or Veremeyev et al. in the response filed on 11/13/2025 (pages 11-12) have been fully considered but they are moot, because the rejections have been withdrawn as indicated above.
Applicant's arguments about the 103 rejection of Claims 7-8 and 25-26 over Stubbers et al. in view of Lockhart et al. and Hesse et al. in page 12/paras 3-4 of the 11/13/2025 response have been fully considered but they not persuasive for the following reasons.
In response to Applicant’s arguments based on the ratio of collagenases vs. neutral protease in para 3 of page 12 of the response, it is noted that regardless of whether collagenases and neutral proteases are derived from the same bacteria/sources, they all have similar proteolytic activities or enzymatic characteristics (for example: collagenases all act on collagen substrate, and neutral proteases all are metalloendopeptidase and operate optimally at neutral pH to hydrolyze peptide bonds). As such, one of ordinary skill in the art would have recognized the ratio of collagenases vs. neutral protease taught by Stubbers et al. is applicable to collagenases and neutral protease from C. histolyticum. Furthermore, specific ratio of Stubbers et al. can be readily optimized for reaching a desirable effect on tissue treatment in view of the fact that collagenases and neutral protease from C. histolyticum are tissue dissociation enzymes commonly used in the art for treating adipose tissue, as supported by Lockhart et al. and Hesse et al. Moreover, it is noted that generally, differences in an enzyme concentration or ratio will not support the patentability of subject matter unless there is evidence indicating such concentration or ratio is critical. "[W]here the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimum or workable ranges by routine experimentation." In re Aller, 220 F.2d 454, 456,105 USPQ 233, 235 (CCPA 1955).
In response to Applicant’s arguments based on unexpected results generated by the claimed ratio of collagenases vs. neutral protease in para 4 of page 12 of the response, these arguments are not persuasive because Applicant failed to provide any factual evidence to support that the claimed ratio generates improvements in SVF yields and these improvements are unexpected to one of ordinary skill in the art.
Overall, the conclusion of the obviousness of the amended claims 1-6, 9-12, 21-24, and 27-28 has been established for all the reasons indicated above.
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.
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Any inquiry concerning this communication or earlier communications from the examiner should be directed to Qing Xu, Ph.D., whose telephone number is (571) 272-3076. The examiner can normally be reached on Monday-Friday from 9:30 AM to 5:00 PM. If attempts to reach the examiner by telephone are unsuccessful, the examiner’s supervisor, Manjunath N. Rao, can be reached at (571) 272-0939. Any inquiry of a general nature or relating to the status of this application or proceeding should be directed to the receptionist whose telephone number is (571) 272-1600.
/Qing Xu/
Patent Examiner
Art Unit 1656
/MANJUNATH N RAO/Supervisory Patent Examiner, Art Unit 1656