DETAILED ACTION
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
This Office Action is in response to the paper filed 26 February 2026. Claims 7, 22, and 25 have been amended. Claims 2, 24, and 26 remain withdrawn. Claim 27 is newly added. Claims 1, 3-11, 13-15, 17, 19-21, 23, 25, and 27 are currently pending and under examination.
This Application is a national phase application under 35 U.S.C. §371 of International Application No. PCT/US2020/059687, filed 9 November 2020, which claims priority to U.S. Provisional Application No. 62/932473, filed 7 November 2019.
Withdrawal of Rejections:
The rejection of claims 7, 23, and 25 under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite, is withdrawn.
Maintenance/Modification of Rejections Necessitated by Amendment:
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.
Claims 1, 3-11, 13-15, 17, 19-21, 23, 25, and 27 are rejected under 35 U.S.C. 103 as being unpatentable over Zhang et al. (IDS; US 2017/0233698, Published 2017).
With regard to claims 1, 23, 25, and 27, Zhang et al. teach preparing a cultivated autologous limbal epithelial cell (CALEC) graft for surgical transplantation (Abs.; Para. 104), the method comprising steps including: providing a tissue from a limbal biopsy or the wet mucosal source (Para. 58, 104); treating the tissue from the limbal biopsy or the wet mucosal source with an enzyme blend thereby isolating limbal epithelial cells (Para. 107, 123); culturing the limbal epithelial cells in the presence of serum-free complete corneal epithelial cell medium for a sufficient period of time until the limbal epithelial cells reach at least about 70%, 80%, or 90% confluence (Para. 109-110, 112-113, 124); detaching the limbal epithelial cells (Para. 124); seeding the limbal epithelial cells on a suitable membrane substrate (Para. 152); growing the cells on the suitable membrane substrate for a sufficient period of time until the limbal epithelial cells reach 70-80% confluence thereby producing a CALEC graft (Para 153, 204); and immersing the CALEC graft in a preservation medium suitable for hypothermic bio-preservation until surgical transplantation (Para. 257-258).
Zhang et al. expressly teach that these steps may be performed in the method. As such, it would have been obvious to one of ordinary skill in the art to perform the expressly taught steps when performing the method, including in a logical order from collection to preservation as desired to prepare the CALEC graft for the end use.
Zhang et al. render obvious the method as claimed, including the components as claimed. As the components cannot be separated from their properties, the result that the limbal epithelial cells are positive for CD49F, CD49E, CD326, CD318, and CD340, and are negative for CD3, CD14, CD16, CD19, CD20, CD56, CD45, CD31, and CD105 naturally flow from performance of the method as rendered obvious by Zhang et al. Further, Zhang et al. teach that the limbal stem cells of the corneal epithelium can be characterized by “the presence, absence and/or expression levels of biomarkers such as…CD105” (Para. 105). This indicates that CD105 can be any of present, absent, or expressed at some level. Thus, Zhang et al. do not require that the cells be CD105+ or that CD105 necessarily be used for sorting.
With regard to claim 3, Zhang et al. teach that the medium used to culture the limbal epithelial cells is free from non-human animal derived products (Para. 56, 65).
With regard to claim 4, Zhang et al. teach the processing of the biopsy material in a sterile manner, and the use of sterile components (Para. 107, 247-248). Zhang et al. further teach placing the biopsy material in preservation medium that is suitable for cryopreservation (Para. 129, 258). As such, it would have been obvious to one of ordinary skill in the art to place the collected biopsy material in a sterile container filled with preservation media suitable for hypothermic bio-preservation.
With regard to claim 6, Zhang et al. teach steps that include washing the limbal epithelial cells, and suspending the cells in culture media at multiple points throughout the taught method (Para. 20, 107, 123-124). As it is taught that the limbal epithelial cells are washed, and that the cells are suspended in culture media, it would have been obvious to one of ordinary skill in the art to utilize the epithelial cell culture media as a washing solution prior to resuspension in the culture media as desired throughout performance of the method.
With regard to claim 7, Zhang et al. teach that the human amniotic membrane, which is a suitable membrane substrate, may be denuded of epithelial cells (Para. 109, 250), which is de-epithelializing the suitable membrane. The human amniotic membrane may be affixed onto a culture plate for culturing the cells (Para. 109), which is deemed to be placing the membrane substate into a cell culture insert in preparation for seeding the limbal epithelial cells.
With regard to claim 8, Zhang et al. teach that the CALEC graft may be immersed in a preservation medium (Para. 257-258). While it is not taught that the preservation medium with the graft is maintained at a temperature of 1-10°C, it is further taught that cryopreservation may be utilized in the method (Para. 129). As such, it would have been obvious to one of ordinary skill in the art to maintain the graft in cold storage until it is needed for use. Additionally, please also note that "the discovery of an optimum value of a variable in a known process is usually obvious." Pfizer v. Apotex, 480 F.3d at 1368. The rationale for determining the optimal parameters for prior art result effective variables "flows from the 'normal desire of scientists or artisans to improve upon what is already generally known.'" Id. (quoting In re Peterson, 315 F.3d 1325, 1330 (Fed. Cir. 2003)). Accordingly, it would have been obvious to optimize the storage temperature of the preservation medium for storage of the graft, including to 1-10°C, to result in an appropriate temperature to maintain the viability of the graft during storage when practicing the taught method.
With regard to claim 9, Zhang et al. teach that 3x104 cells are seeded onto the amniotic membrane (Para. 434).
With regard to claim 10, as Zhang et al. teach the step of treating the tissue from the limbal biopsy or the wet mucosal source with an enzyme blend thereby isolating limbal epithelial cells (Para. 107, 123), the result of this step yielding 3-7x104 limbal epithelial cells would naturally flow from performance of this step.
With regard to claim 11, Zhang et al. teach that time for culturing steps in the method may be from about 3 to about 18 days (Para. 27). Wherein it would have been obvious to an ordinary artisan to utilize a time within the expressly taught range, which includes 6 to 10 days. Additionally, please also note that "the discovery of an optimum value of a variable in a known process is usually obvious." Pfizer v. Apotex, 480 F.3d at 1368. The rationale for determining the optimal parameters for prior art result effective variables "flows from the 'normal desire of scientists or artisans to improve upon what is already generally known.'" Id. (quoting In re Peterson, 315 F.3d 1325, 1330 (Fed. Cir. 2003)). Accordingly, it would have been obvious to optimize the culture time for the cells during the method, including for 6 to 10 days, to result in cells that have reached the desired level of confluence when practicing the taught method.
With regard to claims 13 and 14, Zhang et al. teach that during culturing steps, the culture medium may be changed every other day (Para. 153). As such, it would have been obvious to one of ordinary skill in the art to change the culture medium during the culturing steps every other day, which is every two days.
With regard to claims 15 and 17, Zhang et al. teach that the limbal biopsy originates from a patient suffering from a limbal stem cell deficiency, or from an allogeneic donor, wherein the allogeneic donor is live or cadaveric (Para. 104).
With regard to claim 19, Zhang et al. teach that the wet mucosal source is conjunctiva (Para. 58).
With regard to claims 20 and 21, Zhang et al. teach that the membrane substrate is amniotic membrane, including human amniotic membrane, or a basement membrane (Para. 82, 152, 249).
Claims 1 and 5 are rejected under 35 U.S.C. 103 as being unpatentable over Zhang et al. as applied to claim 1 above, and further in view of Lv et al. (Optimized dissociation protocol for isolating human glioma stem cells from tumorspheres via fluorescence-activated cell sorting, Cancer Letters, Vol. 377, (2016), pp. 105-115).
The teachings of Zhang et al. as applied to claim 1 have been set forth above.
With regard to claim 5, Zhang et al. teach that the enzyme blend may include collagenases, trypsin, and commercial products such as Stem Pro Accutase (Para. 107). However, it is not specifically taught that the collagenases are collagenase I and II, or that the serine protease (e.g. trypsin) is an animal-free protease.
Lv et al. teach that five commonly used digestive agents for dissociating a cell aggregate into single cells include Liberase-TL, trypsin, TrypLE, Accutase, and non-enzymatic cell dissociation solution (NECDS) (Abs.). Liberase-TL contains collagenase I and II (p. 113, Left Col., last para.), and TrypLE contains animal free serine protease (see Specification, p. 9, line 26-28).
It would have been obvious to one of ordinary skill in the art to combine the teachings of Zhang et al. with Lv et al., because both teach digestive agents for dissociating an aggregation of cells into single cells that include collagenases and serine proteases. The use of Liberase-TL and TrypLE for dissociating a cell aggregate into single cells is known in the art as taught by Lv et al. The use of Liberase-TL, which contains collagenases, and TrypLE, which contains a serine protease, in place of the enzyme blend as taught by Zhang et al. amounts to the simple substitution of one known enzyme blend for another, and would have been expected to predictably and successfully dissociate the limbal biopsy tissue into single cells as desired by Zhang et al. Lastly, Lv et al. fails to correct the noted deficiencies.
Response to Arguments
Applicant urges that Zhang et al. do not teach two passage steps as claimed, where cells of the second passage reach 70-80% confluence, instead second passage confluence is taught at 15-20%. Further, Zhang et al. do not teach culturing the limbal epithelial cells in the presence of serum-free complete corneal epithelial cell medium, and instead teach media with 10% fetal bovine serum. Additionally, Zhang et al. do not teach that the cell population is negative for CD105, and instead teach the cells are CD105+. Additionally, Lv et al. do not cure the noted deficiencies of Zhang et al.
Applicant’s arguments have been fully considered, but have not been found persuasive.
With regard to Applicant’s argument that Zhang et al. do not teach two passage steps as claimed, where cells of the second passage reach 70-80% confluence, instead second passage confluence is taught at 15-20%; it is noted that Zhang et al. teach an initial culturing step:
[124] After the limbal tissue is cultured for several days, for example until the cells become confluent, the LSCs can be isolated from the culture. Preferably, in this example, the limbal tissue culture is allowed to grow until it is at least about 50%, 60%, 70%, 80%, 90%, or 95% confluent.
This corresponds to culturing step (c) as set forth by Zhang et al. (see for example para. 151). In paragraph 153 Zhang et al then goes on to teach:
[0153] In another embodiment, the method further comprises step (d), wherein the proliferated LSCs or LSC-like cells are passaged at about 70-90% confluence before passage and about 15-20% confluence after passage.
As “step (d)” is utilized, it is clear that Zhang et al. teaches a second growth step to about 70-90% confluence, which fully encompasses 70-80% confluence. The further step of passaging to about 15-20% confluence is interpreted as an additional step after that second step.
As noted in the rejection, Zhang et al. expressly teach that the steps as taught may be performed in the method. As such, it would have been obvious to one of ordinary skill in the art to perform the expressly taught steps when performing the method, including in a logical order from collection to preservation as desired to prepare the CALEC graft for the end use.
With regard to Applicant’s argument that Zhang et al. do not teach culturing the limbal epithelial cells in the presence of serum-free complete corneal epithelial cell medium, and instead teach media with 10% fetal bovine serum; Zhang et al. specifically teach:
[0112] In a preferred embodiments, the medium used in the invention is a feeder-free, serum-free and xeno-free medium. For example, the media used to prepare the tissue system, including the medium used to transport the limbal tissue biopsies, the medium used to culture the biopsies, the enriched medium used to culture the limbal stem cells, and the medium used to transport the tissue system, do not contain any sera or other factors of animal origin. This will help minimize any risk of contamination of the tissue system with xenogenic components, thereby making the tissue systems safe for human administration. In a most preferred embodiment, the feeder-free, serum-free and xeno-free medium is a chemically defined medium in which all components in the medium are chemically defined and do not contain undefined animal-derived or human-derived products (emphasis added).
It is clear from this teaching, that in a most preferred embodiment, Zhang et al. utilizes serum-free media, which is desirable for use to minimize risk of contamination of the tissue system with xenogenic components, thereby making the tissue systems safe for human administration. Additionally, numerous serum-free complete media are taught by Zhang et al. (see Para. 110).
With regard to Applicant’s argument that Zhang et al. do not teach that the cell population is negative for CD105, and instead teaches the cells are CD105+; as noted in the modified rejection above, Zhang et al. render obvious the method as claimed, including the components as claimed. As the components cannot be separated from their properties, the result that the limbal epithelial cells are positive for CD49F, CD49E, CD326, CD318, and CD340, and are negative for CD3, CD14, CD16, CD19, CD20, CD56, CD45, CD31, and CD105 naturally flow from performance of the method as rendered obvious by Zhang et al. Further, Zhang et al. teach that the limbal stem cells of the corneal epithelium can be characterized by “the presence, absence and/or expression levels of biomarkers such as…CD105” (Para. 105). This indicates that CD105 can be present, absent, or expressed at some level. Thus, Zhang et al. do not require that the cells be CD105+ or that CD105 necessarily be used for sorting.
With regard to Lv et al., the noted deficiencies of Zhang et al. have been addressed above.
Conclusion
No claims are allowable.
Applicant's amendment necessitated the new ground(s) of rejection presented in this Office action. Accordingly, THIS ACTION IS MADE FINAL. See MPEP § 706.07(a). Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action. In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any nonprovisional extension fee (37 CFR 1.17(a)) pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action. In no event, however, will the statutory period for reply expire later than SIX MONTHS from the mailing date of this final action.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to JENNIFER M.H. TICHY whose telephone number is (571)272-3274. The examiner can normally be reached Monday-Thursday, 9:00am-7:00pm ET.
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/JENNIFER M.H. TICHY/Primary Examiner, Art Unit 1653
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