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and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114.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 06/24/2025 has been entered.
Application Status
This action is written in response to applicant’s correspondence received 06/24/2025. Claims 1-2, 4-5, 8, 10-14, 28, 30, 36-38, 41, 60 64, 68, and 91-105 are currently pending. Claim 11 is withdrawn from prosecution as being drawn to non-elected subject matter. Accordingly, claims 1-2, 4-5, 8, 10, 12-14, 28, 30, 36-38, 41, 60 64, 68, and 91-105 are examined herein. The restriction requirement mailed 12/26/2023 is still deemed proper.
Applicant’s arguments, filed 06/24/2025, with respect to the rejection of claim 2 under 35 U.S.C. § 112(d) have been fully considered and are persuasive. The rejection of claim 2 has been withdrawn.
Applicant’s arguments that the method of Wang would exclude such high molecular weight fragments of DNA that are greater than about 10 kb and less than 200 kb, thereby resulting in a fraction having a different structural composition that also affects determination of transgene integration, have been fully considered and are persuasive, with respect to the rejection of claims 1-2, 4-5, 8, 12-14, 28, 30, 36-38, 40-41, 43, 47 and 60 under 35 U.S.C. § 103 over Wang. Therefore, the rejection has been withdrawn. However, upon further consideration, a new ground(s) of rejection is made in view of Lada et al. Additionally, Applicant’s arguments regarding USPAT008 have been fully considered, but are moot because the new ground of rejection does not rely on any reference applied in the prior rejection of record for any teaching or matter specifically challenged in the argument.
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.
The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
Determining the scope and contents of the prior art.
Ascertaining the differences between the prior art and the claims at issue.
Resolving the level of ordinary skill in the pertinent art.
Considering objective evidence present in the application indicating obviousness or nonobviousness.
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, 4-5, 12-14, 28, 30, 36-38, 41, 60, 91-93, and 96-97 are rejected under 35 U.S.C. 102(a)(1) as being anticipated over Lada (Lada et al. Measurement of Integrated HIV DNA by Pulse-Field Gel Electrophoresis and ddPCR. Poster Presentation. CROI conference, Boston, 2016.;of record, applicant’s submission) in view of Charrier (Quantification of lentiviral vector copy numbers in individual hematopoietic colony-forming cells shows vector dose-dependent effects on the frequency and level of transduction. Gene Therapy (2011) 18, 4 79-487.; of record, applicant’s submission)
Regarding claim 1, Lada teaches a method for assessing genomic integration of a transgene, the method comprising separating a high molecular weight fraction of DNA of greater than 10kb (15kb+)from isolated DNA, and from the high weight fraction, determining, by PCR, the copy number of the transgene (i.e., gene which does not naturally occur in that cell) sequence, wherein the PCR is carried out using a pair of primers that are complementary to or are capable of specifically amplifying sequences of the transgene sequence (i.e., viral transgenes such as gag):
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While Lada does not explicitly teach determining the copy number per diploid genome, they do teach calculating the copy number per 1E6 cells using human cells and cell lines. The ordinary artisan would have been able to calculate copies per diploid genome based on the number of cells assayed.
Lada does not teach that, prior to the separating, a polynucleotide comprising the transgene sequence encoding the recombinant protein has been introduced, via transduction with a viral vector, into at least one cell of the one or more cells to produce at least one engineered cell. The broadest reasonable interpretation of an engineered cell encompasses cells which have been genetically engineered to express transgenes, but excludes cells infected with naturally-occurring viruses without human intervention. Lada does not teach engineered cells. Instead, Lada teaches the measurement of integrated HIV DNA in infected CD4+ T cells of patients undergoing antiretroviral therapy (Background).
However, Lada does teach that the method has an advantage over ddPCR alone because ddPCR does not distinguish between integrated and unintegrated molecular forms (Background). Lana further teaches that the method provides a sensitive and precise approach to the measurement of integrated viral (HIV) DNA with sufficient throughput for translational research studies (Cocnlusions). Therefore, Lada provides a teaching, suggestion or motivation to apply the method to other cells and viruses.
Charrier teaches the quantification of lentiviral vector (HIV-1-derived, rHIV) copy numbers in genetically corrected hematopoietic stem cells (Title, p. 479). They further provide a teaching, suggestion or motivation to precisely and sensitively quantify vector copy number, noting (Id.):
The biological potency of the vector is expected to correlate positively with the frequency of transduced cells and also with the number of integration per cell unless transgene silencing is observed, as suggested in some studies. At the same time, genotoxicity related to the number of vector insertions per cell can result from inappropriate transgene expression in cells or from effects of elements contained in the integrated cassette, to thus requiring strict control of the number of gene insertion per cell. It is therefore important to determine the distribution of vector copies in the infected cell population at the single cell level to assess the efficacy and safety, that is, the therapeutic window of integrative vectors.
It would have been prima facie obvious to a person having ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of measuring integrated HIV DNA using PFGE and ddPCR, as taught by Lada, to measure integrated gene therapy viral vectors in engineered cells, as taught by Charrier. The ordinary artisan would have been motivated to do so based on the combination of Lada’s disclosures that the method was sensitive and precise, and on Charrier’s disclosures that sensitive, precise methods of measuring integrated viral vectors were needed in the field of stem cell gene therapy. The ordinary artisan would further have had a reasonable expectation that Lada’s method could successively have been applied to HIV-derived lentiviral vectors based on Lada’s disclosure that the method was effective, precise and sensitive when applied to integrated HIV.
Regarding claim 2, Lada teaches a step of isolating the DNA from the cells (see above in Methods).
Regarding claim 4, by teaching the method being applied to CD4+ T cells of HIV positive patients undergoing antiretroviral therapy, Lada teaches that the one or more cells comprises a population of cells in which a plurality of cells comprises the transgene (HIV) sequence.
Regarding claim 5, Lada teaches calculating the copy number per a certain number of cells, as discussed above, and it would have been well within the means of the ordinary artisan to calculate a mean copy number per genome based on that information.
Regarding claim 12, Lada teaches wherein the one or more cells is a primary cell obtained from a sample from a subject (see above).
Regarding claims 13 and 14, Lada teaches wherein the one or more cells is an immune cell (T cell).
Regarding claims 28 and 91, Lada teaches wherein the separating is carried out by pulse field gel electrophoresis (see above).
Regarding claim 30, the claim recites the limitations of claims 28 and 5 in combination, which are taught by Lada as described above.
Regarding claims 36-38, Lada teaches wherein the cutoff between the low and high molecular weight fractions is about 15 kb, and therefore teaches that the high molecular weight fraction is greater than about 15, about 17.5, or about 20 kb.
Regarding claim 41, Lada teaches wherein the PCR is ddPCR (see above).
Regarding claim 60, Charrier teaches wherein the viral vector is a lentiviral vector (see above).
Regarding claim 92, Lada teaches wherein the copy number is determined per cell expressing the viral protein.
Regarding claim 93, Lada teaches wherein the one or more cells are viable cells (long-lived CD4+ T cells isolated from living subjects, and thus presumed to be viable).
Regarding claim 96, Lada teaches wherein the primary cell is a T cell.
Regarding claim 97, Lada teaches wherein the copy number is determined per cell, as described above.
Claim 8 is rejected under 35 U.S.C. 103 as being unpatentable over Lada and Charrier, as applied to claims 1-2, 4-5, 12-14, 28, 30, 36-38, 41, 60, 91-93, and 96-97 above, further in view of Zhang et al. (The significance of controlled conditions in lentiviral vector titration and in the use of multiplicity of infection (MOI) for predicting gene transfer events. Genetic Vaccines and Therapy 2004, 2:6.).
Lada and Charrier render obvious the invention of claim 1, from which the instantly rejected claims depend, as described above.
Lada and Charrier do not teach wherein the one or more cells has not been incubated at a temperature that is at or about 37C for more than 96 hours following transduction.
Zhang teaches a series of experiments examining various aspects of lentiviral vector transduction, including the determination of transduction efficiency (i.e., transgene integration and expression of EGFP) at 48 hours after transduction (p. 3/10).
It would have been prima facie obvious to a person having ordinary skill in the art before the effective filing date of the claimed invention to have modified the method as taught by Lada and Charrier to comprise a step of determining transduction efficiency (i.e., transgene integration) at least 48 hours after transduction. Based on common sense and sound scientific reasoning, the ordinary artisan would have been motivated to validate transduction efficiency sooner rather than later, to save experimental time and cost. As taught by Zhang, vector integration was measurable 48 hours after transduction, which would have given the ordinary artisan a reasonable expectation that, using known methods, validation would have been effective at that time point.
Claim 10 is rejected under 35 U.S.C. 103 as being unpatentable over Lada and Charrier, as applied to claims 1-2, 4-5, 12-14, 28, 30, 36-38, 41, 60, 91-93, and 96-97 above, further in view of Xu et al. (of record).
Lada and Charrier render obvious the invention of claim 1, from which the instantly rejected claims depend, as described above.
Lada and Charrier do not teach wherein the one or more cells has been cryopreserved prior to the separating of the high molecular weight fraction.
As discussed in the final rejection of 02/25/2025, Xu teaches that cryopreservation of CAR T cells is necessary for their clinical application, and measurement of transduction efficiency can be done after cryopreservation to assess those parameters after resuscitation (Abstract)
It would have been prima facie obvious to a person having ordinary skill in the art before the effective filing date of the claimed invention to have modified the method as taught by Lada and Charrier to comprise a cryopreservation step before assessing the integration of the transgene. The ordinary artisan would have predicted, based on Xu’s teachings, that this would have permitted the successful preservation of the cells without negatively impacting the cells’ functionality, and that it could be applied to CAR T cells, thus facilitating their preservation and functionality until such time as it was appropriate to begin assessment of integration and/or use the cells in a clinical setting.
Claims 64, 68, 94-95, and 98-105 are rejected under 35 U.S.C. 103 as being unpatentable over Lada and Charrier, as applied to claims 1-2, 4-5, 12-14, 28, 30, 36-38, 41, 60, 91-93, and 96-97 above, further in view of Hartmann (Clinical development of CAR T cells—challenges and opportunities in translating innovative treatment concepts. EMBO Mol Med (2017) 9: 1183–1197.).
Lada and Charrier render obvious the invention of claim 1, from which the instantly rejected claims depend, as described above.
Lada and Charrier do not teach wherein the transgene expresses a recombinant receptor (claims 64, 94, 98) which is a CAR T cell receptor (claims 68, 95, 99, 100-105).
Hartmann teaches various factors in the clinical development of CAR T cells (Title). They further teach that, in the manufacture of CAR T cells, the “need for GMP-compliant manufacturing may constitute a specific hurdle in the timely translation to the clinic” (p. 1192). They note that, “Transduction efficiency, transgene expression levels, and copy numbers per cell are additional factors, which can all directly impact on efficacy and safety.”
It would have been prima facie obvious to a person having ordinary skill in the art before the effective filing date of the claimed invention to have modified the method for assessing transgene integration and copy number in engineered cells, as taught by Lada and Charrier, by applying it specifically to engineered CAR T cells for clinical applications. As taught by Hartmann, transduction efficiency (i.e., the number of cells which successfully integrate the transgenic CAR) and copy numbers per cell are important factors in the efficacy and safety of CAR T cells. This would have motivated the ordinary artisan to measure those parameters in CAR T cells, while Lada and Charrier provide a reasonable expectation that the method of Lada could be applied in both primary and engineered immune cells.
Response to Arguments
While it is noted that Applicant’s arguments regarding the previous rejections under 35 U.S.C. 103 are moot in light of the new grounds of rejection, as stated above, Applicant's arguments filed 06/24/2025, on pages 25-29 of the Remarks, with respect to the advantageous results of the claimed invention, are addressed here insofar as they concern objective evidence of nonobviousness provided by the instant specification.
Applicant notes that Example 2 compares two methods for assessing transgene copy number: an exemplary method of the present claims, iVCN, in which a high molecular weight fraction of DNA containing all DNA greater than 15 kb was first separated from lower weight DNA, and ddPCR was performed only on that fraction in order to specifically determine the amount of integrated transgene; and a standard method, which is referred to as the vector copy number or VCN method, which involved performing ddPCR on DNA of all molecular weights, without any prior separation. Applicant draws attention to FIGs. 2A and 2B, showing comparative results between the VCN and iVCN method. Applicant notes that the standard VCB method without separation of the high molecular weight fraction was not accurate until around 96 hours after transduction. Applicant concludes that these results demonstrate that the claimed method provides “unobvious…advantageous properties” by resulting in a more accurate estimate of the amount of transgene integration, particularly at early time points after transduction.
Respectfully, this is not persuasive because, as described in the new grounds of rejection over Lada, Lada’s method includes a separation of 15kb+ DNA prior to ddPCR, and is described as more precise and sensitive than methods which do not include that step. While Lada differs from the claimed method insofar as Lada concerns measuring integrated versus non-integrated viral transgenes in primary cells from living subjects, while the claimed method contemplates applying the same method to engineered cells, Lada applies well-known molecular biology techniques such as PFGE and ddPCR, which have broad applicability to a variety of cells. Therefore, absent evidence to the contrary, the ordinary artisan would reasonably have expected Lada’s iVCN method to generally exhibit greater sensitivity than the VCN method, regardless of the time point.
Conclusion
No claim is allowed at this time.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to AMANDA M ZAHORIK whose telephone number is (703)756-1433. The examiner can normally be reached M-F 8:00-16:00 EST.
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/AMANDA M ZAHORIK/Examiner, Art Unit 1636