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 .
Election/Restrictions
Applicant's election with traverse of Group I, claims 1, 3 and 7-10 and the following species: part (a) in claim 1, HEK263 cells in claim 8 and SEQ ID NO:38 in claim 10 in the reply filed on 05/06/2026 is acknowledged. The traversal is on the ground(s) that Enenkel does not provide or suggest the specific amino acid substitutions recited in claim 1 and does not destroy any special technical feature linking the groups and that nucleic acid sequences of Group II are intrinsically linked to the amino acid sequences of Group I. This is not found persuasive because:
Enenkel (CA 2507664 C on record in IDS) teaches neomycin phosphotransferase gene encoding NPT protein which confers resistance to various aminoglycoside antibiotics such as neomycin, kanamycin and G418 (p. 2, lines 29-32, p. 3, lines 1-2). The amino acid sequence of NPT in Enenkel teaching with SEQ ID NO:2 (p. 16, line 25) is 100% identical to instant sequence with SEQ ID NO:1. Enenkel discloses modification of the wild-type NPT leading to reduction in NPT activity and allowing to increase the expression rates of the co-integrated gene of interest when modified NPT is used as a selectable marker (p. 4, lines 28-32, p. 6, lines 17-28). Enenkel discloses substitution of several amino acid residues, one of which is Glu182Asp, resulting in reduction of NPT activity to 22% (Figure 9B) and in enrichment of transfected mammalian cells with high expression of co-integrated genes (p. 18, lines 22-26) and mentions combination of substitutions (p. 5a, lines 6-7). Enenkel refers to prior art of Shaw for selection of conserved amino acid residues for substitution (p. 17, line 2, p. 19, line 17).
Enenkel does not teach substitution of residue 36 to methionine.
Shaw (Shaw et al. Microbiol. Reviews, 1993, 57, 138-163 on record in IDS) teaches results of substitution of several amino acid residues in APH(3’)-IIa which is the NPT of Enenkel teaching (p. 2, lines 29-31). Shaw discloses that substitution of conserved amino acid residue V36 with methionine results in 20-fold decrease in NPT activity (p. 154, right column, last paragraph).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to try combination of E182D from Enekel teaching and V36M from Shaw teaching to select high producing recombinant cells based on Enenkel teaching. One would have been motivated to do so with reasonably expected success since V36M and E182D substitutions were used for the same purpose and with the same result of reduction of NPT activity and Enenkel mentioned combination of modified conserved amino acid residues.
Since the combination of Enekel and Shaw teaches non-naturally occurring NPT comprising specific amino acid substitutions corresponding to option (b) of claim 1 shared between all Groups, it is not a special technical feature and Groups I-IV lack unity of invention.
The requirement is still deemed proper and is therefore made FINAL.
Claims 1, 3, 7-11, 14, 20, 21, 28, 30, 32, 34, 40, 43, 46, 48, 51, 59 and 60 are pending.
Claims 11, 14, 20, 21, 28, 30, 32, 34, 40, 43, 46, 48, 51, 59 and 60 are withdrawn from further consideration pursuant to 37 CFR 1.142(b), as being drawn to a nonelected invention, there being no allowable generic or linking claim. Applicant timely traversed the restriction (election) requirement in the reply filed on 05/06/2026.
Claims 1, 3 and 7-10 (claim set filed 05/06/2026) are examined on the merits herein.
Priority
This application is a 371 of PCT/US2022/025452 filed 04/20/2022 which claims benefit of 63/177,739 filed 04/21/2021 and claims benefit of 63/177,744 filed 04/21/2021 and claims benefit of 63/177,746 filed 04/21/2021 and claims benefit of 63/177,749 filed 04/21/2021 and claims benefit of 63/177,753 filed 04/21/2021 and claims benefit of 63/177,759 filed 04/21/2021 and claims benefit of 63/177,764 filed 04/21/2021 and claims benefit of 63/177,767 filed 04/21/202. 1Applicant’s claim for the benefit of a prior-filed application under 35 U.S.C. 119(e) or under 35 U.S.C. 120, 121, 365(c), or 386(c) is acknowledged.
Information Disclosure Statement
The information disclosure statements (IDS) submitted on 08/02/2024 and 01/13/2026 comply with the provisions of 37 CFR 1.97. Accordingly, the information disclosure statements are being considered by the examiner.
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, 3 and 7-9 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, as failing to comply with the written description requirement. The claim(s) contains subject matter which was not described in the specification in such a way as to reasonably convey to one skilled in the relevant art that the inventor or a joint inventor, or for applications subject to pre-AIA 35 U.S.C. 112, the inventor(s), at the time the application was filed, had possession of the claimed invention.
Claim 1 is directed to a non-naturally occurring neomycin phosphotransferase (NPT) having NPT activity and comprising a wild-type NPT comprising the recited amino acid substitutions wherein positions of the substituted amino acid residues correspond to the recited positions in SEQ ID NO:1. The specifications defines the “wild-type NPT” as neomycin phosphotransferase which prevails among organisms in nature (paragraph 00176). Claim 1 is interpreted as directed non-naturally occurring variants of NPT having any and unspecified wild-type NPT sequence including any N- and/or C-terminal additions due to transitional phrase "comprises". The wild-type NPT is recited to comprise the recited amino acid substitutions and is interpreted to have the recited substitutions and to include any additional modifications of any other amino acid residues due to transitional phrase "comprises". The specification describes the sequence of APH(3’)-IIa corresponding to SEQ ID NO:1 as a reference sequence to which other wild-type NPT are compared (paragraph 00177). Therefore the amino acid sequence with SEQ ID NO:1 is interpreted as a reference sequence only for identification of amino acid residues in various wild-type NPT sequences corresponding to the recited positions in SEQ ID NO:1 when aligned.
Thus, claim 1 broadly encompasses a genus of NPT variants containing various wild-type NTP sequences with various amino acid substitutions. This would represent a large pool of variant amino acid sequences encoding the NPT proteins which are functional since they are required to have neomycin phosphotransferase activity. The Specification does not provide structure function correlation for NPT proteins and does not describe domains and/or amino acid residues essential for NPT protein and domain and/or amino acid residues which can be modified without loss of NPT activity. Figure 6 provides alignment of various wild-type NPT proteins and shows Motif 1-3, however, the specification does not provide description of the correlation of those motifs with NPT activity. Further, applicants have not shown possession of a representative number of species for variants of various wild type NPT as Specification provides only examples of modification of NPT with SEQ ID NO:1. Table 1 of the specification shows the activity of several single and double mutants of NPT with SEQ ID NO:1. Substitutions encompass 12 amino acid residues (modified as single residues or in combination) from total of 264 amino acid residues for APH(3’)-IIa corresponding to SEQ ID NO:1 which is only 4.5% of possible modifications. Modifications of other amino acid residues and modification of wild-type NPT other than presented by SEQ ID NO:1 are not described.
Therefore, one of ordinary skill in the art would not be able to identify which NPT polypeptide sequences with the recited and possible additional substitutions and belonging to different wild-type NPTs encode for functional NPT protein possessing NPT activity. One of ordinary skill in the art would conclude based on the lack of representative number of species and the lack of describing the domains or amino acid residues of NPT critical for the function of NPT protein, that the Applicant was not in possession of the claimed genus and that the specification fails to satisfy the requirements of written description under 35 U.S.C. 112 (a). Therefore, claim 1 is rejected.
Claims 3 and 7-9, dependent on claim 1, do not resolve the issue mentioned above and are rejected. Claim 10 is directed to specific amino acid sequences and therefore is not rejected.
Claim 9 is 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 the non-natural occurring NPT with V36M/G210A, V36M/E182D and V36M/Y218F double substitutions to produce G418 resistant colonies of mammalian cells with 0.004-1.0% frequency does not reasonably provide enablement for the non-natural occurring NPT to produce G418 resistant colonies with 0.001% or more and less than 0.004% and higher than 1.0% frequency with any substitutions and for NPT with D216G/D261N, V36M/Y218S and V36M/D216G to produce any colonies. The specification does not enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to use the invention commensurate in scope with these claims.
Claim 9 is directed to the non-naturally occurring NPT being able when expressed in the mammalian cells to reduce the number of resistant colonies to frequency from 0.001% to 75% relative to the resistant colonies of mammalian cells expressing wild-type NPT. Claim 9 is dependent on claim 1, reciting six variants of NPT with recited amino acid substitutions. The specification provides working example of the frequency of colony formation of HEK263 cells transfected with mutant NPTs recited in claim 1 (Table 2). Mutant V36M/G210A demonstrated 1.0% frequency of colony formation, mutant V36M/E182D – 0.02% and mutant V36M/Y218F – 0.004%. Mutants D216G/D261N, V36M/Y218S and V36M/D216G could not be evaluated as they did not form any colonies and hence did not provide resistance to G418.
The prior art of Shaw (Shaw et al. Microbiol. Reviews, 1993, 57, 138-163 on record in IDS) teaches effect of substitution of several amino acid residues of NPT, including several instant substitutions, on the resistance to kanamycin or G418 (p. 153, right column, p. 154, p. 155, left column). However, Shaw did not describe the effect of double substitutions on the formation of resistant colonies.
Based on the unpredictability taught by the prior art and absence of working examples and directions provided by inventors, one of ordinary skill in the art would have to undergo undue experimentation to practice the full scope of the invention. Therefore, claim 9 is rejected under 35 U.S.C. 112(a) for failing to disclose sufficient supporting information to enable a person of skill in the art to use D216G/D261N, V36M/Y218S and V36M/D216G variants of NPT to produce the resistant colonies and any recited variants expecting to result in colonies formation with 0.001% or more and less than 0.004% frequency and higher than 1.0% but less than75% frequency. Therefore, claim 9 is rejected.
Claim Rejections - 35 USC § 103
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
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 1, 3, 9 and 10 are rejected under 35 U.S.C. 103 as being unpatentable over Enenkel (CA 2507664 C on record in IDS) in view of Shaw (Shaw et al. Microbiol. Reviews, 1993, 57, 138-163 on record in IDS) and Kocabiyik (Kocabiyik and Perlin Int. J. Biochem., 1994, 26, 61-66 on record in IDS).
Regarding claim 1, Enenkel teaches neomycin phosphotransferase gene encoding amino glycoside-3’-phosphotransferase II (neomycin phosphotransferase, NPT) protein which confers resistance to various aminoglycoside antibiotics such as neomycin, kanamycin and G418 (p. 2, lines 29-32, p. 3, lines 1-2). The amino acid sequence of NPT in Enenkel teaching with SEQ ID NO:2 (p. 16, line 25) is 100% identical to instant sequence with SEQ ID NO:1. Enenkel discloses modification of the wild-type NPT leading to reduction in NPT activity and allowing to increase the expression rates of the co-integrated gene of interest when modified NPT is used as a selectable marker (p. 4, lines 28-32, p. 6, lines 17-28). Enenkel describes the wild-type NPT modification by amino acid substitutions creating non-naturally occurring NPT (p. 5, lines 7-16). Enenkel discloses substitution of several amino acid residues at positions 91, 182, 198, 227, 240 and 261 and mentions combination of substitutions (p. 5, line 9, p. 5a, lines 6-7). Enenkel mentions that the transfection of the modified NPT used as a selectable marker increases productivity of a protein of interest in the cells by a factor of 1.4-14.6 (p. 5, lines 1-5). Enenkel refers to prior art of Shaw for selection of conserved amino acid residues for substitution (p. 17, line 2, p. 19, line 17)
Enenkel does not teach substitution of residue 36 to methionine and residue 210 to alanine (elected species).
Shaw teaches various aminoglycoside phosphotransferases, their origin and resistance mechanism (p. 146-147, section “Phosphorylation”). Shaw presents the sequence alignment for aminoglycoside phosphotransferase family of proteins on Figure 9 (p. 157-158) that identifies common sequence motifs and conserved amino acid residues. Shaw describes results of substitution of several amino acid residues in APH(3’)-IIa which is the instant NPT (specification, paragraph 00176) and NPT of Enenkel teaching (p. 2, lines 29-31). Several conserved amino acid residues, such as D227 and D261, were used in Enenkel teaching and substitution of these residues was shown to result in reduced NPT activity and increased protein of interest productivity (Enenkel, Fig. 6B, Fig. 9B). Shaw discloses that substitution of conserved amino acid residue V36 with methionine results in 20-fold decrease in NPT activity (p. 154, right column, last paragraph). Shaw mentioned mutations on the conserved residues of motif 2, i.e. Asp208/Gly, Gly210/Asp, Arg211/Gln, Asp216/Gly and Asp220/Gly, all of which resulted in less than 6% of the wild-type level of kanamycin phosphorylation and in a similar reduction in aminoglycoside resistance (p. 153, right column).
Kocabiyik teaches substitution of several amino acid residues in APH(3’)-II, including Gly210/Ala substitution (Abstract). The substitutions caused significant reduction in resistance to aminoglycoside antibiotics (including kanamycin, neomycin and G418) (p. 63, Table 3) and reduction in NPT activity compared to the wild-type NPT (p. 63, Table 4).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to follow Shaw teaching and try substitutions of additional conserved amino acid residues of NPT, i.e. V36 and G210, from Shaw teaching to reduce resistance of cells transfected with NPT variant to selection agent and increase expression of co-integrated gene of interest when modified NPT is used as a selectable marker as described by Enenkel. One would have been motivated to do so since Shaw provided sequence alignment of NPT proteins and identification of conserved motifs and amino acid residues and described substitution of residues V36 and G210 to result in reduction in NPT activity and Enenkel taught other conserved residues selected based on Shaw teaching substitution of which decreased NPT activity and increased expression and productivity of cells with modified NPT as a selectable marker. A skilled artisan would have reasonably expected success in the combination because Enenkel and Shaw teach substitution of NPT amino acid residues to reduce NPT activity and resistance to selection agent.
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to substitute Gly 210 of NPT with Ala as taught by Kocabiyik to select high producing recombinant cells based on Enenkel teaching. One would have been motivated to do so since Kocabiyik described that substitution of residues G210 resulted in reduced resistance to aminoglycoside antibiotics (including kanamycin, neomycin and G418) (Table 3) and reduction in NPT activity compared to the wild-type NPT. A skilled artisan would have reasonably expected success in the combination because Enenkel and Kocabiyik teach substitution amino acid residues of NPT to attenuate its activity.
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to try combination of V36M taught by Shaw and G210A taught by Kocabiyik substitutions of the wild-type NPT of Enenkel teaching to select high producing recombinant cells based on Enenkel teaching. One would have been motivated to do so since V36M and G210A were used for the same purpose and with the same result of reduction of NPT activity and Enenkel mentioned combination of modified conserved amino acid residues. A skilled artisan would have reasonably expected success in that because Enenkel, Shaw and Kocabiyik teach APH(3’)-II NPT and attenuation of its activity by amino acid substitutions. Thus, Enenkel, Shaw and Kocabiyik teachings render claim 1 obvious.
Regarding claim 3, Enenkel teaches that modified NPT has reduced activity: “has only 1 to 80%, preferably only 1 to 60%, more preferably only 1.5 to 30%, most preferably only 1.5 to 26% of the activity” and that mammalian cells transfected with modified NPT are cultivated in the presence of agent selectively acting on the growth of mammalian cells and allowing cells expressing NPT to grow and hence modified NPT is used as a selectable marker (p. 6, lines 17-28). Thus, Enenkel, Shaw and Kocabiyik teachings render claim 3 obvious.
Regarding claim 9, Enenkel teaches transfection of mammalian cells with expression vectors carrying variant NPTs and selection with G418 (p. 43, lines 1-2, p. 52, lines 8-9). Enenkel mentions that for cell populations transfected with mutated NPT fewer cells were detected after G418 selection compared to cells with the wild-type NPT (p. 71, lines 7-8). Production of resistant colonies for cell transfected with mutant NPT at frequency from 0.001% to 75% is considered to be property of mammalian cells transfected with mutant NPT. Pursuant to MPEP 2112.01: “Products of identical chemical composition cannot have mutually exclusive properties." In re Spada, 911 F.2d 705, 709, 15 USPQ2d 1655, 1658 (Fed. Cir. 1990). A chemical composition and its properties are inseparable. Therefore, if the prior art teaches the identical chemical structure, the properties applicant discloses and/or claims are necessarily present.” Thus, given the combination of Enenkel, Shaw and Kocabiyik renders the claimed structure of non-naturally occurring NPT with V36M and G210A substitutions of instant SEQ ID NO:1 obvious as described for claim 1 and Enenkel teaches reduction in the colony number for cells transfected with mutant versus wild-type NPT, the combination of prior art would necessarily render the claimed property of production of the resistant colonies for cell transfected with mutant NPT at frequency from 0.001% to 75% obvious as well. Therefore, Enenkel, Shaw and Kocabiyik teachings render claim 9 obvious.
Regarding claim 10, Enenkel teaches modification of NPT (APH(3’)-II ) sequence with SEQ ID NO:2 which has 100% identity to instant SEQ ID NO:1. Shaw teaches substitution of the same NPT protein at position 36, i.e. V36M and Kocabiyik teaches substitution of the same NPT at position 210, i.e. G210A. The sequence of the non-naturally occurring NPT with SEQ ID NO:38 (elected species) is the sequence with SEQ ID NO:1 and V36M and G210A mutations. Therefore, combination of Enenkel, Shaw and Kocabiyik teaches non-naturally occurring NPT with SEQ ID NO:38.
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to substitute V36 with methionine as taught by Shaw and glycine 210 to alanine as taught by Kocabiyik in the wild-type NPT with SEQ ID NO:2 of Enenkel teaching resulting in instant sequence with SEQ ID NO: 38. One would have been motivated to do so to select high producing recombinant cells based on Enenkel teaching since V36M and G210A were shown to reduce the NPT activity and resistance to selection marker allowing to select cells with high level of expression of co-integrated gene of interest. A skilled artisan would have reasonably expected success in that because Enenkel, Shaw and Kocabiyik teach APH(3’)-II NPT and attenuation of its activity by amino acid substitution. Thus, Enenkel, Shaw and Kocabiyik teachings render claim 10 obvious.
Claim 7 is rejected under 35 U.S.C. 103 as being unpatentable over Enenkel (CA 2507664 C on record in IDS) in view of Shaw (Shaw et al. Microbiol. Reviews, 1993, 57, 138-163 on record in IDS) and Kocabiyik (Kocabiyik and Perlin Int. J. Biochem., 1994, 26, 61-66 on record in IDS) as applied to claim 1 above, and further in view of Leff (Leff et al. Appl. Environ. Microbiol., 1993, 59, 417-421).
Teaching of Enenkel, Shaw and Kocabiyik have been set forth above.
Enenkel mentions selection of the cells expressing gene of interest and modified NPT gene with kanamycin (p. 52, lines 8-11), however does not teach bacterial cells as a host (p. 43, lines 7-9). Kocabiyik describes testing resistance of modified NPT compared to the wild-type NPT to kanamycin by determination of the minimum inhibitory concentration for bacterial cells (p. 63, Table 3). However, Enenkel, Shaw and Kocabiyik do not teach colony formation assay performed after growing of bacterial cells for 48 hours on plates with kanamycin.
Leff teaches detection of kanamycin resistant bacteria in the environmental samples (Abstract). Leff mentions that these bacteria contain gene encoding NPT and conveying resistance to kanamycin and neomycin (Abstract). Leff describes that bacteria were seeded on agar plates and incubated for 48 hours in the presence of 50 µg/ml or 200 µg/ml of kanamycin or neomycin. After that colonies were enumerated (p. 417, right column, last paragraph, p. 418, left column, 1st paragraph). Figure 1 shows the difference in the number of resistant colonies between samples (p. 419).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to add colony formation assay with assay conditions including 48 hour incubation of bacterial cells with 50 µg/ml of kanamycin as taught by Leff to method of creating non-naturally occurring NPT based on Enenkel, Shaw and Kocabiyik teachings. One would have been motivated to apply the colony formation assay for evaluation of the resistance level of cells transformed with variant NPT compared to that of the cell with the wild-type NPT since Leff showed difference in the resistant level for samples and provided instructions. A skilled artisan would have reasonably expected success in that because Enenkel, Shaw, Kocabiyik and Leff teach NPT providing resistance to kanamycin.
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention that cells transformed with variant NPT with reduced NPT activity will produce reduced number of colonies in the colony formation assay. One would have been motivated to expect that because Kocabiyik showed bacterial cells with variant NPT having reduced activity to have drastically reduced minimum inhibitory concentration for kanamycin and hence low resistance and therefore those cells will produce less colonies at a fixed kanamycin concentration in colony formation assay. Thus, Enenkel, Shaw, Kocabiyik and Leff teachings render claim 7 obvious.
Claim 8 is rejected under 35 U.S.C. 103 as being unpatentable over Enenkel (CA 2507664 C on record in IDS) in view of Shaw (Shaw et al. Microbiol. Reviews, 1993, 57, 138-163 on record in IDS) and Kocabiyik (Kocabiyik and Perlin Int. J. Biochem., 1994, 26, 61-66 on record in IDS) as applied to claim 1 above, and further in view of Chen (WO 2011014816 A1).
Teaching of Enenkel, Shaw and Kocabiyik have been set forth above.
Enenkel teaches transfection of mammalian cells with expression vectors carrying variant NPTs and selection with G418 at 200-800 µg/ml (p. 43, lines 1-2, p. 52, lines 8-12). Enenkel mentions that for cell populations transfected with mutated NPT fewer cells were detected after G418 selection compared to cells with the wild-type NPT (p. 71, lines 7-8). However, Enenkel, Shaw and Kocabiyik do not explicitly teach colony formation assay performed after 2 weeks growing of mammalian cells with G418.
Chen teaches methods for inhibiting cell proliferation (Abstract). Chen mentions using NPT as selection marker (paragraph 0057). Chen is using colony formation assay to compare proliferation of cells transfected with genes of interest. Chen describes that after transfection mammalian cells (SK-Mel-2 and A375) are cultured at 400 µg/ml or 800 µg/ml of G418 for 2 weeks selection. After that cells are fixed with methanol and stained with 0.1% crystal violet (paragraph 0146). The results of the assay are presented on Figure 6A.
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to follow Chen teaching and apply colony formation assay with assay conditions including 2-weeks selection of mammalian cells with G418 as taught by Chen to method of evaluation of the resistant colonies formed for cell transfected with mutant versus wild-type NPT during selection with 200-800 µg/ml of G418 as described by Enenkel. One would have been motivated to do that since Chen showed difference in the resistant level for samples and provided instructions for the colony formation assay and Enenkel taught cell transfected with mutant NPT to produce less colonies and that can be detected and quantified by the colony formation assay of Chen. A skilled artisan would have reasonably expected success in that because Enenkel and Chen teach mammalian cells resistant to G418 provided by NPT.
Thus, Enenkel, Shaw, Kocabiyik and Chen teachings render claim 8 obvious.
Conclusion
No claims are allowed.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to LIOUBOV G KOROTCHKINA whose telephone number is (571)270-0911. The examiner can normally be reached Monday-Friday: 8:00-5:30.
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If attempts to reach the examiner by telephone are unsuccessful, the examiner’s supervisor, Sharmila G Landau can be reached at (571)272-0614. The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300.
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/L.G.K./Examiner, Art Unit 1653
/SHARMILA G LANDAU/Supervisory Patent Examiner, Art Unit 1653
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