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and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114.
Remarks
The amendments and remarks filed on 08/18/2026 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 Any previously presented objections or rejections that are not presented in this Office Action are withdrawn.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 1-5, 11-12, and 21-24 are pending.
Claims 1, 12, and 21 are amended.
Claims 6-10, 13-20, and 25-28 are canceled.
Claims 1-5, 11-12, and 21-24 have been examined on the merits.
Priority
This application, U.S. Application number 17/782531, is a national stage entry of International Application Number PCT/JP2020/045005, filed on 12/03/2020, which claims for foreign priority under 35 U.S.C. 119 (a)-(d) to JP 2019-219192 filed on 12/04/2019.
Objections - Withdrawn
Objection to Claim 12 is withdrawn due to the amendment to the claim filed on 08/18/2026.
Rejections - Withdrawn
The rejections of claims 1-6, 9-12, 21-24, and/or 27-28 under 35 U.S.C. 103 over Stubbers et al. in view of Lockhart et al., Hesse, Izadpanah et al., and/or Dendo et al. are withdrawn due to the amendment of the claims filed on 08/18/2026.
Claim Rejections - 35 USC § 103
Claims 1-5, 11-12, and 21-24 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), Hesse et al. (US Patent NO: 5853976, 1998, cited in IDS), Izadpanah et al. (US 2013/0190729, 2013, of record), Ilan et al. (US 2013/0224216, 2013), and ALT et al. (US 2017/0182099, 2017), as evidenced by Etchison (Sports Health, 2011, 3(6):499, of record) and ALT-2 et al. (US 2020/0399609, 2020).
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 collagenase is a pure collagenase; wherein the enzyme solution comprises collagenases I and II together with dispase; 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, 0056/lines 6-7, and 0072), and wherein the dispase is a neutral protease (para 0072/second line from bottom) (Note: these meet the claimed limitation of “free of clostripain or thermolysin” in the instant claims 1, 12, and 21). Regarding the ratios of neutral protease activity vs. collagenase activity recited in the instant 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, 12, and 21-23. It is noted that Stubbers et al. do not specifically teach assaying activities of the enzymes with commercial EnzChek collagenase assay kit or 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 of Stubbers et al.
Regarding the limitation of “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 constitutes 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 limitation “free of clostripain or thermolysin” in the claims 1, 12, and 21, the enzyme solution suggested by Stubbers et al. Lockhart et al. and Hesse et al. comprises collagenases I/II and neutral protease from C. histolyticum, which is free of clostripain or thermolysin.
Regarding the limitation of “the enzyme solution has a neutral protease content of not less than 0.05 U per 1 g of the adipose tissue” recited in claims 1, 12, and 21 as well as the further limitation in the claim 5, Stubbers et al. teach using an enzyme solution comprising collagenase (60,000 U) and neutral protease (120 U) to treat/disperse the adipose tissue for preparing a stromal vascular fraction, and Stubbers et al. are silent about a specific weight of the adipose tissue to be treated by these collagenase and neutral protease and do not teach a neutral protease content of not less than 0.05 U per gram of adipose tissue and a collagenase content of not less than 500 U per gram of adipose tissue. However, Stubbers et al. further teach that particular enzyme concentrations (encompassing activity units per gram of tissue) may be optimized to maximize yields of cell subpopulations (para 0056, lines 4-5). It is noted that enzymatic procedures of using a combination of collagenase I/II and neutral protease to treat adipose tissue for preparing a stromal vascular fraction are well established in the art, as supported by Stubbers et al. and as further supported by ALT et al., who teach applying a Matrase enzyme solution (1 unit of tissue mass digestion activity per gram of the tissue) for treating fat tissue and preparing a stromal vascular fraction (para 0102) (NOTE: the Matrase inherently comprises a combination of collagenase I and II and a neutral protease, as evidenced by ALT-2 et al., see para 0103/lines 2-4). Accordingly, a specific ratio of enzymatic activities vs. weight of adipose tissue, based on ratios known in the prior art, is readily applied to and optimized for maximizing yields of stromal vascular fraction and cell subpopulations in the method suggested by the cited prior art. It is further noted that enzymatic activity/tissue weight ratios that fall into the claimed ranges for treating adipose tissue are well known in the art, as supported by Ilan et al., who teach a similar method for preparing a stromal vascular fraction, comprising: treating adipose tissue with collagenase I at a ratio of 840 U/g of the tissue (para 0661: lines 1, 4, 8-9), reading on the claimed range of not less than 500 U/g of tissue; and as further supported by Izadpanah et al., who teach a similar method for preparing a stromal vascular fraction/SVF, comprising: treating ~ 100 ml of adipose tissue/lipoaspirate with an enzyme solution containing a collagenase (60,000 U) and a neutral protease/dispase (120 U), and collecting 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 has a weight of ~ 91.96 g, given adipose tissue has a density of 0.9196 g/mL as evidenced by Etchison (page 499, left col, para 1, line 6). Accordingly, a ratio of neutral protease activity vs. tissue weight taught by Izadpanah et al. is ~ 1.31 U per gram of adipose tissue, reading on the claimed range of not less than 0.05 U/g of tissue. Thus, the claimed ratios are well within the purview of the skilled artisan having the cited prior art as a guide. Examiner notes that generally, differences in an enzyme concentration (e.g. a ratio of enzymatic activity vs. tissue weight) or temperature will not support the patentability of subject matter unless there is evidence indicating such concentration or temperature 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). Therefore, the claimed ratios would have been obvious over the cited prior art, in the absence of evidence to support it is critical.
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 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 effect on dissociating adipose tissues and releasing cells and for maximizing cell yields. 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 Claim 11, Stubbers et al. teach the adipose tissue is human adipose tissue 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., and other cited prior art 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.
Response to Arguments
Applicant's arguments about the objection to claim 12 as well as the rejections of claims 1-6, 9-12, 21-24, and/or 27-28 under 35 U.S. C. 103 over Stubbers et al. in view of Lockhart et al., Hesse, Izadpanah et al., and/or Dendo et al. in the response filed on 08/18/2026 (pages 5-10) have been fully considered but they are moot because the objection and the rejections have been withdrawn as indicated above.
In response to Applicant’s arguments based on the collagenase of Izadpanah contains clostripain in page 7 of the response, Examiner notes that ratios taught by Izadpanah can still be applied to and modified/optimized in the method suggested by the cited prior art even though the collagenase of Izadpanah is slightly different from that of Stubbers et al. with the presence of clostripain. This is because collagenases and neutral proteases all have similar proteolytic activities or enzymatic characteristics (for example: 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 neutral protease vs. adipose weight taught by Izadpanah et al. is applicable to the neutral protease in the method suggested by the cited prior art, and can be modified/optimized for reaching a desirable effect on tissue treatment. In addition, 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).
Overall, the conclusion of the obviousness of the claims 1-5, 11-12, and 21-24 has been established for all the reasons indicated above.
Conclusion
No claim is in condition for allowance.
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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
Prosecution Insights