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 .
Applicant’s amendment and arguments filed on 03-09-2026 has been entered. Claim 22 has been amended. Claims 1-5, 8-10, 17-19 have been canceled. Claims 6-7, 11-16, 20-41 are pending.
This is a Non-Final office action.
Election/Restrictions
Applicant's election of claims 6, 7, and 20-27 (Group II) in the reply filed on 08-15-2024 is acknowledged. Because applicant did not distinctly and specifically point out the supposed errors in the restriction requirement, the election has been treated as an election without traverse (MPEP § 818.0l(a)).
Claims 9-19 are withdrawn from further consideration pursuant to 37 CFR l. l 42(b) as being drawn to a nonelected invention there being no allowable generic or linking claim. Election was made without traverse in the reply filed on 08-15-2024. It is noted that claims 9-10, 17 - 19 have been canceled.
However, in view of applicant amendments and upon further consideration, withdrawn claims 11-16 are hereby rejoined with the elected invention.
Claims 6-7, 11-16, 20-41 are under consideration.
Priority
This application is a 371 of PCT/EP2020/050592 filed on 01/10/2020 that claims priority from a foreign application EP 19151233.4 filed on 01/10/2019. Receipt is acknowledged of certified copies of papers required by 37 CFR 1.55.
Information Disclosure Statement
The information disclosure statements (IDS) submitted on 08-15-2025 is in compliance with the provisions of 37 CPR 1.97. Accordingly, the information disclosure statements have been considered by the examiner.
Maintained -Claim Rejections - 35 USC § 101
35 U.S.C. 101 reads as follows:
Whoever invents or discovers any new and useful process, machine, manufacture, or composition of matter, or any new and useful improvement thereof, may obtain a patent therefor, subject to the conditions and requirements of this title.
Claims 6-7, 20-21, 24-25, are rejected under 35 U.S.C. 101 because the claimed invention is directed to a product of nature without significantly more.
Step 1: Is the claim to a process, machine, manufacture or composition of matter?
Claim 6 is directed to a Drosophila cell, comprising a genetic modification of the FucT6 gene, wherein said modification reduces expression of the FucT6 gene. Claim 7 specifies the Drosophila cell produces N-glycosylated protein comprising less than 2% core α l,6-fucose. Claim 20 specifies the Drosophila cell, wherein expression of an fdl gene has further been disrupted. Claim 21 specifies a cell clone or cell line comprising the Drosophila cell. Claim 24 specifies the Drosophila cell produces N-glycosylated protein with less than 40% FM3/M3 glycan structures. Claim 25 specifies the Drosophila cell produces N-glycosylated protein comprising at least 2% FAl/Al glycan structures. Claim 27 specifies the Drosophila cell produces N-glycosylated protein comprising less than 15% FM3/M3 glycan structures, less than 30% FAl/Al glycan structures, and more than 50% FA2/A2 glycan structures.
Under the broadest reasonable interpretation, the terms of the claim are presumed to have their plain meaning consistent with the specification as it would be interpreted by one of ordinary skill in the art. See MPEP 2111. Broadest reasonable interpretation of the claims encompasses a Drosophila cell. Here, because the insect cells are a composition of matter, the claim is to at least one statutory category of invention (Step 1: YES).
Step 2(A), Prong 1: Does the claim recite an abstract idea, law of nature or natural phenomenon?
The markedly different characteristics analysis is performed by comparing the nature-based product limitation of a Drosophila cell comprising a genetic modification of the FucT6 gene to its naturally occurring counterpart of a Drosophila cell comprising a genetic modification of the FucT6 gene to determine if it has markedly different characteristics from the counterpart. MPEP 2106.04(c) (II).
The claims recite a Drosophila cell comprising a genetic modification on FucT6 gene. The Drosophila cell produces N-glycosylated protein comprising less than 2% core α l,6-fucose. Expression of a fdl gene in the Drosophila cell can be further disrupted. The cells can produce N-glycosylated protein with less than 40% FM3/M3 glycan structures; produce N-glycosylated protein comprising at least 2% FAI/Al glycan structures; produce N-glycosylated protein comprising less than 15% FM3/M3 glycan structures, less than 30% FAI/Al glycan structures, and more than 50% FA2/A2 glycan structures. Here, the closest natural counterpart is naturally occurring Drosophila cell with naturally mutation occurring on FucT6 leading to reduced gene expression. When the claimed Drosophila cell is compared to this counterpart, the comparison indicates that there are no differences in structure, function, or other characteristics which undergo spontaneous null mutation as explained below:
Watanabe et al (Genetics 181: 1035–1043 (March 2009), DOI: 10.1534/genetics.108.093385) teaches Molecular Spectrum of Spontaneous de Novo Mutations in Male and Female Germline Cells of Drosophila melanogaster (Title), and mutation rate and its variability are central to theoretical population genetics and evolutionary studies (Page 1035, left column) and there is significant variability in rate and
pattern of mutation among progenitor genotypes (Page 1035, right column). Keightley et al (Mol. Biol. Evol. 32(1):239–243 doi:10.1093/molbev/msu302, November 3, 2014) estimated the spontaneous mutation rate in Heliconius melpomene by genome sequencing of a pair of parents and 30 of their offspring, based on the ratio of number of de novo heterozygotes to the number of callable site-individuals …… an estimated mutation rate of 2.9x 10-9 (95% confidence interval, 1.3x10-9 –5.5 x10-9), which is similar to recent estimates in Drosophila melanogaster (Abstract). Thus, Watanabe et al and Keightley et al provide evidence that the Drosophila cells of the claimed invention are morphologically and phenotypically similar to Drosophila cells in nature which undergo spontaneous mutation that can encompass null mutations that render the loss of function of genes such as FucT6 and fdl.
Yamamoto-Hino et al (Genes to Cells (2015) 20, 521–542, DOI: 10.1111/gtc.12246, 04 May 2015) teach “Gene silencing in the whole Drosophila body” (Page 522, right column, last para) and “Depigmentation was caused by silencing of da6fut/fucT6, gfr/nac, Csat, and CG33145 (Fig. 6, Table 3). Da6Fut/FucT6 adds a fucose moiety to the core region of N-linked glycans via α1,6- linkage ….” (Page 529, right column, 1st para.).
Yang et al (Entomologia Generalis, Volume 43 (2023), Issue 5, 911–925, DOI: 10.1127/entomologia/2023/2066) provide evidence for a viable FucT6-mutant D. melanogaster larvae: FucT6-mutant D. melanogaster larvae exhibit a somewhat impaired immune response to parasitoid wasp infection, leading to a decreased encapsulation rate (See page 919, right column, 2nd para.).
Zuberova et al (Disease Models & Mechanisms 3, 773-784 (2010) doi:10.1242/dmm.005389) provide evidence for FucT6 on chromosome X can be targeted (insertion) by transposon ( Note: A transposon is a mobile genetic element that can move from one location to another within a genome is a source of mutations that can alter gene function) : Zuberova et al stated that “We screened a total of 49 deletions for chromosome X …… The originally identified deficiency Df(1)Exel9050 in the cytological localization 10D5-10D6 was molecularly defined by the transposon insertions P{XP}d00034 and PBac{RB}FucT6e02394” (Page 774, left column, 4th para.)
It appears that D. melanogaster organism containing FucT6-mutant is viable and the claimed insect cells are identical to what exists in nature (e.g., same genotype and phenotype). There is nothing in the claim that are phenotypically different from what exists in nature or any difference that arose due to applicant's efforts. The claim thus encompasses cells that are identical to naturally occurring cells. Because there is no difference between the claimed and naturally occurring cells for at least some of the embodiments encompassed by the claim, the claimed cells do not have markedly different characteristics, and thus are a "product of nature" exception. In re Roslin Institute (Edinburgh), 750 F.3d 1333, 1338-39 (Fed. Cir. 2014).
Step 2(A), Prong 2: Do the claims recite additional elements that integrate the judicial exception into a practical application?
In view of foregoing analysis, the claims recite a judicial exception of a naturally occurring Drosophila cell with naturally mutation occurring on FucT6 gene, they would still be patent-eligible if “the claims as a whole integrate the recited judicial exception insect cell with naturally mutation occurring on FucT6 gene into a practical application of the exception”. Here the claims recite the insect cell produces N-glycosylated protein comprising less than 2% core α l,6-fucose; Expression of a Fdl gene in the insect cell can be further disrupted; The cells can produce N-glycosylated protein with less than 40% FM3/M3 glycan structures; produce N-glycosylated protein comprising at least 2% FAI/Al glycan structures. However, these are structural limitations which are natural properties of the cells as evidenced by applicant own disclosure: “The FucT6 gene was successfully disrupted …… The polyclonal pool showed an effect and the analyzed clone showed 100% absence of α 1,6-fucose.” (Page 22, lines 2-3) (For claim 7). Also, the additional disruption of expression of Fdl gene can also happen naturally as explained above and mutant flies are viable as evidenced by Sarkar et al (THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 281, NO. 18, pp. 12776–12785, May 5, 2006) who teach fused lobe (fdl) mutant flies : fdl mutants were first identified in an enhancer trap screen for genes that are expressed during late larval development in structures that will give rise to the central complex of the adult brain (Page 12783, left column, 1st para) and Df(2R)achi2 flies (Drosophila melanogaster) have a null mutation in the fdl gene (Page 12783, left column, 2nd para.) (For claim 20). Furthermore, the instant disclosure provides evidence for Drosophila cell lacking fdl with characteristic of producing N-glycosylated protein as cited by the claim: “expression of the fdl gene has been disrupted, which has, as shown herein in the examples, the consequence that the FM3/M3 glycan structures are reduced in proportion on expressed protein …… exhibit less than 40% FM3/M3 glycan structures ……the cell of the first aspect produces N-glycosylated protein comprising at least 2% FA1/A1 glycan structures,” (Page 21, lines 3-11) (For claims 24-25).
Thus, the claim as a whole does not amount to significantly more than each "product of nature" by itself. There is no structural or phenotypic difference in any of the claimed differentiated cells that distinguish them from naturally occurring insect cell with naturally mutation occurring on FucT6 gene. Further, there is no additional element that integrates the recited naturally occurring product (naturally occurring Drosophila cell with naturally mutation occurring on FucT6 gene) into a practical application of the judicial exception. In summary, there is no additional elements recited in the claims, considered individually or in combination, that integrate the recited, naturally occurring Drosophila cell with naturally mutation occurring on FucT6 gene into a practical application of the judicial exception and the claim is therefore directed to the judicial exception (Step 2A Yes).
Step 2(B): Does the claim recite additional elements that amount to significantly more than the judicial exception?
There is no additional element recited in the claims that are sufficient to amount to significantly more than the judicial exception, other than the naturally occurring insect cell with naturally mutation occurring on FucT6 gene. The claims only encompass the Drosophila cell which produces N-glycosylated protein with less than 2% core α 1,6-fucose, produces N-glycosylated protein with less than 40% FM3/M3 glycan structures, produces N-glycosylated protein with at least 2% FA1/A1 glycan structures. The insect cell can have Fdl gene expression further disrupted. Thus, the claims recite a naturally occurring Drosophila cell that do not differ markedly from naturally occurring Drosophila cells.
In summary, the claims as a whole do not amount to significantly more than the judicial exception of “product of nature” itself. Thus, the claims do not qualify as patent eligible subject matter. (Step 2B: NO).
Response to Arguments
Applicant's arguments filed 03-09-2026 have been fully considered but they are not persuasive.
1. Applicant argue that
The assessment as to whether the claimed Drosophila cell is a product of nature improperly uses a fictitious Drosophila cell having a genetic modification of a FucT6 gene as the closest natural counterpart for the markedly different characteristics analysis. Given the lack of evidence for a naturally occurring Drosophila FucT6 genetic mutant, the selection of such a cell as the closest natural counterpart is improper (remarks, page 7).
The teachings of Yamamoto and Zuberova do not disclose or indicate that a Drosophila FucT6 genetic mutant has or would occur in nature. As described more thoroughly below, the newly cited references all describe human-generated Drosophila cells created in an artificial, laboratory environment. Thus, notwithstanding the absence of any Drosophila FucT6 mutant in these references, any conclusion drawn regarding these Drosophila cells or viability thereof cannot be understood as a phenomenon or product of nature lacking human intervention (remarks, page 7-8).
Yamamoto describes that whole-body silencing of FutT6 is lethal. In fact, "spatiotemporal" gene silencing (applied by inducible RNAi expression, see page 529 in Yamamoto), whereby the gene - in a highly artificial manner - is silenced to some degree in certain parts of the animal. Yamamoto describes that D. melanogaster having blocked expression of the FucT6 gene is not viable, which would be understood to be lethal in Drosophila (remarks, page 8)
Zuberova investigates the effects of mutations in phosphorylase kinase on a man-made adgf-a mutant phenotype of D. melanogaster, which is identified as involving 3 genes (PhK-y, CG2444 and FucT6) (see Fig. IA of Zuberova). Importantly, the claimed Drosophila cell exhibits reduced expression of the FucT6 gene. There is nothing in Zuberova that indicates that the adgf-a mutant, which is relied upon by the Examiner, exhibits reduced expression ofFucT6. A review of Fig. I suggests that the gene does not appear to be silenced but rather the transcript could appear to be shorter (remarks, page 8).
Yang does not teach or suggest a FucT6 Drosophila cell having reduced FucT6 expression. In fact, nothing can be deduced about the mutant's effect on glycosylation, its expression level, etc., from the teachings of Yang (remarks, page 9)
Response to Arguments
It is noted that the claims are directed to a Drosophila cell not a Drosophila insect itself, and the Drosophila cell comprises a genetic modification that reduces expression of the FucT6 gene. The claims themselves do not require the entire organism or the Drosophila cell to exist for a period of time. What the claims recites is Drosophila cell comprises a genetic modification that reduces expression of the FucT6 gene with any a period of time no matter how short their existence is. As described above, Watanabe et al teach that molecular spectrum of spontaneous de novo mutations in male and female germline cells of drosophila melanogaster (Title), and mutation rate and its variability are central to theoretical population genetics and evolutionary studies (Page 1035, left column) and there is significant variability in rate and pattern of mutation among progenitor genotypes (Page 1035, right column). Keightley et al estimated the spontaneous mutation rate in Heliconius melpomene by genome sequencing of a pair of parents and 30 of their offspring, based on the ratio of number of de novo heterozygotes to the number of callable site-individuals …… an estimated mutation rate of 2.9x 10-9 (95% confidence interval, 1.3x10-9 –5.5 x10-9), which is similar to recent estimates in Drosophila melanogaster (Abstract). Thus, Watanabe et al and Keightley et al provide evidence that the Drosophila cells of the claimed invention (which are morphologically and phenotypically similar to Drosophila cells in nature ) would undergo spontaneous mutation that can encompass null mutations that render the loss of function of genes such as FucT6 and fdl. There is no evidence in record that FucT6 gene would be absolutely spared or exception from natural phenomenon of spontaneous de novo mutations (genetic modification) that reduces expression of FucT6 gene.
Previously, Applicants argue that silencing fucT6 would not be possible due to there would be no viable fucT6 cell. Yamamoto-Hino et al was cited to teach that “Depigmentation was caused by silencing of da6fut/fucT6, gfr/nac, Csat, and CG33145 (Fig. 6, Table 3). Since depigmentation in Drosophila is a naturally occurring event driven by genetic variation and environmental adaptation, and Yamamoto-Hino et al found that depigmentation was caused by at least in part by silencing of fucT6, these findings support Watanabe et al and Keightley et al teachings for de novo mutations Drosophila Melanogaster. Although Yamamoto-Hino et al stated that “silencing of 56 of these genes resulted in lethality before eclosion” (Page 522, left column, last para), it is clear that the cells with fucT6 silencing exist up to some points before eclosion. In support for this, the above cited reference Yang et al teach silencing FucT6 in D. melanogaster : “Lethal before adult eclosion or specific defects depending on studies” (Page 918, Table 3), and “FucT6-mutant D. melanogaster larvae exhibit a somewhat impaired immune response to parasitoid wasp infection, leading to a decreased encapsulation rate” (See page 919, right column, 2nd para.). Thus, it is clear that mutation in FucT6 exist in cells such as in D. melanogaster larvae. Zuberova et al was cited to provide evidence for FucT6 on chromosome X can be targeted (insertion) by transposon ( Note: A transposon is a mobile genetic element that can move from one location to another within a genome is a natural occurring source of mutations that can alter gene function) : Zuberova et al stated that “We screened a total of 49 deletions for chromosome X …… The originally identified deficiency Df(1)Exel9050 in the cytological localization 10D5-10D6 was molecularly defined by the transposon insertions P{XP}d00034 and PBac{RB}FucT6e02394” (Page 774, left column, 4th para.). Thus, Zuberova et al ’s teachings support Watanabe et al and Keightley et al teachings for de novo mutations Drosophila Melanogaster. It is noted that Watanabe et al teach that “single-gene mutations, including point mutations, insertions, and small deletions” (Page 1037, right column, 3rd para). Thus, there would be modification to reduces expression of the FucT6 gene.
As Per MPEP 2106.04(c) (C) Evaluating Characteristics To Determine Whether They Are "Markedly Different": The courts have emphasized that to show a marked difference, a characteristic must be changed as compared to nature, and cannot be an inherent or innate characteristic of the naturally occurring counterpart or an incidental change in a characteristic of the naturally occurring counterpart. Myriad, 569 U.S. at 580, 106 USPQ2d at 1974-75. Thus, in order to be markedly different, the inventor must have caused the claimed product to possess at least one characteristic that is different from that of the counterpart. In the instant case, there is nothing in the claimed Drosophila cell that possess at least one characteristic that is different from that of the counterpart. There is no evidence in record that FucT6 gene would be absolutely spared or exception from natural phenomenon of spontaneous de novo mutations (genetic modification) that reduces expression of FucT6 gene.
2. Applicant argue that
the instant claims recite a Drosophila cell having a genomic sequence created in the laboratory through human intervention by genetic modification of the FucT6 gene. Accordingly, the instant claims involve a human-directed manipulation of genetic material in a laboratory to generate a modified nucleic acid molecule, i.e., Drosophila cell genome, having an altered structure, to create something new, rather than merely discover what exists in nature (Remarks, page 11-12).
Response to arguments:
Applicants’ arguments are not commensurate to the scope of the claim. There is no limitation in the claims that specify “a genomic sequence created in the laboratory through human intervention by genetic modification of the FucT6 gene”. In fact, the term “genetic modification” is not defined in the instant disclosure. There is no teaching or guidance in the instant disclosure to define the term “genetic modification” to exclude natural occurring phenomenon such as mutation(s) that reduce(s) expression of the FucT6 gene.
Withdrawn - Claim Rejections - 35 USC § 103
Claims 6-7 were rejected under 35 U.S.C. 103 as being unpatentable over Mabashi-Asazuma et al (Pub.No.: US 2019/0359952 A1, Provisional application No. 61/885,294, filed on Oct. 1 , 2013) in view of Collingwood et al (Pub. No.: US 2011/0159541 A1, Pub. Date: Jun. 30, 2011) and Yamamoto-Hino et al (Genes to Cells (2015) 20, 521–542, DOI: 10.1111/gtc.12246, 04 May 2015). Upon further consideration, the previous rejections of claims are hereby withdrawn. Applicants' arguments with respect to the withdrawn rejections are thereby rendered moot. The claims are however subject to new rejections over the prior art of record, as set forth below.
Claims 11-14 and 20-41 were rejected under 35 U.S.C. 103 as being unpatentable over Mabashi-Asazuma et al (Pub.No.: US 2019/0359952 A1, Provisional application No. 61/885,294, filed on Oct. 1 , 2013) in view of Collingwood et al (Pub. No.: US 2011/0159541 A1, Pub. Date: Jun. 30, 2011) and Yamamoto-Hino et al (Genes to Cells (2015) 20, 521–542, DOI: 10.1111/gtc.12246, 04 May 2015) as applied to claim 6-7 above and further in view of Mabashi-Asazuma et al ( hereinafter “Mabashi-Asazuma-2015”, ACS Chem. Biol. 2015, 10, 2199−2208, DOI: 10.1021/acschembio.5b00340) and Jarvis et al (Pub. No.: US 2007/0067855A1, Pub. Date: Mar. 22, 2007). The rejection is withdrawn for the reasons discussed above.
Claims 15-16 are rejected under 35 U.S.C. 103 as being unpatentable over Mabashi-Asazuma et al (Pub.No.: US 2019/0359952 A1, Provisional application No. 61/885,294, filed on Oct. 1 , 2013) in view of Collingwood et al (Pub. No.: US 2011/0159541 A1, Pub. Date: Jun. 30, 2011) and Yamamoto-Hino et al (Genes to Cells (2015) 20, 521–542, DOI: 10.1111/gtc.12246, 04 May 2015) as applied to claim 6-7 above and further in view of Young et al (J. Microbiol. Biotechnol. (2008), 18(2), 383–391). The rejection is withdrawn for the reasons discussed above.
New-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.
Claims 6-7 are rejected under 35 U.S.C. 103 as being unpatentable over Kanda et al (Pub. No.: US 2006/0078991 Al, Pub. Date: Apr. 13, 2006) in view of Johansson et al (Methods Mol Biol. 2012:907:359-70. doi: 10.1007/978-1-61779-974-7_21)
Claim interpretation:
The specification of the claimed invention teaches that "FucT6 gene" is a term which also is meant to cover alpha-(1,6)- fucosyltransferase encoding genes in other insect cells than Drosophila melanogaster derived cells. In fact, the term "FucT6 gene" in the present context covers not only the gene in D. melanogaster, which encodes alpha-(1,6)-fucosyltransferase, but any equivalent gene encoding alpha-(1,6)-fucosyltransferase in other insect cells (see the instant specification Page 22, lines 6-10). Thus, FucT6 gene is interpreted to encompass alpha-(1,6)- fucosyltransferase encoding genes in other insect cells and Drosophila melanogaster derived cells.
Regarding to claims 6, Kanda et al teach “antibody composition-producing cell” and “an isolated host cell which have low or no 1,6- fucosyltransferase activity for adding fucose to N-acetylglucosamine of a reducing terminus of N-glycoside-linked sugar chains by deleting the gene encoding α l,6-fucosyltransferase gene or by adding a mutation to said gene to reduce or eliminate the fucosyltranferase activity” (see claim 1, page 101). Kanda et al teach “As the host cell used for the production of the host cell of the present invention, any cell such as yeast, animal cell, insect cell or plant cell can be used, so long as it has a gene encoding the target enzyme relating to the synthesis of an intracellular sugar nucleotide, GDP-fucose or the target enzyme relating to the modification of a sugar chain wherein 1-position of fucose is bound to 6-position of N-acetylglucosamine in the reducing end through α-bond in the complex N-glycoside-linked sugar chain” ([0342], page 16) and “Examples of the enzyme relating to the modification of a sugar chain wherein 1-position of fucose is bound to 6-position of N-acetylglucosamine in the reducing end through α-bond in the complex N-glycoside-linked sugar chain include α-1,6-fucosyltransferase, α-L-fucosidase and the like ([0438], page 23). Kanda et al teach “The gene disruption method may be any method, so long as it can disrupt the gene of the target enzyme is included” ([0334], page 16). Kanda et al teach antibodies produced from the modified cells have higher ADCC activities than antibodies from parent cells ([0069]-[0070], page 4).
Although Kanda et al teach antibody production in host cell such as insect cell or any cell having a gene encoding enzyme relating to the modification of a sugar chain such as α -1,6-fucosyltransferase can be used for antibody producing cell as described above, Kanda et al do not specify a Drosophila cell. Johansson et al cure the deficiency.
Johansson et al teach “production of recombinant antibodies in drosophila melanogaster s2 cells” (Title). Johansson et al teach “One of the major bottlenecks in antibody discovery for research and therapeutic applications is the need for large quantities of protein in a short amount of time. Here we describe an alternative method using the Drosophila melanogaster S2 expression system to produce high levels of antibodies (both IgG and Fab) with equivalent binding properties to antibodies produced in mammalian cell expression systems. Using the Drosophila S2 expression system for antibody production has many advantages over current mammalian systems making antibody expression, purification, and evaluation a much less time-consuming process” (Abstract)
Therefore, it would have been prima facie obvious for a person of ordinary skill in the art before the effective filing date of the rejected claims to combine the teachings of prior art to modify the method and host cells of Kanda et al by using drosophila melanogaster s2 cells for production of recombinant antibodies, with a reasonable expectation of success. Said modification amounting to combining prior art elements according to known methods to yield predictable results. One of ordinary skill in the art would have been motivated to do so because Johansson et al teach “the Drosophila expression system for antibody production offers the production of large amounts of pure, high quality mAbs or Fabs, utilizing a relatively low-cost and time-effective procedure” (Page 361, 3rd para.), and “Using the Drosophila S2 expression system for antibody production has many advantages over current mammalian systems making antibody expression, purification, and evaluation a much less time-consuming process” (Abstract). One of ordinary skill in the art would have had a reasonable expectation of success in doing so because Johansson et al were successful in production of antibody with Drosophila expression system, and provided detailed protocols with working example and data.
Regarding to claims 7, since Kanda et al teach an isolated host cell which has little or no 1,6- fucosyltransferase activity for adding fucose to N-acetylglucosamine of a reducing terminus of N-glycoside-linked sugar chains by deleting the gene encoding α l,6-fucosyltransferase gene or by adding a mutation to said gene to reduce or eliminate the fucosyltranferase activity (see claim 1, page 101). Thus, it is expected that there is little or no 1,6-fucosyltransferase activity for adding fucose to N-acetylglucosamine of N-glycoside-linked sugar chains (little or no α l,6-fucose). Additionally, if one were to knock out the gene encoding the drosophila 1,6-fucosyltransferase activity in an isolated host cell which has little or no 1,6- fucosyltransferase activity then one would expect that the resulting cells would produce N-glycosylated protein comprising less than 2% core α1,6-fucose. For example, this appears to be what happened when Applicant knocked out activity of the Fuct6 gene in drosophila cells: the instant disclosure teaches that “The FucT6 gene was successfully disrupted in a WT S2 cell line by the use of CRISPR/Cas9. The polyclonal pool showed an effect and the analyzed clone showed 100% absence of αl,6-fucose” (Page 34, lines 1-3) and “secretome glycan patterns with 0% fucose on the glycans upon LC-MS …. This clone shows a complete phenotypical knockout of the fucosyltransferase encoded by FucT6.” (see the instant specification on page 32, lines 17-20)
Claims 11-14 and 20-41 are rejected under 35 U.S.C. 103 as being unpatentable over Kanda et al (Pub. No.: US 2006/0078991 Al, Pub. Date: Apr. 13, 2006) in view of Johansson et al (Methods Mol Biol. 2012:907:359-70. doi: 10.1007/978-1-61779-974-7_21, First Online: 01 January 2012) as applied to claim 6-7 above and further in view of Mabashi-Asazuma et al ( hereinafter “Mabashi-Asazuma-2015”, ACS Chem. Biol. 2015, 10, 2199−2208, DOI: 10.1021/acschembio.5b00340) and Jarvis et al (Pub. No.: US 2007/0067855A1, Pub. Date: Mar. 22, 2007).
The teachings of Kanda et al and Johansson et al above are incorporated herein in their entirety.
The combined references do not teach Fdl gene has been disrupted. However, Mabashi-Asazuma-2015 cures the deficiency.
Regarding to claims 20, 21, 33, 36, and 39 Mabashi-Asazuma-2015 teach that “considering its functional and biotechnological significance, knocking out fdl has been of great interest for some time, as this would provide further information on FDL function and produce a novel insect cell derivative that would be an excellent starting point for efficient, bottom-up glycoengineering of its N-glycan processing pathway” (Page 2200, left column, 3rd para.) and this novel insect cell derivative would be an excellent starting point because Mabashi-Asazuma-2015 teach that “the new FDL-deficient S2 cell derivative produced in this study will enable future bottom-up glycoengineering efforts designed to isolate insect cell lines that can efficiently produce recombinant glycoproteins with chemically predefined oligosaccharide side-chain structures” (Abstract). Mabashi-Asazuma-2015 used the CRISPR-Cas9 system to edit the fdl gene in Drosophila melanogaster S2 cells. CRISPR-Cas9 editing produced a high frequency of site-specific nucleotide insertions and deletions, reduced the production of insect-type, paucimannosidic products (Man3GlcNAc2), and led to the production of partially elongated, mammalian-type complex Nglycans (GlcNAc2Man3GlcNAc2) in S2 cells …… the new FDL-deficient S2 cell derivative produced in this study will enable future bottom-up glycoengineering efforts …. (Abstract).
Therefore, it would have been prima facie obvious for a person of ordinary skill in the art before the effective filing date of the rejected claims to combine the teachings of prior art to modify the method of insect cells of the above references by using CRISPR-Cas9 system to edit the fdl gene as taught by Mabashi-Asazuma-2015, with reasonable expectation of success. Said modification amounting to combining prior art elements according to known methods to yield predictable results. One of ordinary skill in the art would have been motivated to introduce fdl genetic discruption into genetically modified drosophila cells as taught by the above references because Mabashi-Asazuma-2015 teaches that knocking out fdl gene would be an excellent starting point for efficient, bottom-up glycoengineering of its N-glycan processing pathway (Page 2200, left column, 3rd para.) because this approach can be used to isolate insect cell lines that can efficiently produce recombinant glycoproteins with chemically predefined oligosaccharide side-chain structures (Abstract). One of ordinary skill in the art would have had a reasonable expectation of success in doing so because Mabashi-Asazuma-2015 were successful in using CRISPR-Cas9 system to edit the fdl gene with instructions and working examples.
Regarding to claims 22 -23, Mabashi-Asazuma-2015 teach a baculovirus expression vector in Drosophila cells (S2 cells) and method for producing an N-glycosylated polypeptide of interest: “S2R+ cells were routinely maintained at 28 °C as adherent cultures in Schneider’s Drosophila medium …...” (Page 2205, right column, Methods, 4th para.) and “experiments were designed to assess the impact of fdl editing by CRISPR-Cas9 on the glycosylation of a recombinant human glycoprotein expressed in the S2 cell system. To achieve this goal, we transduced S2R+, DmFDLt1, DmFDLt2, and DmFDLt3 cells with a recombinant baculovirus encoding 8X His-tagged human erythropoietin (hEPO) under the control of the ie1 promoter as described in Methods. We subsequently affinity-purified hEPO from the cell-free media, treated equivalent samples with peptide-N4-(N-acetyl-β-glucosaminyl)asparagine amidase-F (PNGase-F) or reaction buffer alone, and analyzed the products by SDS-PAGE with Coomassie Brilliant Blue staining or Western blotting with anti-hEPO, as described in Methods.” (Page 2203, right column, 1st para.). Mabashi-Asazuma-2015 also teach Figure 5. Glycosidase analysis of the impact of fdl editing on hEPO glycosylation (N-glycosylated hEPO) (Page 2203).
Additionally, Mabashi Asazuma et al teach “…baculovirus-insect cell expression systems …… exploited a bacterial enzyme, GDP-4-dehydro-6 -deoxy-D-mannose reductase (Rmd),which consumes the precursor to GDP-L-fucose …... genetically transformed two different insect cell lines …… we constructed a novel baculovirus vector designed to express Rmd immediately after infection and to facilitate the insertion of genes encoding any glycoprotein of interest for expression at a later time after infection …...” ([0031], page 4). Mabashi Asazuma et al also teach “Recombinant Protein Expression and Purification : For small scale recombinant protein expression experiments ….” ([0062], page 8).
Regarding to claims 29, 30, 31, 32, 35, 38, and 41, Mabashi-Asazuma-2015 used the CRISPR-Cas9 system to edit the fdl gene in Drosophila melanogaster S2 cells (Abstract).
The above references do not teach insertion of at least one of a functional GlcNAcTI gene and a functional GlcNAcTII gene. However, Jarvis et al cures the deficiency.
Regarding to claims 26, 28, 34, 37, 40, Jarvis et al teach that the invention relates, e.g., to transgenic insects, or progeny thereof, whose cells contain at least one genomically integrated, expressible, nucleic acid encoding two or more of a set of Nglycosylation enzymes that can glycosylate a heterologous protein with a mammalianized (e.g., humanized) glycosylation pattern. The glycosylation genes are prefer ably expressed in the insect cells in catalytic amounts (Abstract). Jarvis et al teach “we incorporate mammalian glycosylation enzyme genes, including GlcNAc-TII, B4Gal-TI, ST6Gall, ST3GalIV. sialic acid synthase (SAS), and/or CMP-sialic acid synthetase (CMP-SAS) genes, into an insect genome to compensate for the lack of these enzymes in insect larvae.” ([0173], page 12) and “Addition of these transgenically engineered mammalian genes enables transgenic insect larvae to produce complex, terminally sialylated N-glycans ….additional mammalian genes encoding GlcNAc-TI or a CMP-sialic acid transporter into the transgenic insects are incorporated as necessary …...” ([0174], page 12-13). Jarvis et al also teach suitable expression control sequences that can function in insect cells such as Drosophila melanogaster hsp70 ([0102], page 6) and “Example VII : testing transformation efficiency and the effect of transgene expression in the model insect system, Drosophila melanogaster” (Page 15).
Therefore, it would have been prima facie obvious for a person of ordinary skill in the
art to combine the teachings of prior art to modify the insect cells of the above references by using mammalian glycosyltransferase genes such as GlcNAc-TI, GlcNAc-TII to glycosylate a heterologous protein with a mammalianized glycosylation pattern as taught by Jarvis et al as instantly claimed, with a reasonable expectation of success. Said modification amounting to combining prior art elements according to known methods to yield predictable results. One of ordinary skill in the art would be motivated to do so because Jarvis et al provide explicit advantage of using mammalian glycosyltransferase genes: “Partially or completely mammalianized polypeptides exhibit a number of advantages compared to polypeptides produced by an insect that lacks the glycosylation enzymes of the invention. These advantages include, e.g., enhanced stability when introduced into a mammal, altered activities, or the like.” ([0069], page 4). One of skill in the art would have been expected to have a reasonable expectation of success because Jarvis et al provide working example and data for using mammalian glycosyltransferase genes.
Regarding to claim 11, Jarvis et al teach “a transgenic insect, or progeny thereof, whose somatic and germ cells contain recombinant nucleic acid encoding …... a β-1,4-galactosyltransferase (see claim 1, page 23 of Jarvis et al).
Regarding to claims 12 - 13, Jarvis et al teach “ …. sialyltransferase genes are incorporated because ST6GalI and ST3GalTV transfer sialic acids in alpha 2.6- or alpha 2.3-linkages, respectively, …. ” ([0173], page 12).
Regarding to claim 14, Jarvis et al teach “ …. additional mammalian genes encoding GlcNAc-TI or a CMP-sialic acid transporter into the transgenic insects are incorporated as necessary ….” ([0174], page 12-13)
Regarding to claims 24, 25, 27, since the structural limitations of the claimed cells were rendered obvious by Kanda et al, Johansson et al, and Jarvis et al, it would be expected that the cells would produce N-glycosylated protein with less than 40% FM3/M3 glycan structures; produce N-glycosylated protein comprising at least 2% FAI/Al glycan structures; produce N-glycosylated protein comprising less than 15% FM3/M3 glycan structures, less than 30% FAI/Al glycan structures, and more than 50% FA2/A2 glycan structures.
Claims 15-16 are rejected under 35 U.S.C. 103 as being unpatentable over Kanda et al (Pub. No.: US 2006/0078991 Al, Pub. Date: Apr. 13, 2006) in view of Johansson et al (Methods Mol Biol. 2012:907:359-70. doi: 10.1007/978-1-61779-974-7_21) as applied to claim 6-7 above and further in view of Young et al (J. Microbiol. Biotechnol. (2008), 18(2), 383–391).
Claim interpretation:
It is known in the art that MGAT4 and MGAT5 are also synonymous with GNT-IV , GNT-V, respectively (N-Acetylglucosaminyltransferases).
The teachings of Kanda et al, Johansson et al above are incorporated herein in their entirety.
The combined references do not teach expression of functional Mgat4 and Mgat5 genes. However, Young et al cures the deficiency.
Young et al teach “Expression and Characterization of Human N-cetylglucosaminyltransferases
and α2,3-Sialyltransferase in Insect Cells for In Vitro Glycosylation of Recombinant Erythropoietin” (Title). In order to express glycosyltransferases in insect cells, cDNAs encoding human GnT II, GnT IV, GnT V, and ST3Gal IV were inserted into pFastBacMG to yield pFastMG-GnT II, pFastMG-GnT IV, pFastMG-GnT V, and pFastMG-ST3Gal IV, respectively (Fig. 1) (Page 386, left column, last para.).
Therefore, it would have been prima facie obvious for a person of ordinary skill in the art before the effective filing date of the rejected claims to combine the teachings of prior art to modify the method of insect cells of the above references by expressing GnT IV and GnT V (Mgat4 and Mgat5, respectively ) as taught by Young et al, with reasonable expectation of success. Said modification amounting to combining prior art elements according to known methods to yield predictable results. One of ordinary skill in the art would have been motivated to do so because Young et al stated that “GnT II, GnT IV, GnT V, and ST3Gal IV, which play important roles in the synthesis of tetraantennarytype complex glycan structures in mammalian cells, were overexpressed in Trichoplusia ni cells by using a baculovirus expression vector.” (Abstract) and “the above results indicated that glycosyltransferases, expressed in insect cells as N-terminus fusion proteins with IgG-binding domains, can be produced in large quantities as biochemically active forms from limited culture volumes, and they can be purified relatively simply” (Page 388, left column, last para.) and “the construction of glycosyltransferase gene transduced insect cell lines that can express humanized glycoproteins would eventually resolve the current obstacles for the production of complex glycans with highly sialylated forms in insect cells” (Page 390, left column, last para.). One of ordinary skill in the art would have had a reasonable expectation of success in doing so because Young et al were successful in expressing GnT IV and GnT V (Mgat4 and Mgat5, respectively ) in insect cells to produce glycosyltransferases in large quantities, with working examples and data.
Conclusion
No claim is allowed.
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/KHOA NHAT TRAN/Examiner, Art Unit 1632
/PETER PARAS JR/Supervisory Patent Examiner, Art Unit 1632
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