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 .
Claim Rejections - 35 USC § 112
The following is a quotation of the first paragraph of 35 U.S.C. 112(a):
(a) IN GENERAL.—The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor or joint inventor of carrying out the invention.
The following is a quotation of the first paragraph of pre-AIA 35 U.S.C. 112:
The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor of carrying out his invention.
Claims 1, 7-8, 13, 21, 25, 27, and 44-46 as amended, previously presented or newly added, are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, because the specification, while being enabling for:
An in vitro method for enhancing cytolytic activity of a natural killer (NK) cell towards a target tumor cell, the method comprising: (a) transfecting by cytopulse electroporation the primary NK cell with mRNA encoding a TALEN pair that targets exon 2 of a TGFβ receptor in said primary NK cell, wherein expression of the TALEN pair cleaves exon 2 of the TGFβ receptor gene in the genome of the primary NK cell and inactivates TGFβ receptor gene expression in said primary NK cell; and (b) introducing a lentiviral vector comprising a nucleic acid encoding a chimeric antigen receptor (CAR) directed against at least one antigen expressed on the surface the target tumor cell to produce an engineered NK cell comprising (i) a nucleic acid encoding a CAR in its genome; and (ii) a homozygous disruption of the TGFβ receptor in its genome; where the engineered NK cell expresses the CAR and lacks expression of the TGFβ receptor, wherein the engineered NK cell has enhanced cytolytic activity towards the target tumor cell as compared to a NK cell expressing a CAR but has intact endogenous expression of TGFβ receptor.
, does not reasonably provide enablement for:
The TGFβ receptor gene is knockout in the NK cell by any other means that transfecting by cytopulse electroporation said mRNA into the NK cell;
The method that knocks out the TGFβ receptor by an Cas9 RNA guided nuclease;
A method that uses any TALEN pair to have the result of knocking out the TGFβ receptor other than a TALEN pair that targets exon 2 of the TGFβ receptor; and
The NK cell does not comprise the CAR transgene in its genome and does not comprise a homogenous disruption of the TGF TGFβ receptor in its genome.
The specification does not enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the invention commensurate in scope with these claims.
While determining whether a specification is enabling, one considers whether the claimed invention provides sufficient guidance to make and use the claimed invention, if not, whether an artisan would require undue experimentation to make and use the claimed invention and whether working examples have been provided. When determining whether a specification meets the enablement requirements, some of the factors that need to be analyzed are: the breadth of the claims, the nature of the invention, the state of the prior art, the level of one of ordinary skill, the level of predictability in the art, the amount of direction provided by the inventor, the existence of working examples, and whether the quantity of any necessary experimentation to make and use the invention based on the content of the disclosure is “undue”.
Nature of Invention: The claims are directed to a method that improves therapeutic activity of a CAR NK cells by knocking out the TGFβ receptor in a primary NK cell and additionally introducing a CAR coding sequence to the primary NK cell. The claim further states that this modification of the NK cell allows for improved cytotoxic function.
Breadth of the Claims: Claim 1 recited in its preamble “An in vitro method” with the intended use “for enhancing cytolytic activity of a NK cell towards a target tumor cell”. This recitation limits the claims to in vitro methods (i.e. in culture). The body of the claimed method in claim 1 has two active steps: 1) providing a primary NK cell; and 2) generating an engineered CAR NK cell expressing a CAR and having a knockout of the TGFβ receptor gene. The remainder of the claim recites “by” clauses that further specify how the engineered CAR NK cell is produced and “wherein” clauses that specify the functional activity that results from the production of the CAR, TGFβ-receptor-KO NK cell.
Claim 1 further recites, “by knocking out a TGFβ receptor gene…by cleavage of the gene with a TAL-nuclease or Cas9 RNA guided endonuclease encoded by an mRNA introducing into the NK cell”. The claim does not specify how the mRNA is introduced. As such, the breadth of the introduction encompasses any means of introducing including but not limited to electroporation, viral vector delivery, liposome, particle, etc… The claim also does not specify how structural elements of the TAL-nuclease or the Cas9 RNA guided nuclease. As such, the breadth of the TALEN or the Cas9 RNA guided nuclease is one that has any structure, particularly one that targets any nucleic acid sequence that will cause at least one TGFβ receptor to be expressed. This includes TALEN pairs and gRNA that directly target and cleave any sequence in the TGFβ receptor gene including sequences inside and outside the coding sequences. This also includes TALEN pairs and gRNAs that target gene sequences for knockout that are required for the expression of TGFβ receptor gene that when knockout would result in a lack TGFβ receptor.
The structure of the resultant cell is recited as “CAR NK cell expressing the CAR and having a knockout of the TGFβ receptor gene”. The claims does not specify if the CAR is incorporated into the genome of the NK cell. As such, the breadth of the NK cell encompasses one that has episomal existence of the CAR transgene or has the CAR transgene incorporated into its genome. While the claim recites “a knockout of the TGFβ receptor” it does not specify if the knockout is hemizygous or homozygous and does not specify if the knockout results in a full lack of TGFβ receptor expression or a partial expression or reduction of TGFβ receptor. As such, the breadth of the claims encompasses a hemi- or homo-zygous disruption of the TGFβ receptor gene in the genome of the NK cell
Specification Guidance:
The specification contemplates using the cells of the invention as a medicament for treating cancer, infections, GvHD (lines 7-10 and 19). The specification does not provide a definition of “cytolytic function” but only generically describes the CAR NK cells have cytolytic/cytotoxic activity in tumor cells or viral infections (p. 9-10). In Table 1, the specification provide guidance to specific polynucleotide sequences that code TALENs that target exon 2 of TGFβ receptor (SEQ ID NOS:2 and 3; and the targeting sequence (SEQ ID NO:1) for these TALENS. Table 1 also provides specific coding sequences for Beta2M (SEQ ID NOS:20-28). The specification also contemplates other genes involved in cytolytic activity to be inactivated (page 19). Table 2 also provide guidance to Cbl-b targeting TALENS (SEQ ID NOS4-9). This is one of the genes listed as having potential inactivation for its involvement in NK cell cytolytic activity. However, the specification does not provide any guidance to any other TALENs or targeting sequences or any other gene editing guidance.
Regarding Cas9 RNA guided nuclease, the specification generically contemplates its use [0079] but does not provide any specific guidance to any target sequences in the TGFβ receptor gene.
Working Examples:
Example 1 describes transfecting primary NK cells with mRNA encoding with GFP with about 79% of the cells being transfected as determined by FITC FACS analysis. Also see figure 1.
Example 2 describes transfection of primary NK cell with mRNA encoding a TALE-nuclease sequences of SEQ ID NOS:21 and 22 in order to inactivate the gene coding the beta2 microglobulin gene. After 72 hours of transfection, the NK cells were labeled with a beta2 microglobulin antibody and analyzed by FACS analysis. Figure 2 shows that about 11% of the cells were beta2 microglobulin knockout cells.
Example 3 describes a similar method of producing TGFβ receptor knockout NK cells as example 2 using mRNA encoding TALENS specific to target TGFβ receptor (SEQ ID NOS: 2 and 3). Example 4 describes a similar method of producing Cbl-b knockout NK cells as example 2. However, there is no reduction to practice associated with examples 3 and 4.
The specification, while broadly contemplating using any TAL-nuclease having any targeting sequence, solely provides guidance to one species of a TALEN pair targeting exon 2 of the TGFβ receptor gene. The specification fails to provide any guidance to any Cas9 RNA-guided endonucleases that can specifically indirectly or directly target the TGFβ receptor. As such, the specification does not enable the breadth of the claimed TAL-nuclease or RNA guided endonuclease. While contemplating any means of introducing an mRNA encoding the nuclease into the NK cell the specification solely provides specific guidance to electroporation of the mRNA that predictably transfects the NK cell and expression of a TALEN that cleaves the TGFβ receptor and results in enhanced cytolytic activity. Further, while the breadth of the claims encompasses hemizygous disruption of the TGFβ receptor gene that results in reduced expression of the TGFβ receptor, the specification solely provides specific guidance to “impairment of its surface membrane expression…Such inactivation resulted in enhancement of NK cytolytic functions toward target tumor cells” [0273] of the specification. It is further noted that the specification does not provide any guidance to the introduction of a CAR transgenes wherein the CAR transgene exist episomally an not integrated into the genome. Also the specification does not provide guidance to a heterozygous disruption of the TGFβ receptor or reduced expression of the TGFβ receptor as the breadth of the claims embraces.
State of the Art: As stated in the Declaration dated 4/11/2024, Carlsten et al 2015 shows the state of the art in NK cell engineering at the priority date (6/30/2015). In particular, this paper reflects the fact that directed gene inactivation had not been achieved in primary NK cells at that time. Based upon the inventors experience, the stat of the art [of] genetic modification of NK cells was very immature. See section 15-17. Carlsten supports the inventors conclusion by indicating that gene editing in primary NK cells had not been reported. Further Carsten states: Until relatively recently, the genetic manipulation of NK cells has proven challenging. Viral transduction, successfully used for T cells has been associated with low levels of transgene expression and unfavorable effect on cell viability when used with NK cell…Investigators around the world are now exploring the potential of multiple different cell modalities to genetically reprogram with the overall aim of further improving upon their capacity to kill tumor in cancer patients. See section 20. Carlsten further reports on page 6, “To date, studies utilizing CRISPR, ZFN, or TALEN to genetically modify NK cell to silence their inhibitor receptors for the dame purpose of enhancing the anti-tumor capacity of NK cells have not yet been reported.” Also see section 27 of the declaration.
As such, Carlsten while contemplating the use of gene editing for future improvement NK cell function in adoptive therapies, teaches that no gene edited NK cells were achieved at the time of effective filing. This is also consistent with the inventors understanding of the state of the art at the time of effectively filing. Post-filing art also describes continued obstacles to gene editing of NK cells. Afolabi et al. (Immunology 158:63-69, 2019) states, “The discovery and development of the CRISPR/Cas9 technology offer a flexible and efficient gene-editing capability in modulating various pathways that mediate NK cell exhaustion, and in arming NK cells with novel chimeric antigen receptors to specifically target tumour cells. Despite the high efficiency in its gene-editing capability, difficulty in the delivery of the CRISPR/Cas9 system remains a major bottleneck for its therapeutic applications, particularly for NK cells.” See abstract. “NK cells, particularly primary NK cells, are well known to resist ordinary transfection, making the delivery of theCas9 system difficult, and thus representing an important concern in gene-editing of NK cell immunotherapy.” See page 64, paragraph bridging col 1 and 2. Afolabi also reports that although NK cell are relatively resistant to viral transfection, CRISPR/Cas9-mediate genome-editing of human NK cells via electroporation has been reported in two studies post filing Lonza 4D nucleofector system. Paragraph bridging pp. 64-65. However, these two studies disclose new technology developed post-filing and do not enable at the time of effective filing. Afolabi also reports while gene-editing delivery via electroporation may be feasible, poor survival post-electroporation is another hurdle for NK cell gene editing. See p. 65, col 1. Afolabi further reports, “With the advances in CRISPR-base genome-editing, most concerns are on the off-target effects causing undesired modifications to the genome nucleic acids. Effectors have been made to improve the nuclease specificity and precise delivery of the CRISPR/Cas9 System, as well as developing strategies for superior g RNA design and off-target validation to minimize undesired genome-editing effects…In addition to technical barrier, the biggest challenge in NK cell immunotherapy for cancers is the lack of in-depth understanding of the underlying mechanisms of NK cell dysfunction, not only in tumours, but also during adoptive ex vivo expansion before adoptive transfer. “ As such, Afolabi demonstrates that at the time of filing and continuing post-filing effective means of delivering gene-editing nucleases such as CRISPR were hindered by NK cell resistance to viral delivery and post-electroporation cell viability. Additionally gene editing technologies themselves hinder gene editing of NK cells due off target effects causing undesired modification, a lack of specificity and reliable delivery. Additional use of gene-edited NK cells as a therapeutic is hindered by a lack of knowledge of the underlying mechanisms of NK cell dysfunction and expansion, bringing into question the predictability of making an improved therapeutic activity or enhancement of NK cytolytic activity as claimed.
Similarly the more recent post-filing art of Huang et al (Journal of Experimental Medicine 218(3). Pp. 1-20, 2021) reports, “Natural killer (NK) cells are potent immune effectors against cell malignancy, but they are challenging to modify genetically by conventional methods due to the toxicity of DNA when introduced into cells coupled with limited transfection and transduction efficiency”. See abstract. “Genetic engineering of NK cells is challenging using conventional methods. NK cells are highly sensitive and resistant to exogenous DNA that encodes the DNA modifications of interest. Retroviral transduction requires high viral titer and poses concerns of insertional mutagenesis and oncogenesis…. Lentiviral transduction is inconsistent for NK cells even at high multiplicity of infection…. Plasmid transfection has limited efficiency to express transgenes…. A more robust and precise genetic toolkit for NK cells is urgently needed.” See page 1, paragraph briding col 1 and 2. Huang further reports, “Recent advances in CRISPR genome editing technology create exciting new possibilities to genetically study NK cell immunology and engineer NK cells to enhance anticancer capabilities. In this work, we demonstrate a robust CRISPR genome editing platform for primary human NK cells that combines the advantages of feeder-free ex vivo expansion and Cas9 RNP nucleofection to replace inefficient plasmid transfection and viral transduction. There are many opportunities and challenges to extend our proof-of-concept demonstrations toward therapeutic applications of NK cell.” As such, Huang teaches that they have provided new technology around 6 years post-filing that begins to improve the ability to gene edit NK cells but still reports that challenges still remain for therapeutic application of gene edited NK cells, let alone “improving therapeutic activity” as claimed.
As such, the state of gene knock out in NK cells at the time of effective filing was that it had not been established and was hindered by multiple obstacles of to it success. These unpredictability continues well into the post-filing literature until new technologies and unique approaches appear to be advances the state of the art. However, even them the post-filing art teaches that unpredictability persist especially to provide a NK cell of adoptive immunotherapy to treat cancer and to predictable therapeutic phenotypes such as enhanced cytolytic activity capable of a therapeutic effect.
As such, the instant claims lack enablement because the specification while providing some building blocks, such as TALEN development for TGFβ receptor knockout, fall short of providing predictable means of delivering gene editing nuclease, to a CAR NK cell, as well as providing guidance to a means of developing a therapeutic CAR NK cell comprising a TGFβ receptor knockout or said cell with enhanced cytolytic activity. Further, the art at the time of effective filing teaches that no gene-edited NK cells had been reported and that NK cell gene editing as highly unpredictable and had many challenges in gene editing nuclease delivery, NK cell expansion, challenges in gene editing technology itself, as well as hinderances to producing a therapeutic NK cell that would predictable be used in treating cancer. The specification does not provide guidance to means of overcoming these art describe obstacles. As such, neither the specification nor the art provide a predictable means of making and use the claimed method.
Therefore at the time of filing the skilled artisan would need to perform an undue amount of experimentation without a predictable degree of success to implement the invention as claimed.
Response to Arguments
Applicant's arguments filed 11/12/2025 have been fully considered but they are not persuasive.
Applicant submits that the amendment address all the cited deficiencies by the previous office action and therefore is enabled. In response, while the amendments do address many of the issues of enablement they do not address all of the them. In particular, the use of cytopulse electroporation, the use of a Cas/CRISPR system, and targeting any exon in the TGFβ receptor gene. The specification fails to provide specific guidance for the broader embodiments and the prior art provide extensive teachings regarding the unpredictability of gene editing NK cells, particularly with Cas/CRISPR systems. As such, issue of enablement still remain and the rejection is maintained.
The following new objection is necessitated by the amendments to the claims:
Claim Objections
Claims 1, 7, 8, and 13 are objected to because of the following informalities: The amendment to the claims recite “TGFBRII”. This is an abbreviation for “transforming growth factor β receptor type II”. When first reciting an abbreviation, the term must be spelled out followed by the abbreviation in parentheses. Appropriate correction is required.
No claims are allowed.
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 MARCIA STEPHENS NOBLE whose telephone number is (571)272-5545. The examiner can normally be reached M-F 9-5:30.
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MARCIA S. NOBLE
Primary Examiner
Art Unit 1632
/MARCIA S NOBLE/Primary Examiner, Art Unit 1632