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and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114.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 June 11, 2026 has been entered.
Application Status
Applicant’s amendment filed June 11, 2026, amending claims 14, 20, and 25 is acknowledged. Claims 14-16, 18-22 and 24-30 are pending and under examination.
The amendment to claims 14, 20 and 25 overcome the §103 and nonstatutory double patenting rejections in the previous office action because the rejections did not address the newly added limitation HC to LC coding sequence copy ratios.
Applicant’s amendments and arguments have been thoroughly reviewed, but are not persuasive to place the claims in condition for allowance for the reasons that follow.
Claim Interpretation
As indicated in the office action mailed November 12, 2024 (page 5), “incompatible RRSs” is interpreted as “heterospecific RRSs” such that the four RRSs can’t recombine with each other, but can be recognized by the same type of recombinase, e.g., four heterospecific Lox sites that can each be recognized by a Cre recombinase.
Claim Rejections - 35 USC § 112(b)
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.
Claims 25-30 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. This is a new rejection necessitated by amendment.
Claim 25 recites “(b) introducing into the recombinant CHO cell of a) at least three vectors, each vector comprising a pair of incompatible RRSs… and each pair of incompatible RRSs flank one or more SOIs… wherein the SOI comprises…” Part (b) requires at least three vectors each comprising one or more SOIs. Therefore, part B requires at least three SOIs. It is not clear if “the SOI” is referring to just one SOI or all three. Since the claim recites at least three SOIs, it is not clear which SOI “the SOI” is referring to, rendering the claim indefinite.
Claims 26-30 are rejected for depending from claim 25 and not remedying the indefiniteness.
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.
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 25-30 are rejected under 35 U.S.C. 103 as being unpatentable over Ng (WO 2019126634 A2, published June 27, 2019).
For the purposes of compact prosecution, step (b) is interpreted as “wherein one or more SOI comprises at least one HC and at least one LC, wherein the HC:LC copy number is at least 2:1.”
Regarding claims 25-26, step a), Ng teaches TI CHO cells suitable for the expression of recombinant proteins (Abstract). Ng teaches TI CHO cell #4 comprising an integration site at position 79768 of contig NW_003616412.1 (page 101; Table 2). Ng teaches three RRS sites integrated in the targeted integration site of host #4 as illustrated in Figure 4 (page 104, lines 10-15). Ng teaches the three RRS sites, RRS1, RRS2 and RRS3 only recombine with other RRS1, RRS2 and RRS3 sites (Fig 4) and are therefore heterospecific (i.e., incompatible) (page 58, ¶1). Ng teaches in a single embodiment a T1 CHO cell with an exogenous sequence integrated at nucleotides 69303-79768 of NW_003616412.1, wherein the exogenous sequence comprises three heterospecific RRSs (pages 80-83, exemplary embodiment A2/A12/A13/A17/A24). Regarding step b), Ng also teaches introducing into the cell two vectors, wherein each vector comprises two RRSs that match either RRS1/RRS3 or RRS3/RRS2 (i.e., two RRSs matching two of the incompatible RRSs in the integrated exogenous nucleotide sequence) (Fig 4; page 7, lines 2-7). Ng also teaches the two vectors comprise a SOI and/or a different selectable marker flanked by the two RRSs (Fig 4; page 7, lines 5-7). Regarding step c), Ng also teaches introducing one or more recombinases that recognize the RRSs (Fig 4; page 7, lines 7-8). Regarding d), Ng also teaches selecting TI cells that express the second selection marker to isolate a TI host cell expressing the SOIs (page 7, lines 8-10). Ng teaches the SOIs are antibodies (page 36, lines 7-28). Ng teaches individual SOIs encoding heavy and light chain sequences can be integrated into a single exogenous nucleic acid sequence present at a single integration site (page 36, lines 23-26). Ng teaches options for heavy (H) and light (L) chain SOI copy numbers includes HHL at one vector in combination with H, HH, HHL, HHH, HLL, HHHL, HHHH, HHLL, HLLL, LLLL (i.e., at least one SOI comprises at least on antibody HC sequence and at least on antibody LC sequence at a HC:LC ratio of at least 2:1) (page 39, lines 20-26).
Ng does not teach in a single embodiment, the TI CHO cell #4 with four or more incompatible RRSs and introducing a third vector with an SOI and/or selection markers, wherein at least one SOI has at least 2:1 ratio of HC to LC copies.
However, Ng also teaches the exogenous nucleotide sequence can comprise four, five, six, seven or eight RRSs (page 32, line 17-18). Ng teaches there are at least 16 different (i.e., incompatible) RRSs (page 32, lines 25-29). Ng also teaches the cells and method of the invention can be used to integrate/insert four or more sequences of interest (SOIs) into a locus to increase the cell’s production titer (page 2, lines 25-28; page 39, lines 9-12).
It would have been obvious to one skilled in the art before the effective filing date of the claimed invention to have included a fourth heterospecific RRS in the NW_003616412.1 position 79768 integration site of host cell #4 and to have added a third recombination vector harboring either a third SOI or an additional selection marker to the obvious TI CHO cell comprising four RRSs in Ng’s RCME method, and including at least one SOI having the HHL structure. It would have amounted to 1) adding a known RRS in a known site that is known to comprise RRSs in a known cell by known means to yield predictable results, 2) a simple duplication of parts (i.e., the vector) and steps and combination of elements in a known method by known means to yield predictable results. The skilled artisan would have predicted that a fourth RRS could be added that was heterospecific to the first three because Ng teaches that four or more RRSs can be added to an integration site and there are 16 different RRSs to choose from. The skilled artisan would have been motivated to do so for the purpose of allowing the insertion of three or more sequences of interest, and because Ng suggests that four or more RRSs and three or more SOIs can be integrated into a specific locus. Regarding the third vector, “A mere duplication of parts has no patentable significance unless a new and unexpected result is produced” MPEP 2144.04. In this case, Ng suggests that additional RRSs and additional SOIs can be integrated into a single locus, which the skilled artisan would understand requires a third vector with the RRSs that recognize RRS3 and an RRS4. Thus, the addition of another vector of those disclosed in Ng would result in a third integrated antibody gene, which would be an expected result. The skilled artisan would have been motivated to do so to increase the antibody titer of the producing CHO cell as taught in Ng. Finally, the skilled artisan would have predicted that one of the SOIs in one of the three vectors could harbor the HHL construct, and been motivated to do so, because Ng suggests that the HHL construct can be used as an SOI.
Regarding claim 27, Ng teaches the RRS are recognized by a Cre recombinase, a FLP recombinase, a Bxb1 integrase, and a fC31 integrase (page 43, lines 8-11).
Regarding claim 28, Ng teaches the SOIs can be scFvs (page 46, line 20).
Regarding claim 29, Ng teaches the vectors are plasmids (page 103, line 14).
Regarding claim 30, Ng teaches that a TI host cell comprises at least one exogenous nucleotide sequence integrated at one or more integration sites in the genome of the TI host cell (page 31, lines 28-30). Ng teaches additional high-productive integration sites (Table 2).
It would have been obvious to one skilled in the art to have chosen an additional site integration site from Ng’s Table 2 and introduced the obvious exogenous nucleic acid with four RRSs. It would have amounted to the simple duplication of parts of an obvious variant of Ng’s integrated exogenous nucleic acid. “A mere duplication of parts has no patentable significance unless a new and unexpected result is produced” MPEP 2144.04. In this case, Ng suggests that additional exogenous nucleic acid sequences can be integrated into additional loci in the genome. Because Ng teaches specific additional sites, the addition of another landing pad at those sites would result in the claimed starting cell of (a), which would be an expected result. The skilled artisan would have been motivated to do so because 1) Ng suggests it, and 2) to increase the antibody titer of the producing CHO cell as taught in Ng.
Claims 14-16, 18-22 and 24 are rejected under 35 U.S.C. 103 as being unpatentable over Ng (WO 2019126634 A2, published June 27, 2019) as applied to claims 25-30 above, and further in view of Turan (Turan et al., Journal of Molecular Biology (2011), 407: 193-221).
The teachings of Ng are recited above and applied as for claims 25-30. Briefly regarding claims 14 and 20, Ng regarding a T1 CHO-K1M host cell with three integrated heterospecific RRSs at position 69303-79768 of NW_003616412.1 (pages 80-83, exemplary embodiment A2/A12/A13/A17/A24). Ng also teaches b) introducing into the cell two vectors each comprising two RRSs that match two sequentially oriented RRSs in the integrated landing pad and that flank a SOI (Fig 4; page 7, lines 2-7), wherein the SOIs can include an HHL construct (i.e., an HC:LC ratio or at least 2:1) (page 39, lines 21-27). Ng also teaches one of the vectors comprises a selectable marker flanked by the two RRSs (Fig 4; page 7, lines 2-7). Ng also teaches c) introducing one or more recombinases that recognize the RRSs (Fig 4; page 7, lines 7-8). Ng also teaches d) selecting TI cells that express the second selection marker to isolate a TI host cell expressing the SOIs (page 7, lines 8-10). Ng also teaches e) culturing the cell under conditions suitable for expressing the SOI and recovering the polypeptide of interest therefrom (page 7, lines 15-17). Ng also teaches a variety of possible selection markers such that the skilled artisan could choose three different selection markers from the list (page 5, lines 1-4). Ng teaches “the third selection marker can be different from the first and second selection markers” (sentence spanning pages 4-5). Ng teaches the third selection marker can be GFP (page 5, lines 7-8).
The obviousness of adding a fourth heterospecific RRS to the landing pad of Ng, including a third vector comprising a third SOI to the host cell with a 4-RRS landing pad, and choosing the HHL construct for at least one of the SOIs is recited above for claim 25.
Ng does not teach that all the introduced vectors comprise a selection marker and that each selection marker is distinct from at least one other selection marker flanked by a different pair of RRSs.
Turan teaches methods of RMCE, which can use Flp and Cre recombinases (Title, Abstract). Turan teaches “multiplexing RMCE” is a strategy enabling two or more modifications in parallel using multiple heterospecific (i.e., incompatible RRSs) (page 205, ¶4). Turan teaches that multiplexing can use both Flp and Cre sites (page 215, ¶6). Turan teaches that during multiplexing multiple vectors are introduced and each one can carry a selectable marker (Figure 7). Turan teaches that the selectable marker in the first vector is GFP and the other selectable marker in the second vector is RFP (i.e., the selectable markers are different from each other) (Figure 7). Turan also teaches an additional two selection markers already included in the genome that are distinct from GFP and RFP; thus Turan teaches four unique selection markers (Figure 7).
Regarding claims 14 and 20, it would have been obvious to one skilled in the art before the effective filing date of the claimed invention to have included a second and third unique selection marker in the second and third introduced vectors in the RMCE method of Ng. It would have amounted to adding known genetic marker genes to known vectors by known means to yield predictable results. The skilled artisan would have predicted that Ng’s additional vectors could include a selection markers thar are distinct from each other because Turan teaches each vector used in a multiplex RMCE method having a selection marker, while both Ng and Turan each teach at least 4 different selection markers that can be used. The skilled artisan would have been motivated to do so for the purpose of screening for all three cassette integrations simultaneously and to increase the recovery efficiency of clones in which all three recombination events occurred.
Regarding clams 15 and 21, Ng teaches the RRSs are recognized by a Cre recombinase, a FLP recombinase, a Bxb1 integrase, and a fC31 integrase (page 43, lines 8-11).
Regarding claims 16 and 22, Ng teaches the SOIs can be scFvs (page 46, line 20).
Regarding claims 18, Ng teaches the TI host cell #4 is a CHO K1M host cell (page 101, line 6-9).
Regarding claims 19 and 24, Ng teaches the vectors are plasmids (page 103, line 14), as recited above for claim 29.
Response to Arguments - §103
Applicant’s arguments are directed to the withdrawn §103 rejection and are therefore moot. However, to the extent the arguments can be applied to the current §103 rejection above, there are addressed here.
Applicant argues that Ng does not disclose HC and LC ratios (page 8, ¶1). This argument has been fully considered, but is not persuasive because Ng provides many examples of SOI constructs comprising both HC and LC coding sequences integrated via RRS-mediated recombination, many of which comprise an SOI with HHL, which is two copies of the HC coding sequence and one copy of the LC coding sequence.
Applicant argues that the present invention demonstrates an unexpected advantage of increasing the yield of mAbs. Applicant cites to evidence in Example 2 that increasing from 2 copies of HLL to 3 copies of HLL improves yield. Applicant cites to evidence in Example 5 that changing the HC:LC ratio in at least one SOI to HLH (i.e., a 2:1 HC:LC ratio) in conjunction with at least one SOI comprising HLL (a 1:2 HC:LC ratio) also increases yield. Applicant argues that setting a 2:1 ratio in at least one SOI in the RMCE setting is not taught or suggested by Ng (¶ spanning pages 8-9). These arguments have been fully considered but are not persuasive for the following reasons. First, the example provided in Example 2 does not have sufficient nexus with the claimed HC:LC ratios. Each of the SOIs in example 2 have a 1:2 HC:LC ratio. Therefore, except for comparison purposes to example 5, the results from example 3 cannot be used to support unexpected results. Second, in each comparison in example 5, the number of total copies in the CHO genome of LC and HC increases: HLL-HLL (2 HC : 4 LC) to HLL-HLL-HLH (4 HC : 5 LC). In each sample, the ratio of HC to LC is slightly higher - 0.5 to 0.8. However, the bigger difference is the increase in overall copy number: LC copy number increases by 25% and HC copy number increases by 100%. As such, it is not clear if the increased titer is due to the additional copy numbers of both the LC and HC or the fact that a single SOI has a 2:1 ratio of HC to LC. Applicant never does the proper control to see if the ratio within a single SOI can increase yield, which would have needed to compar HLL-HLL-HLL (all 1:2 ratio) to HLL-HLL-HLH (1 SOI having 2:1 ratio) or HLH-HLH-HLH (all three SOIs having 2:1 ratio). It was already well established that increasing copy number of LCs and HCs in CHO cells in generally correlated with increases mAb yield (See e.g., response to arguments in OA mailed December 11, 2025, ¶29). Thus, Applicant’s proffered evidence appears to support increasing overall copy number in general and does not point to any ratio in the SOIs overall or in any specific SOI as causative variable of the increases yield.
Applicant argues that the skilled artisan would not have been motivated by the teachings of Ng to arrive at an HC:LC ratio of 2:1 because Ng teaches an excess of LC gene fragments within a single vector improves antibody yield (page 9, ¶2). This argument has been fully considered but is not persuasive because Ng teaches various combinations that can be used in the SOIs, including many options that include a single SOI having a 2:1 ratio. Ng’s express suggestion is motivation to use at least one HHL SOI. Additionally, the claims only require “at least one SOI” having the claimed ratio. Some of the SOI-SOI combinations in Ng, such as HHL-LLLL would maintain the overall HC:LC ratio suggested by Ng.
Applicant argues that the skilled artisan would have had no expectation that the claimed modification to Ng would yield a technical advantage since the prior art generally understands that an excess of LC relative to HC facilitates proper recombinant protein folding and higher titers of recombinant antibodies. Applicant cites to evidence in Exhibit A in the Reply filed October 31, 2025 that generally increasing LC is known to increase mAb titer (page 9, ¶2). This argument and evidence has been fully considered but is not persuasive. First, the evidence is not commensurate in scope with the claims. It appears that Figure 3 in exhibit A only shows results of expressing HC and LC from just two SOIs since there is only one dash. Second, it appears from the evidence that the effect of HC and LC ratios on mAb titer depends on the specific mAb produced. For instance, when changing from HLL-HHL to HLL-HLL, mAb 1 titer increased (i.e., more LCs provide higher titer), mAb 3 titer decreased (i.e., more LCs provide lower titer), and mAb 2 titer production was not significantly different. The claims do not recite a specific mAb produced, and thus the proffered evidence does not support the nonobviousness of choosing one specific HC:LC ratio in an SOI when Ng provides many HC:LC combinations as suitable for the SOIs.
Applicant argues that Turan does not remedy the deficiencies of Ng (Remarks, page 10, ¶2-3). This argument has been fully considered but is not persuasive because a prima facie case of obviousness is established for claims 20-25 for the reasons set for in the §103 rejections of record and the response to Applicant’s arguments above.
Double Patenting
The nonstatutory double patenting rejection is based on a judicially created doctrine grounded in public policy (a policy reflected in the statute) so as to prevent the unjustified or improper timewise extension of the “right to exclude” granted by a patent and to prevent possible harassment by multiple assignees. A nonstatutory double patenting rejection is appropriate where the conflicting claims are not identical, but at least one examined application claim is not patentably distinct from the reference claim(s) because the examined application claim is either anticipated by, or would have been obvious over, the reference claim(s). See, e.g., In re Berg, 140 F.3d 1428, 46 USPQ2d 1226 (Fed. Cir. 1998); In re Goodman, 11 F.3d 1046, 29 USPQ2d 2010 (Fed. Cir. 1993); In re Longi, 759 F.2d 887, 225 USPQ 645 (Fed. Cir. 1985); In re Van Ornum, 686 F.2d 937, 214 USPQ 761 (CCPA 1982); In re Vogel, 422 F.2d 438, 164 USPQ 619 (CCPA 1970); In re Thorington, 418 F.2d 528, 163 USPQ 644 (CCPA 1969).
A timely filed terminal disclaimer in compliance with 37 CFR 1.321(c) or 1.321(d) may be used to overcome an actual or provisional rejection based on nonstatutory double patenting provided the reference application or patent either is shown to be commonly owned with the examined application, or claims an invention made as a result of activities undertaken within the scope of a joint research agreement. See MPEP § 717.02 for applications subject to examination under the first inventor to file provisions of the AIA as explained in MPEP § 2159. See MPEP § 2146 et seq. for applications not subject to examination under the first inventor to file provisions of the AIA . A terminal disclaimer must be signed in compliance with 37 CFR 1.321(b).
The filing of a terminal disclaimer by itself is not a complete reply to a nonstatutory double patenting (NSDP) rejection. A complete reply requires that the terminal disclaimer be accompanied by a reply requesting reconsideration of the prior Office action. Even where the NSDP rejection is provisional the reply must be complete. See MPEP § 804, subsection I.B.1. For a reply to a non-final Office action, see 37 CFR 1.111(a). For a reply to final Office action, see 37 CFR 1.113(c). A request for reconsideration while not provided for in 37 CFR 1.113(c) may be filed after final for consideration. See MPEP §§ 706.07(e) and 714.13.
The USPTO Internet website contains terminal disclaimer forms which may be used. Please visit www.uspto.gov/patent/patents-forms. The actual filing date of the application in which the form is filed determines what form (e.g., PTO/SB/25, PTO/SB/26, PTO/AIA /25, or PTO/AIA /26) should be used. A web-based eTerminal Disclaimer may be filled out completely online using web-screens. An eTerminal Disclaimer that meets all requirements is auto-processed and approved immediately upon submission. For more information about eTerminal Disclaimers, refer to www.uspto.gov/patents/apply/applying-online/eterminal-disclaimer.
Claims 14-16, 18-22 and 24-30 are rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1-84 of U.S. Patent No. 12129480 in view of Ng (WO 2019126634 A2, published June 27, 2019) and Turan (Turan et al., Journal of Molecular Biology (2011), 407: 193-221).
Patented claims 1-2 recite a TI host cell comprising an exogenous nucleotide sequence integrated at an integration site within a locus is at least about 90% homologous to a sequence selected from 41190-45269 of NW_006874047.1 (SEQ ID NO: 8), nucleotides 63590-207911 of NW_006884592.1 (SEQ ID NO: 9), nucleotides 253831-491909 of NW_006881296.1 (SEQ ID NO: 10), nucleotides 69303-79768 of NW_003616412.1 (SEQ ID NO: 11), nucleotides 293481- 315265 of NW_003615063.1 (SEQ ID NO: 12), nucleotides 2650443-2662054 of NW_006882936.1 (SEQ ID NO: 13), and nucleotides 82214-97705 of NW_003615411.1 (SEQ IDNO: 14); or (b) sequences within 3,000 base pairs from nucleotide 45269 of NW_006874047.1 (SEQ ID NO: 8), nucleotide 207911 of NW_006884592.1 (SEQ ID NO: 9), nucleotide 491909 of NW_006881296.1 (SEQ ID NO: 10), nucleotide 79768 of NW_003616412.1 (SEQ ID NO: 11), nucleotide 315265 of NW_003615063.1 (SEQ ID NO: 12), nucleotide 2662054 of NW_006882936.1 (SEQ IDNO: 13),and nucleotide 97705 of NW_003615411.1 (SEQ ID NO: 14). Patented claims 18-19 recite wherein the TI host cell is a mammalian host cell including a hamster host cell. Patented claims 20-22 recite wherein the exogenous nucleotide sequence comprises at least two recombination recognition sequences (RRSs) recognized by a recombinase including Cre recombinase, an FLP recombinases. Patented claims 23-29 recite the exogenous nucleic acid comprises a third heterospecific RRS, wherein two selection markers, one or two SOIs, and a IRES operably linked to a third selection marker are in between the heterospecific RRSs. Patented claims 30 and 32 recite the SOIs are antibody light chains, heavy chains or scFVs. Patented claim 66 recites a method for expressing two polypeptides of interest comprising a) providing the cells of patented claim 1-2, (b) introducing into the cell provided in a) a first vector comprising two RRSs matching the first and the third RRS on the integrated exogenous nucleotide sequence and flanking at least one first exogenous SOI and at least one second selection marker; (c) introducing into the cell provided in a) a second vector comprising two RRSs matching the second and the third RRS on the integrated exogenous nucleotide sequence and flanking at least one second exogenous SOI; (d) introducing one or more recombinases, or one or more nucleic acids encoding one or more recombinases, wherein the one or more recombinases recognize the RRSs; and (e) selecting for TI cells expressing the second selection marker to thereby isolate a TI host cell expressing the first and second polypeptides of interest.
The patented claims do not recite the host cells comprising a fourth heterospecific RRS, a third vector or a specific ratio of LC and HC in at least one SOI (all claims), wherein each vector comprises a selection marker that is different from at least one other selection marker (claims 14-16, 18-22 and 24). The patented claims do not recite specific CHO cells (claim 18).
The teachings of Ng are recited above in paragraphs 15, 17, 19-22 and 25 and incorporated here. The teachings of Turan are recited above in paragraph 28 and incorporated here.
Regarding claims 14-16, 18-22 and 24-30, it would have been obvious to one skilled in the art before the effective filing date of the claimed invention to have included an additional RRS to the patented CHO cell with the integrated landing pad, and 1) at least one SOI having HC and LC coding sequences in a 2:1 ratio and 2) a selection marker in the second and third introduced vectors in the patented RMCE method. It would have amounted to adding a known RRS in a known site that is known to comprise RRSs in a known cell by known means to yield predictable results and to have additionally added known genetic marker genes to known vectors by known means to yield predictable results. The skilled artisan would have predicted that a fourth RRS could be added that was heterospecific to the first three patented RRSs because Ng and Turan both teach that four or more RRSs can be added to a genome and there are 16 different RRSs to choose from. The skilled artisan would have been motivated to do so for the purpose of allowing the insertion of three or more sequences of interest, and because Ng suggests that four or more RRSs and three or more SOIs can be integrated into a specific locus. The skilled artisan would have also predicted that an additional vector with a selection marker could be added to the patented method because Turan teaches each vector used in a multiplex RMCE method having a selection marker. The skilled artisan would have been motivated to do so for the purpose of screening for all three cassette integrations simultaneously and to increase recovery efficiency of clones in which all three recombination events occurred. Finally, the skilled artisan would have predicted that one of the SOIs in one of the obvious variant of the patented method could harbor Ng’s HHL construct, and been motivated to do so, because Ng suggests that the HHL construct can be used as an SOI in a similar method.
Regarding claim 18, it also would have been obvious to have used CHO K1M cells because Ng teaches 1) that such CHO cells have RRSs landing pads integrated at the patented and instantly claimed sites, and 2) multiplexed RMCE is operational in those cells.
Claims 14-16, 18-22 and 24-30 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claims 89-91 of copending Application No. 18883322 in view of Ng (WO 2019126634 A2, published June 27, 2019) and Turan (Turan et al., Journal of Molecular Biology (2011), 407: 193-221).
Copending claim 89 recites a method for expressing two polypeptides of interest comprising:(a) providing a TI host cell comprising an exogenous nucleotide sequence integrated at a site within a locus of the genome of the host cell, wherein the locus is at least about 90% homologous to a sequence selected from contig NW_006874047.1 (SEQ ID NO: 8),contig NW_006884592.1 (SEQ ID NO: 9), contig NW_006881296.1 (SEQ ID NO: 10),contig NW_003616412.1 (SEQ ID NO: 11), contig NW_003615063.1 (SEQ ID NO: 12), contig NW_006882936.1 (SEQ ID NO: 13), and contig NW_003615411.1 (SEQ ID NO:14), wherein the exogenous nucleotide sequence comprises a first and a second RRS flanking at least one first selection marker, and a third RRS located between the first and the second RRS, and all the RRSs are heterospecific; (b) introducing into the cell provided in a) a first vector comprising two RRSs matching the first and the third RRS on the integrated exogenous nucleotide sequence and flanking at least one first exogenous SOI and at least one second selection marker; (c) introducing into the cell provided in a) a second vector comprising two RRSs matching the second and the third RRS on the integrated exogenous nucleotide sequence and flanking at least one second exogenous SOI; (d) introducing one or more recombinases, or one or more nucleic acids encoding one or more recombinases, wherein the one or more recombinases recognize the RRSs; and (e) selecting for TI cells expressing the second selection marker to thereby isolate a TI host cell expressing the first and second polypeptides of interest.
The copending claims do not recite the host cells comprising a fourth heterospecific RRS in the genome of the host cell or introducing a third vector (all claims) wherein each vector comprises a selection marker that is different from at least one other selection marker (claims 14-16, 18-22 and 24). The copending claims types of recombinases (claims 15, 21, and 27). The copending claims do not recite specific SOIs (claims 16, 22 and 28) or specific CHO cell types (claim 18). The copending claims do not recite types of vectors (19, 24, and 29).
The teachings of Ng are recited above in paragraphs 15, 17, 19-22 and 25 and incorporated here. The teachings of Turan are recited above in paragraph 28 and incorporated here.
Regarding claims 14-16, 18-22 and 24-30, the obviousness of having included an additional RRS to the copending CHO cells with the integrated landing pad and having included selection markers in the second and third introduced vectors, and at least one SOI having an HC:LC ratio of 2:1 in the copending RMCE method is recited above in paragraph 45 and incorporated here.
Regarding claim 18, the obviousness of having used CHO K1M cells is recited above in paragraph 46 and incorporated here.
Regarding claims 15, 21 and 27, it would have been obvious to one skilled in the art to have specifically used Cre and FLP recombinases because Ng teaches that such recombinases can be used to introduce transgenes into RRS-containing landing pads via an RMCE method.
Regarding claims 16, 22 and 28, it would have been obvious to one skilled in the art to have specifically included antibody light and heavy chains as the SOIs in the copending method because Ng teaches that antibody light and heavy chains can be produced in CHO cells and the genes encoding them can be integrated into the CHO genome via an RMCE method.
Regarding claims 19, 24 and 29, it would have been obvious to one skilled in the art to have specifically used plasmids as the vectors to integrate the SOI in the copending method because Ng teaches that plasmids can be used to introduce transgenes into RRS-containing landing pads via an RMCE method.
This is a provisional nonstatutory double patenting rejection.
Response to Arguments - NSDP
Applicant argues that the ‘480 patent is a national stage application of the applied Ng reference and the ‘322 copending application is a continuation of the ‘480 patent. Applicant argues for the reasons argued against the §103 rejections, the examined claims are nonobvious over the patented and copending claims (Remarks, pages 11-12). This argument has been fully considered but is not persuasive for the reasons explained above in paragraphs 34-39.
Conclusion
No claims are allowable.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to CATHERINE KONOPKA whose telephone number is (571)272-0330. The examiner can normally be reached Mon - Fri 7- 4.
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/CATHERINE KONOPKA/Primary Examiner, Art Unit 1635
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