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 .
Applicants’ response of 2/10/2026 has been received and entered into the application file. Claims 1, 2, 5, 6, 9-11, 19, and 23 are pending, all of which have been considered on the merits.
Status of Prior Rejections/Response to Arguments
RE: Rejection of claims 1, 2, 5, 6, 9-11, 19, and 23 under 35 U.S.C. 103 over Alkema et al (Scientific Reports, 2015) in view of Shabihkhani et al (Clin Biochem, 2014), and Nalge Nunc International (1998).
Applicants traversed the rejection of record on the grounds that Alkema and Shabihkhani are directed entirely different fields of endeavor than that of the instant application. Specifically, applicants argue Alkema is directed to cryopreservation of tumor tissue for the purpose of reimplantation as patient derived xenografts and Shabihkhani reviews best practices for preserving DNA, RNA, and protein quality in frozen biospecimen whereas the instant application is drawn to cryopreservation for the manufacture of TILs.
In response to applicant's argument, “[F]or the manufacture of tumor infiltrating lymphocytes” is an intended use, not a claimed step. The claimed method only recites steps drawn to cryopreservation of tumor tissue. It has been held that a prior art reference must either be in the field of the inventor’s endeavor or, if not, then be reasonably pertinent to the particular problem with which the inventor was concerned, in order to be relied upon as a basis for rejection of the claimed invention. See In re Oetiker, 977 F.2d 1443, 24 USPQ2d 1443 (Fed. Cir. 1992). In this case, Alkema, Shabihkhani, and the instant application are all drawn to cryopreservation of tissue samples.
Additionally, applicants assert a person of ordinary skill would understand that optimizing a method for tumor cell survival could produce entirely different results for lymphocytes resident within the same tissue. These assertions are unsupported by evidence and drawn to limitations which are not claimed (e.g. cryoprotectant effects on TILs). Alkema, Shabihkhani, and Nalge Nunc render the method as claimed obvious.
Applicants further argue, the examiner has not explained why POSITA would have selected the cryopreservation method of Alkema and modified the method in view of Shabihkhani and Nalge Nunc. In response, Alkema and the instant application are both drawn to methods of cryopreserving tumor tissue. Shabihkhani and Nalge Nunc teach methods of improving cryopreserved samples. A detailed explanation of why it would have been obvious to modify the method of Alkema using the teachings of Shabihkhani and Nalge Nunc can be found in the rejection below.
Applicants further traversed the rejection of record on the grounds that Alkema does not teach incubation in cryopreservation medium at 2° C to 8° C and Shabihkhani teaches to eliminate time before freezing. Specifically, applicants argues Shabihkhani teaches holding tissue at 4° C to mitigate cold ischemia and recommends snap freezing within 20 minutes which is different from a deliberate 30-80 minute hold step.
In response, the argument has been fully considered but is not found convincing. The rejection of record relies on the teachings of Alkema, Shabihkhani, and Nalge Nunc. Shabihkhani teaches keeping biospecimen on ice or in a 4°C refrigerator after resection but before fixation or freezing can limit cellular changes and degradation. This is a desirable effect that a person of ordinary skill in the art would have taken into consideration while performing the method of Alkema in order to prevent degradation of DNA and proteins during the cryopreservation process.
Applicants further traversed the rejection of record on the grounds that Nalge Nunc is inapplicable to solid tissue fragments and teaches away from the claimed 2-8° C range. Specifically, applicants argue Nalge Nunc’s equilibration guidance is directed to individual cells, not tissue fragments and the diffusion of a cryoprotected agent through a solid tumor fragment of 1.5 to 6mm in diameter is different from the diffusion across a single-cell membrane. Additionally, Nalge Nunc teaches equilibration at ambient temperature, not 2-8° C.
In response, the argument has been fully considered but is not found persuasive. A person of ordinary skill in the art would have recognized that it would take longer for a cryoprotectant to diffuse through the multiple layers of cells as compared to a single layer of cells. The absence of mention of intact tissue samples does not constitute a teaching away. Thus, a person of ordinary skill in the art would have been motivated to optimize the incubation time in the cryoprotectant. Additionally, while Nalge Nunc teaches equilibration at ambient temperature, a person of ordinary skill in the art would have been motivated to perform a longer equilibration step at 4° C based on the teachings of Shabihkhani.
Additionally, applicants argue unexpected and advantageous results. Specifically, applicants point to Fig. 10 which compares IFNγ production by TILs derived from fresh and frozen tumor tissue. Applicants point to the data labeled Gen 2 as teaching higher production of IFNγ production by samples which were exposed to 30 min and 60 min incubations compared to control samples and samples which were flash frozen. Furthermore, applicants rebut the examiners interpretation of the data presented in Fig. 10. Applicants state that the fresh control sample for the Gen 2 process in Example 7 was not stored in Hypothermosol and 10 mL of CryoStor10 was added to all frozen samples. Additionally, the data cited by the examiner referenced example 8 which describes a general process description and is not the data represented by Fig. 10.
However, this is not convincing in view of Fig. 11. While applicants state Example 8 teaches a general process, the general process describing Gen 2.2 is the claimed process. Furthermore, the description of results in Fig. 10 in ¶0914 states “TILS expanded from frozen tissue first incubated for 30 minutes in storage medium expressed the highest level of IFN-γ.” The described results correlate with the results in the graph labeled “Gen2.2” in Fig. 10. The Gen2.2 graph further teaches higher IFNγ production by TILs that were flash frozen compared to Gen 2 controls as well as “24 post HTS” samples. Therefore, the argument that the cryopreservation method of the instant application results in an unexpected increase in IFNγ production is not convincing because one could not conclude that the results are not based on the use of CS10 cryopreservation medium as opposed to other cryopreservation mediums. Additionally, the claimed method of the instant application does not specifically require CS10, therefore, one could not conclude that performing the claimed method with a different cryopreservation media would yield the same results.
The rejection is therefore maintained.
Maintained Rejections
Claim Rejections - 35 USC § 103
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
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.
This application currently names joint inventors. In considering patentability of the claims the examiner presumes that the subject matter of the various claims was commonly owned as of the effective filing date of the claimed invention(s) absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and effective filing dates of each claim that was not commonly owned as of the effective filing date of the later invention in order for the examiner to consider the applicability of 35 U.S.C. 102(b)(2)(C) for any potential 35 U.S.C. 102(a)(2) prior art against the later invention.
Claims 1, 2, 5, 6, 9-11, 19, and 23 are rejected under 35 U.S.C. 103 as being unpatentable over Alkema et al (Scientific Reports, 2015) in view of Shabihkhani et al (Clin Biochem, 2014), and Nalge Nunc International (1998).
Alkema et al teaches a method of cryopreserving tumors comprising harvesting and dissecting, primary tumors and tumors harvested from patient derived xenografts, cutting the tumor into 3 x 3 x 3 mm pieces, transferring the pieces into tubes containing 95%FCS/5% DMSO, placing the tubes in an isopropanol freezing container placed at -80° C overnight, then transferring the tubes to liquid nitrogen (See pg. 9, Sec. Preservation using FCS/DMSO and Thawing procedure). “For the manufacture of tumor infiltrating lymphocytes” is an intended use and does not alter the structure or composition of the tumor fragments.
Regarding claim 1: Alkema et al teaches a method of cryopreserving tumor tissue comprising a step of cutting the tumor into 3 x 3 x 3mm pieces, using a surgical instrument, which reads on fragmenting the tumor tissue. The cryopreservation method of Alkema et al further comprises steps of placing tumor pieces in tubes containing 95% FCS/5% DMSO which reads on incubating the fragments in a single cryopreservation medium. Furthermore, the cryopreservation method of Alkema et al comprises a step of transferring tubes from -80°C to liquid nitrogen which reads on a step of flash freezing from -80°C to approximately -196°C.
The method of Alkema et al differs from the method of claim 1 in that Alkema et al does not teach 1) incubating in a cryopreservation medium for 30 to 80 minutes; 2) incubating in cryopreservation medium at a temperature of 2°C to 8°C; and 3) flash freezing using the vapor phase of liquid nitrogen.
Regarding the first, Nalge Nunc teaches cells in suspension should be mixed with a cryoprotectant for at least 15 minutes, to allow the preservation medium to penetrate the cells, thereby equilibrating the cells in the cryopreservation medium (See pg. 4, Sec. Equilibration). Nalgene Nunc further teaches the optimal equilibration time should be determined empirically for cells being cryopreserved to maximize recovery and that larger, less permeable cell require longer equilibration periods (See pg. 4, Sec. Equilibration).
Given that Alkema et al are cryopreserving tumor pieces, and Nalge Nunc discloses the length of time in which cells are equilibrated in cryopreservation media can be affected by cell size and permeability and should therefore be optimized, it would have been prima facie obvious to optimize the amount of time required to equilibrate the tumor fragments in cryopreservation medium, in the method of Alkema, and arrive at 30-80 minutes through routine experimentation. Where 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. See MPEP2144.05(II)
Regarding the second, Shabihkhani et al reviews experimental data on procurement, storage, and quality assurance regarding the handling of frozen biospecimen (See abstract). In this review, Shabihkhani et al discusses optimal and practical approaches to reducing ischemia during tissue procurement including placing the specimen on ice immediately and freezing the specimen within 20 minutes to mitigate artifactual gene expression and phosphorylation aberrations (See pg. 11, Conclusions). Additionally, keeping the biospecimen on ice or in a 4°C refrigerator after resection but before fixation or freezing can limit cellular changes and degradation (See pg. 4, sec. 2.1 Procurement of tissue).
Given that Alkema et al teaches incubating tumor pieces in a cryopreservation media prior to freezing and Shabihkhani et al teaches keeping resected tissue on ice or at 4°C prior to freezing limits cellular changes and degradation of the tissue, it would have been prima facie obvious to incubate the tumor pieces of Alkema et al in a cryopreservation solution at a temperature of 4°C (reads on 2°C to 8°C) in order to limit cellular changes and degradation of the tumor pieces. One would have a reasonable expectation of success because Shabihkhani et al teaches a temperature of 4°C can be achieved using a refrigerator.
Regarding the third, Shabihkhani et al discloses -80°C to -150°C as standard temperatures for long term storage of biospecimen (See pg. 5, sec. 3.1 Storage of frozen tissue). Optimally, temperatures below -137°C should be used as -137°C is the glass transition temperature of water below which biochemical activity that might degrade intracellular contents are thought to be inert (See pg. 5, sec. 3.1 Storage of frozen tissue). Temperatures below -137°C can be easily achieved through storage in liquid nitrogen tanks which contain a vapor phase (-150°C) and a liquid phase (-196°C) (See pg. 5, sec. 3.1 Storage of frozen tissue). Preferably, the tissue is stored in the vapor phase of a liquid nitrogen tank because of the risk of contamination by errant floating tissue fragments in liquid nitrogen (See pg. 5, sec. 3.1 Storage of frozen tissue).
Given that Alkema et al discloses a step of immersing tumor fragments in liquid nitrogen (reads on flash freezing) and Shabihkhani et al discloses using the vapor phase of liquid nitrogen is preferable over the liquid phase, it would have been prima facie obvious to perform the flash freezing step of Jin et al in the vapor phase of liquid nitrogen as disclosed by Shabihkhani et al. One would have been motivated to perform the flash freezing step in the vapor phase of liquid nitrogen over the liquid phase because the liquid phase may contain floating tissue fragments which can contaminate samples. One would have a reasonable expectation of success because Shabihkhani et al teaches that biospecimen should be frozen below -137°C and the preferable method to achieve this temperature is by using the vapor phase of liquid nitrogen which has a temperature of -150°C.
Regarding claims 2, 5, and 6: Following the discussion of claim 1 above, Alkema et al discloses cutting tumors with a surgical instrument into 3 x 3 x 3 mm pieces. Given that the tumor pieces of Alkema et al are cut manually with a surgical instrument, the fragments do not have a defined shape, and rather fall on a spectrum between spherical and cuboidal. The tumor pieces measuring 3 x 3 x 3 mm can thus be considered “approximately spherical” with a diameter of 3 mm (reads on 1.5 mm to 6 mm), “generally rectangular” with a shortest edge length of 3 mm (reads on at least 1.5 mm), and cubical with edge length of 3 mm (reads on between 1.5mm and 6 mm). The exact shape of the tumor tissue does not affect the properties of the tissue, and thus is not a patentable distinction (See MPEP2144.04(IV)(B), In re Dailey, 357 F. 2d 699, 149 USPQ 47(CCPA 1966).
Furthermore, because the tumor pieces are cut manually, it is unlikely the tumor pieces measure exactly 3 x 3x 3 mm and therefore fall within a range of sizes with a longest length of approximately 3 mm. Where the claimed ranges “overlap or lie inside ranges disclosed by the prior art” a prima facie case of obviousness exists (See MPEP2144.05(I); In re Wertheim, 541 F.2d 257, 191 USPQ 90 (CCPA 1976): In re Woodruff, 919 F.2d 1575, 16 USPQ2d 1934 (Fed. Cir. 1990)). In the instant case because the prior art range overlaps the claimed range (3mm) a prima facie case of obviousness exists.
Regarding claim 9: Following the discussion of claim 1 above, Alkema et al discloses harvesting and dissecting tumors which reads on the tumor tissue is from a dissected tumor.
Regarding claim 10: Following the discussion of claims 1 and 9 above, Alkema et al discloses a method for cryopreserving dissected tumor tissue.
Alkema et al does not disclose how long ago the tumor tissue was dissected.
Shabihkhani et al teaches biospecimen should be frozen within 20 minutes (reads on less than eight hours old) of procurement to mitigate artifactual gene expression and phosphorylation. Shabihkhani et al also discloses that some biobanks send a technician to the operating room with liquid nitrogen, to collect samples, in order to reduce time, the sample undergoes ischemia (See pg. 3, sec. 2.1 Procurement of tissue).
Given that Alkema et al teaches a freezing step and Shabihkhani et al teaches freezing within 20 minutes from tissue procurement mitigates artifactual gene expression and phosphorylation and the amount of time a sample undergoes ischemia should be reduced, it would have been prima facie obvious to one of ordinary skill in the art to optimize the length of time between the dissection step and freezing step of Alkema et al, in order to minimize the length of time the sample undergoes ischemia and in order to mitigate artifactual gene expression and phosphorylation. Where 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. See MPEP2144.05(II)
Regarding claim 11: Following the discussion of claim 1 above, Alkema et al discloses a cryopreservation Media comprising 95% FBS and 5% DMSO which reads on the cryopreservation medium comprises 2% v/v to 15% v/v DMSO.
Regarding claim 19: Following the discussion of claim 1 above, Alkema et al discloses freezing tumor pieces in liquid nitrogen. Shabihkhani et al discloses the vapor phase of liquid nitrogen has a temperature of 150°C and the liquid phase has a temperature of -196°C. The instant claim recites the freezing takes place at a temperature in the range of -125°C to about -196°C. In the case where the claimed ranges “overlap or lie inside ranges disclosed by the prior art” a prima facie case of obviousness exists (See MPEP2144.05(I); In re Wertheim, 541 F.2d 257, 191 USPQ 90 (CCPA 1976): In re Woodruff, 919 F.2d 1575, 16 USPQ2d 1934 (Fed. Cir. 1990)). In the instant case because the prior art range overlaps with the claimed range (-150° to -196°C) a prima facie case of obviousness exists.
Regarding claim 23: Following the discussion of claim 1 above, Alkema et al discloses storing tumor pieces in liquid nitrogen.
Alkema et al does not disclose the tumor pieces are stored in the vapor phase of liquid nitrogen.
Shabihkhani et al discloses that biospecimen should preferably be stored in the vapor phase of liquid nitrogen because there is a risk of contamination from floating tissue fragments in liquid nitrogen.
Given that Alkema et al discloses storing the tumor slices in liquid nitrogen and Shabihkhani et al discloses using the vapor phase of liquid nitrogen is preferable over the liquid phase, it would have been prima facie obvious to store the tumor pieces of Alkema et al in the vapor phase of liquid nitrogen as disclosed by Shabihkhani et al because the liquid phase may contain floating tissue fragments which can contaminate samples. One would have a reasonable expectation of success because Shabihkhani et al teaches that biospecimen should be frozen below -137°C and the preferable method to achieve this temperature is by using the vapor phase of liquid nitrogen which has a temperature of -150°C.
Conclusion
THIS ACTION IS MADE FINAL. 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 MARISOL A O'NEILL whose telephone number is (571)272-2490. The examiner can normally be reached Monday - Friday 7:30 - 5:00 EST.
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/MARISOL ANN O'NEILL/ Examiner, Art Unit 1633
/ALLISON M FOX/ Primary Examiner, Art Unit 1633