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 .
1. The Amendment filed October 6, 2025 in response to the Office Action of April 7, 2025, is acknowledged and has been entered. Claims 1-4, 6, 8-10, 12-15, 17-24 are pending and being examined. Claims 5, 7, 11, and 16 are canceled. Claims 1, 12, 13 are amended. Claims 1-4, 6, 8-10, 12-15, 17-24 are being examined as drawn to the elected species of:
B. Claim 6, host cell further comprising a second targeted integrated exogenous nucleic acid SOI encoding a second polypeptide of interest and a second selection marker…; and
C. hamster host cell (claim 9).
New Rejections
(necessitated by amendments)
Claim Rejections - 35 USC § 112
The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
2. Claims 1-4, 6-10, and 12 are rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention. Claim 1 recites a targeted integrated exogenous nucleic acid sequence of interest (SOI) encoding a first polypeptide of interest and a first selection marker in part a), and a randomly integrated exogenous nucleic acid SOI encoding a second polypeptide of interest and a second selection marker in part b). Claim 1 concludes with the phrase “wherein the targeted integration of the SOI and selection markers are promoted by an exogenous nuclease”, where the claim identifies a singular SOI and plural selection markers, therefore it is unclear which SOI and which markers the phrase is referring back to.
Examiner Suggestion: Amend the last phrase of claim 1 to recite: “wherein the targeted integrated exogenous nucleic acid SOI encoding a first polypeptide of interest and a first selection marker was integrated within the targeted locus by an exogenous nuclease.”
3. Claims 13-15 and 17-24 are rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention. Claim 13 recites the limitation "d) introducing into the host cells provided in (a) a nucleic acid…”. There is insufficient antecedent basis for this limitation in the claim because there is no part (a). It appears Applicants changed the lettering of parts without indicating such an amendment.
Examiner Suggestion: Amend claim 13 to change parts c) – i) to (a) – (g).
Maintained Rejection
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.
4. Claim(s) 13-15 and 17-24 remain rejected under 35 U.S.C. 103 as being unpatentable over Gaidukov et al (Nucleic Acids Research, May 4, 2018, 46:4072-4086) and Supplemental Information.
Gaidukov teaches a CHO-K1 cell capable of expressing a polypeptide of interest, the cell comprising two integration sites (“landing pads” or LPs) at different loci (i.e., cell clones “dLP1” and “dLP2”), that are integration sites for exogenous nucleic acids, wherein:
(a) the first integration site comprises an integrated exogenous nucleic acid (LP2-LA) encoding a first polypeptide (i.e., EYFP fluorescent protein) and a first selection marker (i.e., Hygro = hygromycin antibiotic resistance), flanked by two recombination recognition sites (RRS) (i.e., att), wherein the integrated exogenous nucleic acid sequence is integrated within a targeted locus genome of the CHO cell; and
(b) the second integration site comprises an integrated exogenous nucleic acid (LP15-LA) encoding a second polypeptide (i.e., EBFP fluorescent protein) and a second selection marker (i.e., Bla = blasticidin antibiotic resistance). See Materials and Methods and Supplementary Figure S8 below.
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Gaidukov teaches these LPs, or integration sites at loci, were found by random integration of exogenous genes and screening for loci that supported stable-long term expression of the exogenous genes. Gaidukov constructed LPs at these loci for targeted gene integration/exchange. Therefore, the LPs taught by Gaidukov are inherently both random integration sites and targeted integration sites. Claim 1 recites that the integration sites were “targeted” or “randomly” integrated, however, the process by which the exogenous nucleic acid sequences were integrated into the genome does distinguish the claimed host cell and integrated exogenous nucleic acid sequence from the prior art host cell and integrated exogenous nucleic acids. MPEP 2113 states: The patentability of a product does not depend on its method of production. If the product in the product-by-process claim is the same as or obvious from a product of the prior art, the claim is unpatentable, even though the prior product was made by a different process.
Gaidukov further teaches integrating a single exogenous sequence (“mAb payload”) encoding a light chain (LC) and heavy chain (HC) of a monoclonal antibody (polypeptide of interest) and Puro (Puromycin antibiotic resistance) selection marker, into the two different LP integration sites comprising LP2-LA and LP15-LA (Figure 3):
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Therefore, Gaidukov produced a CHO-K1 host cell comprising:
a) an integrated exogenous nucleic acid sequence encoding a first monoclonal antibody heavy and light chain, and a first Puro selection marker flanked by two RRS, wherein the integrated exogenous nucleic acid sequence is integrated within a targeted locus of the genome of the host cell; and
b) an integrated exogenous nucleic acid sequence encoding a second same monoclonal antibody heavy and light chain and second same Puro selection marker, that is integrated at least once in the genome of the host cell (Figure 3).
With regard to claim 6, Gaidukov teaches a CHO-K1 host cell comprising three LPs (three integration sites) (i.e., “tLP1” clone). The tLP1 clone was produced by adding an LP2-LA LP cassette to the biLP20 clone that already comprised two separate integration site cassettes named “LP20-LA”. The three LPs each comprise integrated exogenous nucleic acid sequences encoding a fluorescent protein (wherein LP20-LA and LP-20 LA encode the same protein) and selectable marker of Bla or Hygro (wherein LP20-LA and LP-20 LA encode the selectable marker), flanked by RRS attL and attR. See Materials and Methods and Supplemental Figures S8 and S9A:
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Gaidukov further teaches integrating a single exogenous sequence encoding a light chain (LC) and heavy chain (HC) of a monoclonal antibody (polypeptide of interest) and Puro (Puromycin antibiotic resistance) selection marker, into the three different LP integration sites. See Supplementary Figure S11 and Figure S12B:
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The host cells taught by Gaidukov and summarized above comprise two or three integrated exogenous nucleic acid sequences.
Gaidukov teaches targeted integration and selection of markers promoted by an exogenous nuclease CRISPR/Cas9 (p. 4074, col. 2 to p. 4075, col. 1, “Landing pads construction by homologous recombination with CRISPR/Cas9”).
Gaidukov further teaches a method comprising:
(a) providing a CHO-K1 host cell comprising one, two, or three LPs, the LPs comprising an exogenous nucleotide sequence integrated at a targeted locus of the genome of the host cell, wherein the exogenous nucleotide sequence comprises two RRSs (att) flanking a first selection marker of Hygro or Bla; wherein the LPs were integrated by exogenous nuclease CRISPR/Cas9;
(b) introducing into the host cell a “payload” nucleic acid comprising matching RRSs flanking a first exogenous nucleic acid sequence encoding a monoclonal antibody heavy chain and light chain and a second selection marker of Puro;
(c) introducing a recombinase BxB1 that recognizes the att RSSs;
(d) selecting for host cells expressing the second Puro selection marker; and culturing the selected host cells to express and recover the monoclonal antibody (see rejection above, Materials and Methods, and Figure 3 above).
With regard to claims 23 and 24, Gaidukov teaches the expression of the exogenous nucleotide sequences are controlled by regulatable promoters including hEF1a and CMV (Figures 1-4; Supplemental Figures S4A, S8, S9).
Gaidukov does not teach the method further comprising:
(e) introducing via random integration, a second exogenous nucleic acid sequence encoding the monoclonal antibody heavy chain and light chain and a third selection marker into the genome of the host cell;
(f) selecting for cells expressing the third selection marker; and
(g) culturing the host cell under conditions sufficient to express the first and second polypeptides.
However, Gaidukov does teach and successfully demonstrate known methods for introducing exogenous nucleic acid sequences encoding polypeptides and selectable markers by random integration into CHO-K1 cells, selecting the cells by their marker, and culturing the cells to express the protein (Figure 1). Gaidukov successfully performed this method to identify loci that supported stable long-term expression of exogenous genes encoding antibodies. Gaidukov identified 21 genomic sites using this method and produced CHO cells with multiple LPs (integrated exogenous nucleic acid sequences encoding polypeptide and selectable marker) located at two or three of these identified sites (abstract; p. 4074, col. 1-2; p. 4076, col. 1-2; Table 1; Figures 1-4).
Gaidukov also teaches and successfully demonstrates:
(a) providing a CHO cell clone “biLP20” already comprising two LP integration site cassettes “LP20-LA” wherein each LP comprises an exogenous sequence encoding a first fluorescent protein EYFP and first selectable marker Hygro,
(b) adding a third LP integration site cassette “LP2-LA” to the host cell that comprises an exogenous sequence encoding a second fluorescent protein EBFP and second selectable marker Bla, thereby making clone “tLP1” having three LPs;
(c) culturing the cells to produce the fluorescent proteins from all three LPs (see supplementary Figure S9);
(d) further comprising introducing into the cell a “payload” nucleic acid comprising matching RRSs and encoding monoclonal antibody heavy and light chain sequences and a selectable marker Puro; wherein when the payload integrates into the LPs, the resulting CHO cells can be selected by any of the single or combinations of the expressed antibiotic resistance and/or fluorescent proteins to purify cells having one, two, or all three LPs swapped to the payload exogenous nucleic acid sequence encoding monoclonal antibody + Puro. Gaidukov teaches and demonstrates successfully selecting for and utilizing these multi-LP cells to increase production of monoclonal antibodies, because more copies of genes producing the expressed protein or antibody results in higher yield (abstract; Figure 4; Supplemental Figures S11 and S12; p. 4081, col. 1-2; p. 4084, col. 1-2).
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It would have been prima facie obvious to one of ordinary skill in the art at the time the invention was filed to further introduce another LP integration site or integrated exogenous sequence encoding the monoclonal antibody by random integration into the CHO-K1 cell of Gaidukov that already comprises other LPs encoding proteins or antibody and selection markers, and to select a cell comprising the random integration, and to produce the protein/antibody encoded by the multiple integrated nucleic acid sequences. One would have been motivated to, and have a reasonable expectation of success to, because: (1) Gaidukov teaches the need to identify multiple loci integration sites in CHO-K1 cells that allow for stable, long-term expression of exogenous genes, and the need to produce CHO cells having multiple stable integration sites in order to increase production of a protein/payload/antibody encoded by the integrated exogenous sequences; (2) Gaidukov demonstrates random integration is a known and successful method for integrating exogenous genes, expressing the encoded protein and selectable markers, and for identifying multiple integration sites; (3) Gaidukov teaches the need to produce CHO cells having multiple integration sites in order to increase copy numbers of the genes expressed and to increase yield of the expressed protein/payload/antibody; (4) Gaidukov successfully demonstrates adding new integration sites to CHO cells already comprising LPs in order to increase copy number and yield of the expressed protein/payload/antibody; and (5) Gaidukov successfully demonstrates engineering CHO cells to have multiple integration sites with the same or different protein and selection marker expressed; demonstrates routine methods of selecting for or excluding cells expressing the selectable markers; and demonstrates routine methods of culturing the selected cells to produce and recover the expressed protein/antibody.
In the test of whether it is “obvious to try” there must be:
(1) a finding in the art at the time of filing of the invention that there had been a recognized problem or need in the art;
(2) a finding that there had been a finite number of identified, predictable potential solutions to the recognized need or problem;
(3) a finding that one of ordinary skill in the art could have pursued the known potential solutions with a reasonable expectation of success.
Gaidukov recognizes the need to identify stable integration sites for protein production in CHO cells, and to produce CHO cells comprising multiple stable integration sites in order to engineer CHO cells harboring multiple copies of genes, to result in higher protein/payload/antbody production.
Gaidukov demonstrates the known solutions and successful methods for identifying and creating multiple stable integration sites supporting long term expression of exogenous genes via random integration. Gaidukov demonstrates known methods for adding integration sites to cells that already harbor multiple integrations sites. Gaidukov successfully demonstrates routine methods for selecting cells by the different or same selectable markers expressed by the multiple integrated nucleic acid sequences, and routine methods for culturing cells to express the encoded proteins, and recover the proteins, such as antibodies.
One of skill in the art could have pursued additional steps to introduce another integration site or integrated exogenous sequence encoding a protein/payload/antibody and selectable marker by random integration into the CHO-K1 cells of Gaidukov that already comprises other LPs encoding protein/payload/antibody and selection markers, and to select a cell comprising the additional random integration, and to produce the protein/payload/antibody encoded by the multiple integrated nucleic acid sequences with a reasonable expectation of success.
Response to Arguments
5. Applicants argue that Examiner states Gaidukov does not teach random integration of SOI as required by the claims. Applicants argue that Gaidukov randomly integrated landing pads (LPs) as targets for targeted integration into the CHO cells’ genome. Applicant argue this technique was not used to randomly insert antibody sequences into the cells’ genome. Applicants argue that Examiner misunderstands the claimed invention that requires randomly integrated SOI or antibody sequence, and not a landing pad. Applicants argue that Gaidukov teaches away from random integration because Gaidukov teaches host cells comprising multiple landing pads for targeted integration of antibody sequences at the landing pad sites. Applicants argue that one would not be motivated to combine random integration with targeted integration based on the disclosure of Gaidukov. Applicants argue that Gaidukov provides motivation to add additional landing pads for additional targeted integration.
6. The arguments have been carefully considered but are not persuasive. Applicants are arguing limitations not recited in the claims. Claim 13 recites: “g) introducing into the host cells, via random integration, a second exogenous SOI encoding a second polypeptide of interest and a third selection marker into the genome of the host cell”. Therefore, step g) requires use of random integration process in the method of integrating the second exogenous SOI and selection marker and does not specify how or when random integration is used. Gaidukov teaches and renders obvious introducing into the host cells a second exogenous SOI encoding a polypeptide and a selection marker into the genome of the host cell via random integration. In other words, by way of random integration of landing pads (via random integration), Gaidukov introduced into the host cells a second exogenous SOI encoding a polypeptide and a selection marker into the genome of the host cell. Therefore, Gaidukov teaches and renders obvious utilizing random integration during their method of integrating the second exogenous SOI and selection marker. Further, as admitted by Applicants, Gaidukov exemplifies using random integration and targeted integration together to integrate genes encoding antibodies and selection markers into CHO host cell genome.
7. All other rejections recited in the Office Action mailed April 7, 2025 are hereby withdrawn in view of amendments. The rejection of claims as being anticipated by US Patent 12,129,480 under 35 USC 102 is withdrawn in view of the exception filed under 35 USC 102(b)(2)(C). The rejection of claims as being anticipated by Gaidukov (2018) under 35 USC 102 is withdrawn in view the amendment to claim 1 requiring target locus at least 95% homologous to a sequence selected from SEQ ID NOs:1-7, which Gaidukov does not teach or render obvious. The obviousness double patenting rejection over US Patent 12,129,480 has been withdrawn in view of claim amendments requiring target locus at least 95% homologous to a sequence selected from SEQ ID NOs:1-7, which the US Patent does not claim. Although US Patent 12,129,480 claims targeting locus SEQ ID NO:8 (727,214 nucleotides long) for targeted gene integration, and SEQ ID NO:8 comprises instant SEQ ID NO:1 (2,974 nucleotides long matching nucleotides 41,517-45,490 of SEQ ID NO:8), the claims of US Patent 12,129,480 do not recite or suggest selecting nucleotides 41,517-45,490 specifically from the 727,214 nucleotides of SEQ ID NO:8 for targeted locus.
8. Conclusion: No claim is allowed.
Conclusion
9. 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.
10. Any inquiry concerning this communication or earlier communications from the examiner should be directed to LAURA B GODDARD whose telephone number is (571)272-8788. The examiner can normally be reached Mon-Fri, 7am-3:30pm.
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/Laura B Goddard/Primary Examiner, Art Unit 1642