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and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114.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 11/03/2025 has been acknowledged. Applicant's amendment and response filed on 09/30/2025 has been received and entered into the case.
Amendments
In the reply filed on 09/30/2025, Applicant has amended claim 116.
Claim Status
Claims 116-117 and 119-134 are pending.
Claims 124-125 and 128-129 have been withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to non-elected inventions, there being no allowable generic or linking claim. Election was made with traverse in the reply filed on 05/04/2023.
Claims 116-117, 119-123, 126-127 and 130-134 are considered on the merits.
Withdrawn Claim Rejections - 35 USC § 103
The prior rejections of claims 116-117, 119-123, 126-127 and 130-134 under 35 U.S.C. 103 set forth in the prior Office action mailed on 07/01/2025 are withdrawn in light of Applicant’s amendment to claim 116 to recite new limitation wherein the CAR polypeptide comprises “a NKG2D transmembrane”, which is not taught by the cited art.
New 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:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. 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 116, 119-123, 126-127, 130 and 133 are rejected under 35 U.S.C. 103 as being unpatentable over Kararoudi et al., (bioRxiv preprint doi: https://doi.org/10.1101/743377; published August 22, 2019. Downloaded on 2/2/2024. P. 1-16. Prior art of record) in view of Tang et al., (Am J Cancer Res. 2018;8(6):1083-1089. Prior art of record) and Li et al., (Cell Stem Cell. 2018; 23: 181-192, cited in IDS 07/26/2022).
With respect to claim 116, Kararoudi teaches a method of site-directed gene insertion in primary human natural killer cells, thus teaches the preamble of claim 116 a method of genetically modifying a primary natural killer (NK) cell.
In regard to step a) introducing an RNP into the NK cell, Kararoudi teaches the NK cells are electroporated with Cas9/RNP targeting AAVS1 (p. 5, para 2), and teaches the pre-complexed Cas9/RNP comprises Cas9 nuclease (S.p. HiFi Cas9 Nuclease V3) and one guide RNA comprising a CRISPR-Cas9 crRNA and a CRISPR-Cas9 tracrRNA (p. 3, last full para), in which the crRNA comprises a sequence targeting AAVS1 site in NK cells (p. 3, para 2). Thus, Kararoudi teaches step a) introducing an RNP complex comprising a class 2 CRISPR/Cas endonuclease (i.e., Cas9) complexed with a CRISPR/Cas guide RNA into the NK cell, wherein the CRISPR/Cas guide RNA comprises a crRNA comprising a nucleotide sequence that is complementary to a target sequence within the genomic DNA of the NK cell.
In regard to step b) subsequently infecting the NK cell with an AAV, Kararoudi teaches thirty minutes after electroporation, the NK cells are collected, resuspended and transduced with AAV (p. 5, para 2). Kararoudi teaches the AAV comprises an AAV vector (scAAV6) to deliver CRISPaint DNA encoding a gene of interest such as mCherry (p. 5, para 2), and teaches in the CRISPaint DNA templates, a single (PAMg) or double (PAMgPAMg) Cas9-targeting sequences are incorporated around the mCherry transgene (p. 4, last para). Thus, Kararoudi teaches step b) subsequently infecting the NK cell with an AAV comprising an AAV vector comprising a polynucleotide sequence encoding a gene of interest, wherein the polynucleotide sequence is adjacent to one PAM and one polynucleotide sequence encoding the crRNA (i.e., a PAMg), or flanked by two PAMs and two polynucleotide sequences encoding the crRNAs (i.e., PAMgPAMg, also see Fig 9b). It is also noted that Fig 9a indicates the polynucleotide sequence encodes a CAR as the gene of interest.
In regard to the NK cell being incubated between 3 to 7 days with IL-2 or IL-21 post-infection with the AAV, Kararoudi teaches “[t]he day after electroporation and transduction, we added 150ul of fresh media containing 100 IU of IL2 … . The cells were kept in culture for 48 hours after electroporation and were then restimulated with K562 feeder cells” (p. 5, para 2. Note that these K562 feeder cells are irradiated mbIL21-expressing K562 feeder cells, see e.g., p. 6, 1st para), thus teaches the NK cells are incubated with IL-2 or IL-21 post-infection with AAV. Regarding the incubation time, Kararoudi teaches in Figure 14 that “[w]e electroporated NK cells with Cas9/RNP and transduced with 300K MOI or 150K MOI of ssAAV or scAAV6 delivering HR or CRISPaint DNA template. Six days post CRISPR modification, flow cytometry results show that…” (see Figure 14 legend in page 14), thus teaches the NK cells are incubated between 3 to 7 days (e.g., 6 days) post-infection with the AAV.
In regard to the RNP complex hybridizing to the target genome sequence and the polynucleotide being inserted into the genome, Kararoudi teaches the RNP complex hybridizes to the target sequence within the genomic DNA of the NK cell, and DNA repair enzymes of the NK cell insert the polynucleotide encoding the gene of interest into the genome of the NK cell at the target sequence thereby creating a modified NK cell (see Fig 9a showing the process of gene integration into genome of the NK cell, and see p. 5, para 1 for the DNA repair enzyme, LIG-4, in the NK cell that mediates NHEJ-directed gene insertion through CRISPaint). It is also noted that Fig 9a indicates the polynucleotide sequence encodes a CAR as the gene of interest.
However, Kararoudi teaches the genetic material in the site-directed insertion into the NK cells is a polynucleotide encoding mCherry (abstract and e.g., p. 5, para 2), but does not specifically teach a polynucleotide encoding a CAR in the working example, nor teach the structure of the CAR.
Nevertheless, regarding genetically modifying NK cells with a CAR, Kararoudi teaches “modifying NK cells with a chimeric antigen receptor (CAR) can improve their targeting and increase specificity. However, genetic modification of NK cells has been challenging due to the high expression of innate sensing mechanisms for viral nucleic acids” (abstract), and teaches “we successfully generated highly efficient and stable transgene-modified NK cells using mCherry as a proof of concept” (p. 2, last sentence). Furthermore, as stated supra, Kararoudi teaches in Fig 9a a polynucleotide sequence encoding a CAR is site-specifically inserted into the genome of the NK cells.
Tang teaches a method of genetically modifying an NK cell by introducing a third generation CD33-CAR vector into the NK cell for cancer immunotherapy in patients in a clinical trial (abstract and Fig 1 legend. The clinical trial identification number is NCT02944162, see p. 1084, right col, para 1). Tang teaches the anti-CD33 CAR polypeptide comprises a single-chain variable fragment (scFV) that specifically binds to a CD33 receptor on a target tumor cell (see Fig 1A for diagram and Fig 1C for specific lysis of CD33-expressing tumor cells), and teaches the structure of the CAR is a third generation CAR lentiviral construct containing both CD28 and 4-1BB costimulatory molecules and a CD3ζ signaling domain (Fig 1 legend and p. 1084, left col, last para). Tang teaches the anti-CD33 CAR transduced NK cells effectively treat human patients with relapsed and refractory AML (abstract, p. 1085-1086, see Fig 2).
Li acknowledges that the clinical trials using CAR-expressing NK cells, e.g., NCT02944162 (i.e., the clinical trial of Tang) uses CARs designed for T cells and not optimized for NK cell signaling (p. 182, left col, para 2), therefore evaluates different CAR constructs comprising NK cell activation domains on NK cell-mediated killing (p. 182, left col, para 3, and abstract). Li identifies a CAR containing the transmembrane domain of NKG2D, the 2B4 co-stimulatory domain, and the CD3ζ signaling domain to mediate strong antigen-specific NK cell signaling (abstract, see Fig 1B and Fig 2). Li teaches NK cells expressing this CAR demonstrate improved antitumor activity, significantly inhibited tumor growth and prolonged survival compared with T-CAR-expressing NK cells (abstract, see Fig 3B for in vitro and Fig 4 for in vivo results).
Therefore, it would have been obvious for one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of genetically modifying an NK cell with CRISPR/Cas9 mediated targeted integration of a gene-of-interest (e.g., mCherry as a proof of concept) disclosed by Kararoudi, by substituting the mCherry with an anti-CD33 CAR as suggested by Kararoudi and taught by Tang with a reasonable expectation of success. Since Kararoudi suggests modifying NK cells with a CAR can improve their targeting and increase specificity, and further reduces to practice a method of generating highly efficient and stable transgene-modified NK cells (abstract, p. 2, last sentence, also see Fig 9a), and since Tang teaches the anti-CD33 CAR transduced NK cells effectively treat human patients with relapsed and refractory AML (abstract, p. 1085-1086, see Fig 2), one of ordinary skill in the art would have had a reason to substitute the reporter gene with Tang’s anti-CD33 CAR in the method of Kararoudi in order to improve targeting and specificity of the NK cells to tumor cells to effectively treat human patients with relapsed and refractory AML (Kararoudi, abstract).
Furthermore, one of ordinary skill in the art would have substituted the third generation CAR construct of Tang with an NK-CAR containing the transmembrane domain of NKG2D, the 2B4 co-stimulatory domain, and the CD3ζ signaling domain taught by Li with a reasonable expectation of success. Since Li acknowledges that Tang’s CAR is designed for T cells but not optimized for NK cell signaling and teaches NK cells expressing this NK-CAR demonstrate improved antitumor activity, significantly inhibited tumor growth and prolonged survival compared with T-CAR-expressing NK cells (see above), one of ordinary skill in the art would have had a reason to substitute with Li’s NK-CAR construct combining Tang’s anti-CD33 scFV to generate an anti-CD33 NK-CAR-modified NK cell in order to take the advantages of improved antitumor activity, significantly inhibited tumor growth and prolonged survival as suggested by Li.
With respect to claim 119 directed to the primary NK cell being incubated for about 4 to 10 days in the presence of IL-2 prior to infection or the primary NK cell being expanded for about 4 to 10 days in the presence of irradiated feeder cells prior to infection, and claim 120 directed to the irradiated feeder cells expressing membrane-bound IL-21, Kararoudi teaches NK cells are isolated from PBMC (i.e., primary NK cells) and are stimulated and cultured with irradiated membrane-bound IL-21 (mbIL-21)-expressing K562 feeder cells for 7 days in expansion medium containing IL-2 before electroporation (p. 3, 1st para, i.e., expanded NK cells).
With respect to claim 121 directed to the method further comprising expanding the modified NK cell with IL-2 or with irradiated feeder cells following infection wherein the irradiated feeder cells express membrane-bound IL-21, as stated supra, Kararoudi teaches the day after electroporation and transduction, fresh media containing IL-2 is added to the cells (p. 5, para 2), and the cells are continued expanding using irradiated mbIL21-expressing feeder cells (p. 6, 1st para).
With respect to claim 122 directed to the NK cell being infected with about 5 to 500K MOI of the AAV, Kararoudi teaches the electroporated NK cells are transduced with 300K or 150K MOI of AAV and alternatively with 500K MOI of AAV (p. 5, para 2).
With respect to claim 123 directed to the RNP complex being introduced into the NK cell via electroporation, as stated supra, Kararoudi teaches the NK cells are electroporated with Cas9/RNP complex (p. 5, para 2).
With respect to claim 126 directed to the AAV comprising serotype AAV6, as stated supra, Kararoudi teaches transducing the cells with several serotypes of AAVs and teaches that NK cells transduced with AAV6 have the highest expression level of gene of interest and the AAV6 viral genome could be detected up to 48 hours post-transduction, which is critical for the endonuclease function of Cas9 protein (p. 4, para 2). Thus, Kararoudi teaches the AAV comprising serotype AAV6.
With respect to claim 127 directed to the AAV vector being ssAAV or scAAV, Kararoudi test both ssAAV and scAAV for DNA template delivery into NK cells (p. 2, para 1) and teaches using scAAV6 to deliver CRISPaint (p. 5, para 2).
With respect to claim 130 directed to the class 2 CRISPR/Cas endonuclease being a Cas9 endonuclease, as stated supra, Kararoudi teaches the RNP complex comprises a Cas9 nuclease (S.p. HiFi Cas9 Nuclease V3) (p. 3, last full para).
With respect to claim 133 directed to the polynucleotide sequence being flanked by two PAMs and two polynucleotide sequences encoding the crRNA, as stated supra, Kararoudi teaches in the CRISPaint DNA templates, a double (PAMgPAMg) Cas9-targeting sequences are incorporated around the transgene (p. 4, last para, see Fig 9b), thus teaches the polynucleotide sequence being flanked by two PAMs and two polynucleotide sequences encoding the crRNA.
Hence, the claimed invention as a whole was prima facie obvious to a person of ordinary skill before the effective filing date of the claimed invention in the absence of evidence to the contrary.
Response to Traversal:
Applicant’s arguments filed on 09/30/2025 are acknowledged.
Applicant argues that neither cited art in the prior Office action discloses the new limitation that the CAR polypeptide having a NKG2D transmembrane domain following an anti-CD33 scFv domain, thus it would not have been obvious to one of skill in the art to generate a CD33CAR NK cell, wherein the CAR polypeptide contains a NKG2D transmembrane domain following an anti-CD33 scFv (Remarks, p. 8-9).
Applicant’s arguments have been fully considered and they are persuasive. Therefore, the prior rejection has been withdrawn. However, as necessitated by amendment, a new ground of rejection has been made over Kararoudi, Tang and Li. Specifically, Li teaches an NK-CAR having a NKG2D transmembrane domain, a 2B4 co-stimulatory domain and a CD3ζ signaling domain, and the NK-CAR-expressing NK cells show improved anti-tumor activity compared with T-CAR-expressing NK cells.
Claim 117 is rejected under 35 U.S.C. 103 as being unpatentable over Kararoudi et al., (bioRxiv preprint doi: https://doi.org/10.1101/743377; published August 22, 2019. Downloaded on 2/2/2024. P. 1-16. Prior art of record) in view of Tang et al., (Am J Cancer Res. 2018;8(6):1083-1089. Prior art of record) and Li et al., (Cell Stem Cell. 2018; 23: 181-192, cited in IDS 07/26/2022), as applied to claim 116 above, and further in view of Suzuki et al., (Nature. 2016; 540: 144-149 and Extended Data Figure 1. Cited in IDS 07/26/2022).
Claim 117 is directed to an order PAM-crRNA-CAR-PAM-crRNA.
As stated supra, Kararoudi teaches in the CRISPaint DNA templates, a double (PAMgPAMg) Cas9-targeting sequences are incorporated around the transgene (p. 4, last para, see Fig 9b). Regarding the order of the element arrangement, on one hand, Kararoudi teaches the double targeting sequences as “PAMgPAMg”, indicating the order is likely as a first PAM and a first crRNA sequence (i.e., a first PAMg), the reporter, and a second PAM and a second crRNA sequence (i.e. a second PAMg), which is the claimed order. On the other hand, Kararoudi also illustrates the CRISPaint vector containing two Cas9 targeting sites in Fig 9b as PAM-crRNA-mCherry-crRNA-PAM, a different order as claimed.
Thus, Kararoudi does not clearly teach the claimed order.
Nevertheless, Kararoudi clearly suggests it is preferable to obtain a fragment of crRNA-CAR-PAM after Cas9 cleavage (see attached Fig 9a lower row, 2nd left panel), because this will ensure successful gene insertion in the forward direction that cannot be recut by Cas9 thus results in stable gene integration (see Fig 9a right part, middle row), while either the gene integration in reverse direction (Fig 9a right part, upper row), or genome repair without gene integration (Fig 9a right part, lower row), undergoes additional cleavage by Cas9 to re-generate double-stranded break in genome DNA and the fragment of crRNA-CAR-PAM (illustrated by the arrows in the modified Fig 9a attached). This will repeat until the Cas9-uncleavable, correct forward insertion is obtained (i.e., Fig 9a right part, middle row).
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It is noted that this donor template (crRNA-gene of interest-PAM) can only be obtained when the double targeting sequences are arranged as PAM--crRNA-Gene of interest-PAM--crRNA, since the cutting sites of Cas9 are around the junction between PAM and crRNA (referred to by “--"). Furthermore, Kararoudi refers to reference #4, for integrating CRISPaint donor template using non-homology repair machinery (Ref. #4, p. 2, lines 2-3 from bottom).
Suzuki, being the reference #4 of Kararoudi, teaches a method of genome editing via CRISPR/Cas9 mediated homology-independent targeted integration (HITI, abstract). Suzuki teaches an HITI donor template comprising two CAS9/gRNA target sequences flanking the sequence of gene-of-interest (IRESmCherry-2c, note that “2c” refers to 2-cut by Cas9 so that the inserted DNA is devoid of bacterial backbone) has high knock-in efficiency and less pronounced transgene silencing than DNA carrying bacterial sequences (p. 144, right col, para 1, see Fig 1a). Suzuki teaches the donor template comprising two target sequences (“2cut”) is arranged as PAM-crRNA-Gene Of Interest-PAM-crRNA (see attached modified Extended Data Figure 1 below, upper right panel, note that the PAM site (underlined “NGG”) is on the triangle side of the blue blocks, and the crRNA site is on the rectangle side of the blue blocks, the black line in the blue blocks refers to the Cas9 cutting site), which facilitates gene integration in the correct forward direction (lower left panel), because the reverse direction integration (lower right panel) results in Cas9 re-cut that regenerates genome break and donor fragment, similar to the suggestion of Kararoudi (Kararoudi, Fig 9a, see above).
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Therefore, it would have been obvious for one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of genetically modifying an NK cell with homology-independent targeted integration using a donor template comprising two targeting sequences flanking the CAR sequence suggested by Kararoudi in view of Tang and Li, by choosing an arranging order as PAM-crRNA-CAR-PAM-crRNA as suggested by Kararoudi and taught by Suzuki with a reasonable expectation of success. Since Kararoudi suggests a fragment of crRNA-CAR-PAM is preferable to ensure gene integration in the correct forward direction (see Fig 9a attached and discussion above), which can only be obtained by arranging the elements as PAM-crRNA-CAR-PAM-crRNA, and since Suzuki teaches the donor template comprising 2 cuts is arranged as PAM-crRNA-Gene Of Interest-PAM-crRNA that results in high knock-in efficiency in the correct direction and less pronounced transgene silencing (p. 144, right col, para 1, see Fig 1a, and modified Extended Data Figure 1 attached above), one of ordinary skill in the art would have had a reason to choose the order of PAM-crRNA-CAR-PAM-crRNA as suggested by Kararoudi and taught by Suzuki in order to ensure gene integration in the correct forward direction and reduce transgene silencing (Kararoudi, Fig 9a and Suzuki, p. 144, right col, para 1).
Hence, the claimed invention as a whole was prima facie obvious to a person of ordinary skill before the effective filing date of the claimed invention in the absence of evidence to the contrary.
Response to Traversal:
Applicant’s arguments filed on 09/30/2025 are acknowledged and have been discussed above.
Claims 119-121 and 131-132 are rejected under 35 U.S.C. 103 as being unpatentable over Kararoudi et al., (bioRxiv, doi: https://doi.org/10.1101/743377; published August 22, 2019. Downloaded on 2/2/2024. P. 1-16. Prior art of record) in view of Tang et al., (Am J Cancer Res. 2018;8(6):1083-1089. Prior art of record) and Li et al., (Cell Stem Cell. 2018; 23: 181-192, cited in IDS 07/26/2022), as applied to claim 116 above, and further in view of Oyer et al., (Cytotherapy. 2016; 18: 653–663. Prior art of record).
As stated supra, Kararoudi teaches the primary NK cell is expanded for about 4 to 10 days with irradiated feeder cells expressing membrane bound IL-21 prior to infection (p. 3, 1st para), and teaches expanding the modified NK cell following infection with irradiated feeder cells expressing membrane bound IL-21 (p. 6, 1st para).
However, Kararoudi, Tang and Li do not teach the expansion is with plasma membrane particles (as the elected species by Applicant in the reply filed on 05/04/2023) expressing membrane bound 4-1BBL and membrane-bound IL-21 in claims 119-121 and 131-132.
Oyer teaches a method of expanding NK cells with plasma membrane (PM) particles expressing membrane bound 4-1BBL and membrane-bound IL-21 (PM21 particles, abstract). Oyer teaches PM21 particles are prepared from plasma membranes of K562-mb21-41BBL cells expressing 41BBL (note that 4-1BBL is a membrane protein) and membrane-bound IL-21, and teaches presence of IL-21 and 41BBL on PM particles is confirmed by enzyme-linked immunosorbent assay and Western blot (abstract, p. 655, left col, para “Preparation and characterization of plasma membrane particles). Oyer teaches PM21 particles specifically expand NK-cell ex vivo from PBMCs from healthy donors and patients with AML (p. 656), and NK cells stimulated with PM21 particles expanded exponentially, reaching more than 100,000 fold expansion over the period of 28 days (p. 656, Fig 1). Importantly, the NK cells expanded by this method biodistribute out from the abdominal cavity to peripheral blood and multiple organs that are potential sites of various other cancers such as marrow, spleen, lung, liver and brain (p. 659, Fig 5), while in contrast, long-term ex vivo culturing of NK cells with feeder cells causes loss of ability to home to the site of disease, such as bone marrow (p. 654, left col, para 3) and the in vivo persistence of the feeder-cell-expanded NK cells is not optimal (p. 660, section “Discussion”, para 1).
Therefore it would have been obvious for one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of genetically modifying an NK cell comprising expanding the NK cell with feeder cells expressing membrane-bound IL-21 prior to and following infection suggested by Kararoudi in view of Tang and Li, by substituting the feeder cells with plasma membrane particles expressing membrane-bound 4-1BBL and membrane-bound IL-21 as taught by Oyer with a reasonable expectation of success. Since Oyer teaches ex vivo expanding NK cells with feeder cells causes loss of ability to home to the site of disease such as bone marrow and has less optimal in vivo persistence (p. 654, left col, para 3 and p. 660, section “Discussion”, para 1), while NK cells expanded ex vivo with plasma membrane particles expressing membrane-bound 4-1BBL and membrane-bound IL-21 obtain exponential expansion to 100,000 fold over 28 days (p. 656, Fig 1), have high in vivo persistence (p. 657-658, Figs 2-4), and biodistribute to peripheral blood and multiple organs that are potential sites of various cancers such as marrow, spleen, lung, liver and brain (p. 659, Fig 5), one of ordinary skill in the art would have had a reason to substitute with plasma membrane particles as taught by Oyer in the method of Kararoudi in view of Tang and Li in order to obtain a large number of gene-modified NK cells at clinical scale and achieve biodistribution to the sites of diseases with high in vivo persistence to treat cancer (Kararoudi, 1st para).
Hence, the claimed invention as a whole was prima facie obvious to a person of ordinary skill before the effective filing date of the claimed invention in the absence of evidence to the contrary.
Response to Traversal:
Applicant’s arguments filed on 09/30/2025 are acknowledged and have been discussed above.
Claim 134 is rejected under 35 U.S.C. 103 as being unpatentable over Kararoudi et al., (bioRxiv preprint doi: https://doi.org/10.1101/743377; published August 22, 2019. Downloaded on 2/2/2024. P. 1-16. Prior art of record) in view of Tang et al., (Am J Cancer Res. 2018;8(6):1083-1089. Prior art of record) and Li et al., (Cell Stem Cell. 2018; 23: 181-192, cited in IDS 07/26/2022), as applied to claim 116 above, and further in view of Lee et al., (US PGPub No. 2018/0163176. Cited in IDS 07/26/2022).
Claim 134 is directed to the polynucleotide encoding the scFv comprising at least 70% sequence identity to SEQ ID NO: 29.
However, Kararoudi, Tang and Li are silent on the sequence of the anti-CD33 scFv that comprises at least 70% sequence identity to SEQ ID NO: 29.
Lee teaches an NK cell genetically modified with a CAR for treating cancer that results in greater than 80% cytotoxicity following treatment (abstract, see Example 3 for an anti-CD33 CAR modified NK cell). Lee further teaches the CAR targets a tumor-associated antigen such as CD33 (see Lee claim 48) and teaches the CD33 CAR polynucleotide being Lee’s SEQ ID NO: 10 ([0078]). The sequence (nucleotide 48-812) in Lee’s SEQ ID NO: 10 comprises 626 nucleotides identical to the instant SEQ ID NO: 29 (which consists of 801 nucleotides). Thus, the overall sequence identity between Lee’s SEQ ID NO: 10 (nucleotide 48-812) and the instant SEQ ID NO: 29 is 78% (626/801=0.78), with a best local similarity of 81% (see SCORE search 06/19/2025, “20250618_152511_us-17-511-187-29.rni” file, result #1).
Therefore, it would have been obvious for one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of genetically modifying an NK cell with an anti-CD33 CAR comprising a scFv that specifically binds to CD33 suggested by Kararoudi in view of Tang and Li, by choosing a polynucleotide encoding the scFv comprising at least 70% sequence identity to SEQ ID NO: 29 as taught by Lee with a reasonable expectation of success. Since Tang acknowledges that changes in antigen binding can have a large impact on the effectiveness of CAR-mediated therapy and suggests enhancing CD33-CAR binding will improve the efficacy of the CAR NK cells (p. 1087, end of left column to the beginning of the right column), and since Lee reduces to practice the polynucleotide sequence of anti-CD33 CAR and teaches the anti-CD33 CAR NK cells have greater than 80% cytotoxicity against NK-resistant tumor cell lines (see Example 3), one of ordinary skill in the art would have had a reason to choose the polynucleotide encoding the scFv that comprises at least 70% sequence identity to instant SEQ ID NO: 29 as taught by Lee in order to obtain high cytotoxicity against tumor cells.
Hence, the claimed invention as a whole was prima facie obvious to a person of ordinary skill before the effective filing date of the claimed invention in the absence of evidence to the contrary.
Response to Traversal:
Applicant’s arguments filed on 09/30/2025 are acknowledged and have been discussed above.
Conclusion
No claims are allowed.
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/JIANJIAN ZHU/Examiner, Art Unit 1631