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 .
Application Status
This action is written in response to applicant’s correspondence received on 6/1//2026. Claims 38, 40-45, 47-50, and 52-59 are pending. Claims 1-37, 39, 46, and 51 have been cancelled. Claim 38 has been amended. Claim 59 is newly added All pending claims are currently under examination.
Any rejection or objection not reiterated herein has been overcome by amendment. 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. This Office Action is Final.
Claim Interpretation
The following claim interpretation is given with regards to the use of the term “exogenous” in the claims of the present application. Guidance for the definition and use of this term is offered in the specification of the present application in paragraph 36. Paragraph 36 of the specification states that:
“The term "exogenous protein chaperone" generally refers to a protein chaperone (e.g., a recombinant protein chaperone) that is not normally expressed by the bacterial strain used to prepare the bacterial extract, or a recombinant protein chaperone that is expressed by a nucleic acid construct that is not present in the native bacterial strain. For example, if the native bacterial strain used to prepare the bacterial extract naturally expresses low levels of the endogenous protein chaperone (e.g., at levels not sufficient to improve the expression levels of a biologically active protein of interest), the exogenous protein chaperone can be expressed from a non-native nucleic acid construct, such that the nucleic acid sequences encoding the exogenous protein chaperone are under the control of different regulatory sequences than the endogenous sequences encoding the chaperone. For example, the protein chaperones DsbC and FkpA are naturally occurring E. coli proteins, but their expression levels are below the limit of detection using the ELISA assays described herein to detect proteins in bacterial extracts. Thus, the term "exogenous" is synonymous with "heterologous," which refers to a protein chaperone not normally expressed by the bacterial strain used to prepare the bacterial extract, or a nucleic acid encoding the protein chaperone that is not present in the native bacterial strain”
Thus, the specification offers the proteins DsbC and FkpA, which are expressed in E. coli cells endogenously as examples of proteins which can be called “exogenous” if their expression levels are increased beyond their native expression levels, according to paragraph 36 of the specification. Thus, the term “exogenous “is interpreted in the context of the present application to include proteins that are native to a bacterial cell but are expressed using different, non-native expression systems. For example, an expression construct in E. coli controlling the expression of the endogenous DsbC gene would be considered “exogenous” if an inducible, non-native promoter were controlling the expression of the DsbC gene because such a construct would be a “recombinant protein chaperone that is expressed by a nucleic acid construct that is not present in the native bacterial strain.” This interpretation appears to align with the intention of the inventors who demonstrate, in Example 6 of the specification, the overexpression of the DsbC protein in E. coli cells. Furthermore, paragraph 60 of the specification states that the exogenous protein chaperone can be the DsbC gene and protein from E. coli. In addition, the overexpression of a disulfide isomerase (such as DsbC) is claimed in claim 38, and the bacterial host of claim 38 is recited as E. coli in claim 45. The claims recite the DsbC and FkpA genes, which are endogenous to E. coli, and also an E. coli host. Thus, it appears from the specification and claims that a reasonable interpretation of the term “exogenous” can refer to a protein that is in fact “endogenous” to a bacterial cell, but which is being expressed from non-native expression constructs, promoters, or other nucleic acid sequences.
Claim Rejections - 35 USC § 112 – New Matter Rejection
Claims 38, 40-45, 47-50, and 52-59 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.
Regarding claim 38 has been amended to currently recite the limitation “the bacterial strain comprises native genes encoding native disulfide isomerase and/or prolyl isomerase, and the native genes comprise altered codons to suppress recombination with the exogenous genes,” (final lines of the claim). Newly recited claim 59 also recites the same limitation. Thus, independent claim 38 and newly recited claim 59 recite that the bacterial strain comprises native genes encoding the isomerases, where the native gene has “altered” codons to suppress exogenous genes. This claim limitation is broadly and reasonably interpreted to mean that the isomerase genes which are naturally present in the bacterial strain (i.e., “native genes”) have themselves undergone sequence mutations to render “altered codons,” where the native chromosomal genes have had their codon sequence changed. This claim language is problematic because it does not appear to be present in the original application, drawings, or claims, as filed.
Regarding the specification, the Applicant has stated that the support for the present amendment can be found in paragraphs 167-173 and 176-179 (see Remarks filed 6/1/2026, first paragraph of page 7). With regards to codon alterations to suppress homologous recombination, the specification offers the following recitations:
“SBMT095 was made competent and then transformed with pACYC-Pc0-2xFkpA, a medium copy plasmid with two copies of FkpA behind a constitutive promoter. Both copies coded for wild type E. coli FkpA, but one gene had been synthesized to reduce nucleotide homology to the WT gene,“ (paragraph 167)
and:
“This strain has dual tandem copies of dsbC integrated into the bacterial galK locus, constitutively expressed using a modified MTcons-10 promoter (Thouvenot B. et al. Biochem J2004; 383:371-82). This is in addition to the wild type gene at the normal dsbC locus. The dual tandem gene cassette contains one copy of the parental dsbC gene, and one copy of a synthetic version of the dsbC gene designed to encode the wild type protein, but with altered codons to suppress unwanted sequence recombination with other versions of dsbC gene elsewhere in the genome. This DsbC overexpressing strain was transformed with the 2xFkpA plasmid to produce strain '2xD + 2xF',” (paragraph 176).
Thus, with regards to the specification as filed and its teachings with respect to “altered codons,” the specification recites that it is not the “native genes” which comprise “altered codons” to suppress recombination with the exogenous gene because the “native genes” have not undergone any alteration, as presently recited in claims 38 and 59. Rather, the sections of the specification referenced by the Applicant in support of the amendments (paragraphs 167-179) in fact recite that it is the copy of the genes present on the cassettes that are introduced into the cell which have “altered codons” relative to the wildtype gene (paragraphs 167 and 176, above). Thus, the specification does not recite or teach that the codons of native isomerase genes have been altered. The presently recited subject matter is therefore new matter, and is rejected under 112(a).
Claims 40-45, 47-50, and 52-59 depend from claim 38 and do not resolve this issue and are therefore also rejected. Furthermore, claim 59 recites claim language with the same issue as claim 38 and is rejected for the same reasons given above.
Claim Rejections - 35 USC § 103 – Maintained/Updated in Response to Amendments
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.
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 38, 40, 45, 47-48, 52, and 57-59 are rejected under 35 U.S.C. 103 as being unpatentable over Chatterjee (WO 2004/081033 A2, of record) in view of Anderson (US 2011/0136169), Kurokawa (Kurokawa Y et al. Appl Environ Microbiol. 2000 Sep;66(9):3960-5, of record), Ow (Ow DS et al. Microb Cell Fact. 2010 Apr 13;9:22, of record), Choi (Choi et al. Chemical Engineering Science, Volume 61, Issue 3, 2006, 876-885, of record), and Madden (WO 2010/085731 A2). The rejection is further evidenced by UniProt (UniProt Accession numbers, sequences, and alignments for UniProt accession numbers of E. coli K12 dsbC and DsbG genes, published 2010) and Shao (Shao F et al. J Biol Chem. 2000 May 5;275(18):13349-52).
Interpretation of Claim 38
As an initial matter, claims 38 and 59 can broadly be interpreted in the following manner: claims 38 and 59 recite “the bacterial strain comprises native genes encoding native disulfide isomerase and/or prolyl isomerase, and the native genes comprise altered codons to suppress recombination with the exogenous genes,” (step “b”, final lines of the claim). This claim language does not specify or establish a connection between the “native” isomerase genes and the “exogenous” disulfide and prolyl isomerases. In other words, step “b” of claims 38 and 59 do not establish or recite that the isomerase gene must be the same/encode the same protein as the exogenous disulfide and/or prolyl isomerase recited previously as the “exogenous” isomerases recited in step “i” of the claims. The “native” isomerase genes recited in the claims are therefore only structurally defined in that they comprise “altered” codons compared with the exogenous isomerases. Thus, claim 38 broadly reads on E. coli strains which natively comprise different isomerases with respect to exogenously introduced isomerases, which would comprise “altered” codons relative to the exogenous isomerases (see below).
Rejection
Regarding claim 38, Chatterjee is a patent document which focuses on the synthesis of proteins by cell-free protein expression (Abstract and throughout). Chatterjee teaches that the addition of molecular chaperones or foldases can be applied to their cell-free synthesis methods/systems, including the addition of both the DsbC and FkpA proteins as part of a protein cocktail (page 7, third paragraph, page 24-25, beginning with the final paragraph of page 24, page 45 first paragraph, and claims 43 and 44). Note that DsbC is a disulfide isomerase and FkpA is a prolyl isomerase (e.g., as defined by instant claim 40). Chatterjee teaches that the addition of the proteins DsbC and FkpA to the systems that they teach are useful because they improve expression of proteins (bottom of page 24 into page 25). Chatterjee teaches cell-free systems for protein synthesis, that such systems have commercial advantages, and furthermore that the addition of the chaperone/folding proteins DsbC in combination with FkpA to their systems offers advantages because they improve protein production (Background, first paragraph, and bottom of page 24 into page 25). Chatterjee therefore teaches a motivation to produce DsbC and FkpA proteins, so that they can be added to their cell-free synthesis systems.
Furthermore, Chatterjee also teaches a direct motivation and suggestion to produce/express the enzymes DsbC and FkpA in a cell because they teach that components of their synthesis system can include cellular extracts to supply enzymes used in the system, where such cellular extract are from E. coli (i.e., the foldase cocktail that they teach on page 7, third paragraph, for instance, which includes both DsbC and FkpA, and page 15, 3rd paragraph), thereby teaching a suggestion to make a bacterial strain comprising expression cassettes to make DsbC and FkpA to supply these advantageous proteins in cellular extracts to help improve their systems (e.g., page 6 second paragraph, page 9 second paragraph, claim 10).
Chatterjee teaches that their constructs can be made in E. coli cells such as K12 strains of E. coli (e.g., Example 6 page 46). With respect to strain K12, as evidenced by UniProt, E. coli strain K12 comprises two disulfide isomerases: dsbG and dsbC (see pages 2 and 6 of UniProt). As evidenced by Shao, DsbG is a disulfide isomerase (Title, Abstract) Furthermore, an alignment of E. Coli K12 isomerase DsbG and DsbC is given on page 1, where the region of closest identity is 27% (page 1 of UniProt). Thus, E. Coli strain K12 encodes multiple isomerase genes which encode protein sequences which are significantly different (UniProt pages 1-2 and 6). Thus, the “native” isomerase DsbG is broadly interpreted to comprise “altered codons” relative to the isomerase DsbC. Such differences in the gene encoding DsbG and an exogenously introduced DsbC would inherently “suppress recombination” between the two gene sequences. Thus, the E. coli strains taught by Chatterjee inherently comprise a “native” disulfide isomerase gene that has “altered codons” relative to DsbC with suppressed recombination as a result of an “altered codon” sequence (i.e., K12 encodes the disulfide isomerase DsbG).
Chatterjee, while teaching both the usefulness of DsbC and FkpA in an enzyme cocktail to improve expression of proteins in cell-free systems and a motivation and suggestion to express DsbC and FkpA in a single bacterial cell to use as a cellular extract in their methods, does not teach that the expression cassettes for these genes are linked to constitutive promoters. Furthermore, Chatterjee does not reduce to practice such cells. Chatterjee does not teach that the intracellular concentration of DsbC and FkpA is at least 1 mg/mL, or the integration of the exogenous genes into the galK loci.
Anderson is a patent document which focuses on expression of recombinant proteins in E. coli cells (Abstract and throughout). Anderson and Chatterjee therefore directly overlap in subject matter and endeavor because they both teach the expression of recombinant proteins in bacterial cells. Regarding DsbC and FkpA as well as their expression and relationship to proper protein folding, Anderson teaches that:
“not all host cells or organisms are equipped with the requisite chaperone proteins or express such proteins at adequate levels. In such instances, the chaperones may be co-expressed with the instant expression system as additional components to the expression vector above, or, alternatively, on a second “helper vector.” Such chaperones may include any elements commonly known in the art to assist with proper protein folding of the target protein, such as, but not limited to, heat shock proteins, isomerases, or the like. In certain embodiments, the proteins include one or a combination of DsbA, DsbC, FkpA, and/or Sura. In further embodiments, the helper plasmid which may be provided on the helper plasmid pTUM4,” (paragraph 88).
Thus, Anderson teaches a combination of DsbC and FkpA (selected from a list of only four foldases), where Anderson also teaches and understood that there is a motivation to overexpress such proteins to fit the desired design of a practitioner to help assist with the proper folding of proteins, teaching that host cells may require additional expression of such “commonly known” proteins to fit the adequate level desired by an artisan. Anderson therefore teaches the co-expression of DsbC and FkpA from the same cell, where furthermore Anderson also teaches that it is advantageous to overexpress these folding chaperones so that such chaperones can be produced at “adequate levels” (i.e., overexpressed) to ensure proper protein folding.
Regarding the reduction to practice of such cells, Kurokawa is a research article which focuses on the overexpression of the protein disulfide isomerase DsbC on a plasmid cassette system in E. coli (Abstract, Title, and see document). Kurokawa therefore teaches a bacterial strain (E. coli) comprising an expression cassette that expresses a high level of the disulfide isomerase DsbC (Abstract, Title, and see document). The DsbC gene was expressed in a non-native, arabinose-inducible expression construct (Materials and Methods, sections “Bacterial Strains and Plasmids” and “Culture Conditions and Protein Expression”), and thus the DsbC gene was an “exogenous” disulfide isomerase following the claim language interpretation outlined in the above “Claim Interpretation” section. Note also that the title of Kurokawa identifies DsbC as a “disulfide isomerase.” The Abstract of Kurokawa states that the DsbC proteins in their study were “overexpressed.” Furthermore, Kurokawa teaches that the overexpression of DsbC increased the expression of a protein of interest severalfold (Abstract), and further teaches that such overexpression is essential in stabilizing and solubilizing the expression of recombinant proteins of interest (Abstract and page 3960 in its entirety to 3961, left column, first paragraph). Kurokawa therefore also teaches a motivation to overexpress DsbC in E. coli (Abstract and page 3960 in its entirety to 3961, left column, first paragraph).
Ow teaches the overexpression of FkpA from an arabinose-inducible, non-native cassette in E. coli cells (Abstract Materials and Methods section entitled “Cloning and bacterial strains”), wherein the FkpA gene is under the control of an inducible promoter (i.e., it is overexpressed). Ow teaches that FkpA is a prolyl isomerase (background, second paragraph). Ow therefore teaches a bacterial strain (E. coli) comprising an expression cassette that expresses an exogenous (see “Claim Interpretation” section, above) prolyl isomerase. Furthermore, Ow teaches that the overexpression of FkpA is known to alleviate the stress response of E. coli during the accumulation of misfolded proteins, suppresses the formation of inclusion bodies, promotes proper protein folding, and significantly improves the solubility and functional expression of recombinant protein expression such as the expression of recombinant antibodies (scFv) (page 2, left column, second paragraph). Furthermore, Ow teaches and reduced to practice the expression of FkpA to increase scFv solubility and cell viability in a dose dependent manner (page 10, left column, second paragraph). Thus, Ow teaches a motivation to overexpress FkpA, as the benefits of FkpA in helping cell viability and proper protein folding are taught by Ow to be dose-dependent (page 10, left column, second paragraph).
Kurokowa and Ow have therefore reduced to practice the overexpression of both DsbC and FkpA separately in E. coli cells.
Choi is a review article that teaches the production of recombinant proteins in E. coli (Title, Abstract, and see document). Choi teaches that E. coli is widely used for the production of recombinant proteins for industrial purposes (Abstract). Choi teaches a number of constitutively expressed systems in industrial E. coli (Table 2). Choi teaches that inducible expression systems such as those taught by Kurokowa and Ow can be expensive, and that constitutive expression systems are a ready alternative (page 882, left column, first paragraph). Choi therefore teaches that the use of constitutive promoters to express proteins in E. coli is a known method in the industry that can also have advantages over inducible systems for proteins that are intended to be overexpressed (page 882, left column, first paragraph).
In addition, Madden is a patent document which teaches the development of recombinant microorganisms for the production of desired pathways (Title, Abstract, and throughout). Madden and Chatterjee therefore directly overlap in subject matter and field of endeavor because both teach the creation of recombinant microorganisms and methods of making such microorganisms. Madden teaches that it is a known and useful technique to integrate heterologous/exogenous genes into the chromosome of a desired microorganism (page 66, final paragraph). Furthermore, Madden teaches that “Genes cloned into MB5718 can be inserted into the chromosome at the galK locus allowing them to be stably expressed without the requirement for antibiotic selection,” (page 67, first paragraph). Thus, Madden teaches that, when creating recombinant microorganisms, the galK locus is a known safe harbor site for introducing heterologous genes into a bacterial chromosome, where furthermore there is an advantage to doing so because 1) galK is a known site to receive heterologous genes and 2) introducing heterologous genes into the galK locus allows for the stable introduction of heterologous genes into a chromosome (page 67, first paragraph).
It would have been obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to modify the bacterial cells comprising DsbC and FkpA as taught by Chatterjee to further include the linking of such expression to overexpressed promoters as suggested by Anderson, Kurokowa, Ow, to include constitutive promoters such as those taught by Choi, as such a combination is the simple combination of prior art elements according to a known method to yield predictable results. In the present case, Chatterjee has already taught a motivation to express DsbC and FkpA in the same cell, where Anderson has also taught the overexpression of the same proteins in a single cell. Thus, a practitioner would be motivated to overexpress the isomerases DsbC and FkpA in a cell to be used in the methods of Chatterjee, because Chatterjee directly teaches that such a cocktail is useful in their methods and can be paired with a cell extract comprising said cocktail. Apart from the motivation to create such a cell which expresses DsbC and FkpA from a constitutive promoter, the results were predictable because such overexpression of these proteins has already been accomplished and reduced to practice by both Kurokowa and Ow, who teach similar advantageous benefits as both Chatterjee and Anderson to overexpressing DsbC and FkpA. Furthermore, Choi teaches a motivation to make the promoters constitutive promoters because constitutive promoters can be cheaper. The results were predictable because Kurokowa, Ow, Court, and Choi are all discussing the genetic engineering of E.coli specifically, where overexpression has already been accomplished by Kurokowa and Ow. Therefore, as discussed above, the art is replete with motivational teachings and knowledge concerning the isomerases DsbC and FkpA, where Chatterjee, Anderson, Kurokowa, and Ow each teach these well-known folding chaperone proteins and each individually and in totality teach highly motivational suggestions to overexpress these proteins in a cell as the advantages are well known in the art. Furthermore, reduction to practice is predictable because to accomplish such reduction to practice would require only routine tools known in the art such as expression cassettes/plasmids, and promoters as taught by Anderson, Kurokowa, Ow, and Choi.
With regards to the claim limitation that the intracellular concentration be at least 1 mg/mL, although this specific value is not taught in Chatterjee, Anderson, Kurokowa, Ow, and Choi, a practitioner of ordinary skill in the art could arrive at this value by routine optimization and lab work. Furthermore, a practitioner would be motivated to overexpress the recited proteins especially in light of the fact that Ow teaches that the efficacy of the overexpressed proteins is in direct proportion to a dosage dependence (page 10, left column, second paragraph). A practitioner would therefore be motivated to optimize the expression levels of the beneficial proteins DsbC and FkpA taught by Chatterjee and Anderson, Kurokowa and Ow through routine methods to arrive at the presently claimed invention. Furthermore, MPEP 2144.05 Section II(A) specifically states that differences in concentration fall under routine optimization and will not support patentability unless there is evidence indicating such concentration is critical.
Additionally, the galK locus is already a known safe harbor expression site for bacterial gene integration, as taught by Madden. Thus, the combination including Madden is the simple combination of known prior art elements with predictable success. Furthermore, the practitioner is motivated to integrate heterologous genes into the galK locus because Madden teaches that such integration is useful, as it stabilizes offers stable integration into the chromosome.
Regarding claim 40, Chatterjee, Anderson, Kurokowa and Ow teach the DsbC and FkpA genes, respectively (see rejection of claim 38).
Regarding claim 45, Chatterjee teaches that the cells can be E. coli cell extracts (e.g., page 15, third paragraph, Example 6, page 46).
Regarding claim 47, Chatterjee teaches cellular extracts of the cells of claim 38 (e.g., page 15, second paragraph and throughout).
Regarding claim 48, Chatterjee teaches obtaining cell extracts from E. coli cells used for their methods, which reasonably includes culturing of the bacterial strain rendered obvious by the combination of Chatterjee/Anderson/Kurowa/Ow/Choi of claim 38. Furthermore, such culturing techniques are known. For instance, Kurokowa teaches culturing their bacterial strains and inducing said strains to overexpress DsbC (“Culture conditions and protein expression,” page 3961, left column, second paragraph). Ow also teaches culturing their bacterial strains and inducing the overexpression of FkpA (“Shake flask and bioreactor culturing,” page 11, left column, final paragraph).
Regarding claim 52, as discussed above in the rejection of claim 47, both Kurokowa and Ow teach the generation of cell free extracts ( Kurokowa “Fractionation and Analysis of Proteins,” page 3961, left column, third paragraph, and Ow, page 11, right column, third paragraph). Chatterjee also teaches that cell free extracts are used in their cell-free synthesis systems and methods (e.g., page 33, second paragraph). Chatterjee further teaches that their cell-free synthesis systems comprise one or more energy sources providing chemical energy for protein or biological macromolecule synthesis (claim 1 of Chatterjee), wherein the one of more energy sources generates or regenerates high-energy triphosphate compounds (claim 2 of Chatterjee, i.e., Chatterjee teaches an energy source for active oxidative phosphorylation), tRNAs (e.g., claim 11 and page 8, third paragraph), amino acids and ribosomes (page 8, third paragraph), a DNA template for protein synthesis (page 8, first paragraph). With regards to the claim limitations that the protein chaperone be expressed in the bacterial strain at a level of at least 1 gm/liter or extract, and that the protein of interest be expressed at a concentration of at least 100 mg/L, Chatterjee teaches that “the skilled artisan will recognize that concentrations of the various components of the incubation medium can be adjusted as is known in the art while still maintaining the synthetic function”, (page 33, second paragraph). Thus, the exact concentration of the protein chaperone and its expression could be determined through routine experimentation and optimization. Similarly, the concentration of the production of a protein of interest could be determined through routine optimization and experimentation of the methods of Chatterjee.
Regarding claim 57, Chatterjee teaches that the purpose of the inclusion of chaperones is to ensure expression and proper folding of proteins in their cell-free synthesis system (pages 24-25 and Example 4, which discusses the use of chaperones and folding in their methods). Example 4 of Chatterjee details a cell-free synthesis method where bacterial extracts are incubated under conditions to permit proper protein folding of a protein of interest (Example 4).
Regarding claim 58, Chatterjee teaches the importance of producing the isomerases DsbC and FkpA and therefore provides a motivation for the production of these proteins, which can be used in their commercially relevant cell-free synthesis systems (bottom of page 24 and into page 25, and see rejection of claim 41).
The prior art references cited do not explicitly teach that the isomerase is produced with a concentration of at least 1g/L.
It would have been obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to arrive at the presently recited claim limitation where the isomerase is present at a concentration of at least 1 gm/liter, because such a concentration could be arrived at through routine experimentation given that Chatterjee has already taught that the production of the recited isomerases has commercial value (see rejection of claim 41). A practitioner would therefore be motivated to optimize the expression of the recited isomerases in the extracts of the strains, and the recited concentration range could be arrived at through routine experimentation. See MPEP 2144.05, section II(A) for a discussion on routine optimization.
Regarding claim 59, these claim limitations are addressed in the rejection of claim 38, where both the galK locus is a known integration site and also bacterial cells inherently encode “native” isomerase genes which comprise “altered codons,” such as the dsbC and DsbG genes, where such coding sequence differences would inherently “suppress recombination” with the exogenous isomerase gene.
Claims 41-44, 49-50, and 53-56 are rejected under 35 U.S.C. 103 as being unpatentable over Chatterjee (WO 2004/081033 A2, of record) in view of Anderson (US 2011/0136169), Kurokawa (Kurokawa Y et al. Appl Environ Microbiol. 2000 Sep;66(9):3960-5, of record), Ow (Ow DS et al. Microb Cell Fact. 2010 Apr 13;9:22, of record), Choi (Choi et al. Chemical Engineering Science, Volume 61, Issue 3, 2006, 876-885, of record), and Madden (WO 2010/085731 A2) as applied to claims 38, 40, 45, 47-48,52, and 57-59 above, and further in view of Court (Court D et al. Annu Rev Genet. 2002;36:361-88, of record) and Jonasson (Jonasson P et al. Biotechnol Appl Biochem. 2002 Apr;35(2):91-105, of record).
Regarding claim 41, the rejection of claims 38 and 40 are discussed above. As discussed above, Chatterjee teaches that protein cocktails containing DsbC and FkpA are known to have beneficial properties used in systems such as cell free extracts (page 25 first paragraph).
None of Chatterjee, Kurokowa, Ow, Court, or Choi teach or suggest that their bacterial strains comprise two copies of the dsbC gene integrated into the chromosome.
Court is a review article that focuses on genetic engineering strategies in E. coli (throughout). Court teaches that “in vivo technologies have emerged that, due to
their efficiency and simplicity, may one day replace standard genetic engineering techniques. Constructs can be made on plasmids or directly on the Escherichia coli chromosome from PCR products or synthetic oligonucleotides by homologous recombination”, (Abstract). Court therefore teaches that, when manipulating E. coli strains, it was a known technique in the art that constructs can exist either on plasmids or as genomic integrations for the purposes of genetic engineering, and also that such techniques are efficient and simple (Abstract).
Jonasson is a review article focused on the facilitated production and recovery of recombinant proteins from E. coli (Title, Abstract, and throughout). Jonasson teaches the strategy of gene multimerization for increased protein yields in recombinant E. coli (page 100, left column, second paragraph). Jonasson teaches that using multiple copies of a gene can increase the yield of a target protein (page 100, left column, second paragraph).
It would have been obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to combine the modify the bacterial cells overexpressing DsbC and FkpA rendered obvious by Chatterjee, Anderson, Kurokowa, Ow, and Choi with multi-gene copy number integrated into the chromosome as taught and suggested by Jonasson and Court to arrive at a bacterial strain comprising two copies of the dsbC gene integrated into its chromosome because such a combination is simply the combination of prior art elements according to known methods to yield predictable results. Furthermore, a practitioner would be motivated to create a bacterial strain containing two copies of the dsbC gene because 1) multiple copies of a gene or sequence can improve yield of the protein product, as taught by Jonasson (page 100, left column, second paragraph) and 2) dsbC as a protein product was known to have value in the cell-free protein synthesis method taught by Chatterjee (Background, first paragraph, and bottom of page 24 into page 25). A practitioner would be motivated to produce as much dsbC as possible in order to use in the cocktail described by Chatterjee, and would therefore be motivated to create a bacterial strain capable of producing as much dsbC as possible. Inserting multiple copies of the dsbC gene into the genome of a bacterial strain would be an obvious solution to the need taught by Chatterjee as taught by the combination of Court, and Jonasson.
Regarding claim 42, as discussed above, Chatterjee also teaches that FkpA is an important gene product in the cell-free synthesis cocktails that they teach (Background, first paragraph, and bottom of page 24 into page 25). Kurokowa, Ow, Court, Choi, Chatterjee, and Jonasson do not teach the strain according to claim 41 further comprising a plasmid with two copies of the FkpA gene linked to a promoter.
It would have been obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to further modify the strains of E. coli rendered obvious by Kurokowa, Ow, Court, Choi, Chatterjee, Anderson, and Jonasson to create a bacterial cell strain capable of producing an abundance of FkpA as presently recited. A practitioner would be motivated to combine the teachings of Kurokawa, Ow, Court, Choi, Chatterjee, and Jonasson to include two copies of FkpA within the cell recited in claim 41, because FkpA enhances protein expression in the methods taught by Chatterjee, and FkpA is therefore a valuable protein to produce (Background, first paragraph, and bottom of page 24 into page 25). A practitioner would therefore be motivated to produce as much FkpA as possible, so that it could be used in a cell-free synthesis system taught by Chatterjee. To accomplish the production of FkpA, a practitioner could use the overexpression system of Ow and the suggestion to use multiple copies of the gene as suggested by Jonasson (page 100, left column, second paragraph). Furthermore, Court teaches that such genes can be introduced either on a plasmid or in the chromosome, thereby teaching ready alternatives to how exogenous genes can be expressed in a cell (Abstract).
Regarding claim 43, as discussed above, Court teaches integrating genes into a bacterial chromosome, and the advantages of multiple copies of FkpA in a bacterial strain are rendered obvious by the combination of Chatterjee, Anderson, Kurokowa, Ow, Choi, Court, and Jonasson (Court, Abstract).
Regarding claim 44, as discussed above, Court teaches integrating gene copies into a bacteria’s chromosome, and Jonasson teaches that including multiple copies of a target protein can increase yield (Court, Abstract, Jonasson page 100, left column, second paragraph). Chatterjee teaches that dsbC and FkpA are important proteins in their cell-free synthesis systems (Background, first paragraph, and bottom of page 24 into page 25). A practitioner would therefore be motivated to create a bacterial strain which overexpressed dsbC and FkpA, so that these proteins could be used in the systems taught by Chatterjee. Claim 44 is therefore simple combination of known prior art elements.
Regarding claims 49 and 50, these claim limitations are addressed in the rejections of claims 41-44. As discussed above, Court teaches both the expression of genes from the genome and from a plasmid (Abstract). A bacterial strain including two copies of DsbC and FkpA, wherein the gene copies are either in the chromosome or on plasmids is the simple combination of prior art elements to yield predictable results. The prior art elements (DsbC, FkpA, two copies of each gene, plasmid and/or chromosome integration), are discussed in the above rejections of claims 38, 40-45 and 47-48.
Regarding claim 53, a bacterial cell comprising two copies of the dsbC gene is discussed in the rejection of claim 41. Furthermore, Chatterjee teaches the use of cell extracts to be used in their cell-free synthesis systems (page 28, final paragraph).
Regarding claim 54, these claim limitations are addressed above in the rejection of claim 42. Furthermore, Chatterjee teaches the use of cell extracts to be used in their cell-free synthesis systems (page 28, final paragraph).
Regarding claim 55, these claim limitations are addressed above in the rejection of claim 43. Furthermore, Chatterjee teaches the use of cell extracts to be used in their cell-free synthesis systems (page 28, final paragraph).
Regarding claim 56, these claim limitations are addressed above in the rejection of claim 44. Furthermore, Chatterjee teaches the use of cell extracts to be used in their cell-free synthesis systems (page 28, final paragraph).
Additional 103 Rejection – Updated/Maintained
Claims 38, 40, 45, 47, 48 and 59 are rejected under 35 U.S.C. 103 as being unpatentable over Schlapschy (Schlapschy M et al. Protein Eng Des Sel. 2006 Aug;19(8):385-90) and Madden (WO 2010/085731 A2). The rejection is further evidenced by UniProt (UniProt Accession numbers, sequences, and alignments for UniProt accession numbers of E. coli K12 dsbC and DsbG genes, published 2010) and Shao (Shao F et al. J Biol Chem. 2000 May 5;275(18):13349-52).
Interpretation of Claim 38
As an initial matter, claims 38 and 59 can broadly be interpreted in the following manner: claims 38 and 59 recite “the bacterial strain comprises native genes encoding native disulfide isomerase and/or prolyl isomerase, and the native genes comprise altered codons to suppress recombination with the exogenous genes,” (step “b”, final lines of the claim). This claim language does not specify or establish a connection between the “native” isomerase genes and the “exogenous” disulfide and prolyl isomerases. In other words, step “b” of claims 38 and 59 do not establish or recite that the isomerase gene must be the same/encode the same protein as the exogenous disulfide and/or prolyl isomerase recited previously as the “exogenous” isomerases recited in step “i” of the claims. The “native” isomerase genes recited in the claims are therefore only structurally defined in that they comprise “altered” codons compared with the exogenous isomerases. Thus, claim 38 broadly reads on E. coli strains which natively comprise different isomerases with respect to exogenously introduced isomerases, which would comprise “altered” codons relative to the exogenous isomerases (see below).
Regarding claim 38, Schlapschy teaches a bacterial strain comprising one or more expression cassettes which comprise genes encoding an exogenous disulfide isomerase and prolyl isomerase, operably linked to constitutive promoters, where the expression cassettes express the isomerases (see Abstract for description of the pTUM4 plasmid, see page 387, left column, final paragraph, “constitutive expression” of several protein folding catalysts, including DsbC and FkpA). Thus, the pTUM4 plasmid taught by Schlapschy reads on the isomerases recited in claim 38. Furthermore, Schlapschy teaches that the pTUM4 is a useful helper vector which improves protein expression and folding, and therefore teaches that such overexpressed proteins as those on the plasmid (i.e., isomerases such as DsbC and FkpA) should be overexpressed to desired concentrations.
Furthermore, Schalpshcy teaches that they used E. coli K-12 strains (page 387, right column, final paragraph). With respect to strain K12, as evidenced by UniProt, E. coli strain K12 comprises two disulfide isomerases: dsbG and dsbC (see pages 2 and 6 of UniProt). As evidenced by Shao, DsbG is a disulfide isomerase (Title, Abstract) Furthermore, an alignment of E. Coli K12 isomerase DsbG and DsbC is given on page 1 of UniProt, where the region of closest identity is 27% (page 1 of UniProt). Thus, E. Coli strain K12 encodes multiple isomerase genes which encode protein sequences which are significantly different (UniProt pages 1-2 and 6). Thus, the “native” isomerase DsbG is broadly interpreted to comprise “altered codons” relative to the isomerase DsbC. Such differences in the gene encoding DsbG and an exogenously introduced DsbC would inherently “suppress recombination” between the two gene sequences. Thus, the E. coli strains taught by Schlapschy inherently comprise a “native” disulfide isomerase gene that has “altered codons” relative to DsbC with suppressed recombination as a result of an “altered codon” sequence (i.e., K12 encodes the disulfide isomerase DsbG).
Schlapshcy, while clearly teaching overexpression of disulfide/prolyl isomerase proteins such as DsbC and FkpA (see e.g., Figure 2) does not teach that the concentration is 1 mg/mL, or that the exogenous gene is integrated into the galK locus.
In addition, Madden is a patent document which teaches the development of recombinant microorganisms for the production of desired pathways (Title, Abstract, and throughout). Madden and Schlapshchy therefore directly overlap in subject matter and field of endeavor because both teach the creation of recombinant microorganisms and methods of making such microorganisms. Madden teaches that it is a known and useful technique to integrate heterologous/exogenous genes into the chromosome of a desired microorganism (page 66, final paragraph). Furthermore, Madden teaches that “Genes cloned into MB5718 can be inserted into the chromosome at the galK locus allowing them to be stably expressed without the requirement for antibiotic selection,” (page 67, first paragraph). Thus, Madden teaches that, when creating recombinant microorganisms, the galK locus is a known safe harbor site for introducing heterologous genes into a bacterial chromosome, where furthermore there is an advantage to doing so because 1) galK is a known site to receive heterologous genes and 2) introducing heterologous genes into the galK locus allows for the stable introduction of heterologous genes into a chromosome (page 67, first paragraph).
It would have been obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to optimize the pTUM4 plasmid already taught by Schlapschy to produce such useful folding proteins such as the isomerases DsbC and FkpA at a concentration of 1 mg/ml, because a practitioner of ordinary skill in the art could arrive at this value by routine optimization and lab work. Furthermore, a practitioner would be motivated to overexpress the recited proteins to a high concentration especially in light of the fact that Schlapschy teaches overexpressing this protein cocktail improves folding of proteins during recombinant expression (Abstract). A practitioner would therefore be motivated to optimize the expression levels of the beneficial proteins DsbC and FkpA taught by Schlapschy through routine methods to arrive at the presently claimed invention. Furthermore, MPEP 2144.05 Section II(A) specifically states that differences in concentration fall under routine optimization and will not support patentability unless there is evidence indicating such concentration is critical.
Additionally, the galK locus is already a known safe harbor expression site for bacterial gene integration, as taught by Madden. Thus, the combination including Madden is the simple combination of known prior art elements with predictable success. Furthermore, the practitioner is motivated to integrate heterologous genes into the galK locus because Madden teaches that such integration is useful, as it stabilizes offers stable integration into the chromosome.
Regarding claim 40, Schlapschy teaches that the isomerases are DsbC and FkpA (Abstract).
Regarding claim 45, Schlapschy teaches that the strain is E. coli (Title and throughout).
Regarding claim 47, Schlapschy teaches a cell free extract prepared from the strain (see for instance Figure 4 and caption).
Regarding claim 48, Schlapschy teaches a method of culturing the cells of claim 38 to overexpress the chaperones (page 387, left column, paragraphs 2-4).
Regarding claim 59, these limitations are addressed above in the rejection of claim 38.
Claims 41-44 and 49-50 are rejected under 35 U.S.C. 103 as being unpatentable over Schlapschy (Schlapschy M et al. Protein Eng Des Sel. 2006 Aug;19(8):385-90) and Madden (WO 2010/085731 A2) as applied to claims 38, 40, 45, 47, 48, and 59 above, and further in view of Court (Court D et al. Annu Rev Genet. 2002;36:361-88, of record) and Jonasson (Jonasson P et al. Biotechnol Appl Biochem. 2002 Apr;35(2):91-105, of record).
The teachings of Schlapschy/Madden are discussed above and incorporated herein.
Regarding claim 41, as discussed above, Schlapschy teaches the overexpression of genes FkpA and DsbC, as well as motivational teachings to overexpress such proteins within a cell (see above). Schlaphschy used a plasmid construct and constitutive promoters to express the DsbC and FkpA genes, and therefore does not teach chromosomal integration or the introduction of multiple copies of the DsbC and FkpA genes.
Court is a review article that focuses on genetic engineering strategies in E. coli (throughout). Court teaches that “in vivo technologies have emerged that, due to
their efficiency and simplicity, may one day replace standard genetic engineering techniques. Constructs can be made on plasmids or directly on the Escherichia coli chromosome from PCR products or synthetic oligonucleotides by homologous recombination”, (Abstract). Court therefore teaches that, when manipulating E. coli strains, it was a known technique in the art that constructs can exist either on plasmids or as genomic integrations for the purposes of genetic engineering, and also that such techniques are efficient and simple (Abstract).
Jonasson is a review article focused on the facilitated production and recovery of recombinant proteins from E. coli (Title, Abstract, and throughout). Jonasson teaches the strategy of gene multimerization for increased protein yields in recombinant E. coli (page 100, left column, second paragraph). Jonasson teaches that using multiple copies of a gene can increase the yield of a target protein (page 100, left column, second paragraph).
It would have been obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to modify the plasmid construct taught by Schlapschy by integrating multiple copies of the DsbC gene into the chromosome as such a combination is the simple combination of known prior art elements to yield predictable results. Furthermore, a practitioner would be motivated to combine the teachings of Schlapschy with Court and Jonasson to include two copies of FkpA within the cell recited, because FkpA enhances protein expression in the methods taught by Schlapschy (Abstract). A practitioner would therefore be motivated to produce as much FkpA as possible, as the overexpression of FkpA and DsbC were known to improve protein folding and expression, per Schlapschy. To accomplish the production of FkpA, a practitioner could use the overexpression system of Schalpschy and the suggestion to use multiple copies of the gene as suggested by Jonasson (page 100, left column, second paragraph). Furthermore, Court teaches that such genes can be introduced either on a plasmid or in the chromosome, thereby teaching ready alternatives to how exogenous genes can be expressed in a cell (Abstract).
Regarding claims 42-44 and 49-50, these claims recite similar inventinve concepts to claim 41, where multiple copies of the genes already taught by Schlapschy (i.e., DsbC or FkpA) are introduced either in multiple copies on plasmids or integrated into the chromosome. As discussed above, such integration techniques and methods of overexpression are already known and taught by Court/Jonasson, respectively. Therefore a practitioner could immediately envision switching plasmid expression for chromosomal integration to express either DsbC or FkpA, as taught by Court. Furthermore, the introduction of genes DsbC and FkpA in multiple copies is rendered obvious by the teachings of Jonasson, who teaches such an approach for an artisan to overexpress a desired protein, such as the beneficial and useful DsbC and FkpA proteins taught by Schlapschy.
Claims 52-58 are rejected under 35 U.S.C. 103 as being unpatentable over Schlapschy (Schlapschy M et al. Protein Eng Des Sel. 2006 Aug;19(8):385-90) and Madden (WO 2010/085731 A2) in view of Court (Court D et al. Annu Rev Genet. 2002;36:361-88, of record) and Jonasson (Jonasson P et al. Biotechnol Appl Biochem. 2002 Apr;35(2):91-105, of record) as applied to claims 38, 41-44 and 49-50, above, and further in view of Chatterjee (WO 2004/081033 A2, of record).
A discussion of the combination of Schlapschy, Court, and Jonasson is given above, the discussion incorporated herein.
Schlapschy, Court, and Jonasson do not teach a method involving cell-free synthesis such as that recited in claim 52.
Regarding claim 52, Chatterjee is a patent document that teaches cell-free synthesis methods (Title, Abstract, and throughout). Furthermore, Chatterjee also teaches that foldase cocktails containing DsbC and FkpA, such as the proteins taught by Schlapschy, should be used in their methods. Thus, the methods of Schlapschy and Chatterjee overlap in field of endeavor and area because both concern the production of proteins using chaperone cocktails including the same proteins (i.e., FkpA and DsbC). Furthermore, Chatterjee teaches that such cocktails which contain DsbC and FkpA are useful in their methods (page 24 final paragraph into page 25 first paragraph).
Chatterjee also teaches that cell free extracts are used in their cell-free synthesis systems and methods (e.g., page 33, second paragraph). Chatterjee further teaches that their cell-free synthesis systems comprise one or more energy sources providing chemical energy for protein or biological macromolecule synthesis (claim 1 of Chatterjee), wherein the one of more energy sources generates or regenerates high-energy triphosphate compounds (claim 2 of Chatterjee, i.e., Chatterjee teaches an energy source for active oxidative phosphorylation), tRNAs (e.g., claim 11 and page 8, third paragraph), amino acids and ribosomes (page 8, third paragraph), a DNA template for protein synthesis (page 8, first paragraph). With regards to the claim limitations that the protein chaperone be expressed in the bacterial strain at a level of at least 1 gm/liter or extract, and that the protein of interest be expressed at a concentration of at least 100 mg/L, Chatterjee teaches that “the skilled artisan will recognize that concentrations of the various components of the incubation medium can be adjusted as is known in the art while still maintaining the synthetic function”, (page 33, second paragraph). Thus, the exact concentration of the protein chaperone and its expression could be determined through routine experimentation and optimization. Similarly, the concentration of the production of a protein of interest could be determined through routine optimization and experimentation of the methods of Chatterjee.
It would have been obvious to a person of ordinary skill in the art before the effective filing date of the invention to modify the teachings of Schlapschy to apply their construct to the cell-free synthesis methods also taught by Chatterjee, as such a combination is the simple combination of prior art elements with predictable results. In the present case, Chatterjee teaches that cell-free synthesis are useful methods which have been reduced to practice, and furthermore teaches that the same proteins which Schlapschy teaches are useful for protein folding and expression are also useful in their methods. Thus, a practitioner is reasonably motivated to combine the teachings of Schlapshcy with the teachings of Chatterjee because they both teach the same isomerase proteins to be used with their methods, and both teach the benefits of using such isomerase cocktails. Furthermore, the results are predictable because Schlaspchy has already shown that such protein cocktails have been reduced to practice and are useful for helping proper protein folding and expression (Abstract).
Regarding claims 53-56, these claims recite various embodiments where DsbC and FkpA genes are introduced using either plasmids or chromosomal integration and in various copy numbers; these limitations are addressed in the rejection of claims 41-44, where claims 53-56 are rejected using the same rationale in that such minor variations could be envisioned by a person of ordinary skill in the art given the teachings of Court/Jonasson in light of the motivational teachings to include DsbC/FkpA per Schlapshcy/Chatterjee.
Regarding claim 57, Chatterjee teaches that the purpose of the inclusion of chaperones is to ensure expression and proper folding of proteins in their cell-free synthesis system (pages 24-25 and Example 4, which discusses the use of chaperones and folding in their methods). Example 4 of Chatterjee details a cell-free synthesis method where bacterial extracts are incubated under conditions to permit proper protein folding of a protein of interest (Example 4).
Regarding claim 58, Chatterjee teaches the importance of producing the isomerases DsbC and FkpA and therefore provides a motivation for the production of these proteins, which can be used in their commercially relevant cell-free synthesis systems (bottom of page 24 and into page 25, and see rejection of claim 41).
The prior art references cited do not explicitly teach that the isomerase is produced with a concentration of at least 1g/L.
It would have been obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to arrive at the presently recited claim limitation where the isomerase is present at a concentration of at least 1 gm/liter, because such a concentration could be arrived at through routine experimentation given that Chatterjee has already taught that the production of the recited isomerases has commercial value (page 24, final paragraph into page 25, first paragraph). A practitioner would therefore be motivated to optimize the expression of the recited isomerases in the extracts of the strains, and the recited concentration range could be arrived at through routine experimentation. See MPEP 2144.05, section II(A) for a discussion on routine optimization.
Response to Arguments
The Applicant’s arguments filed 6/1/2026 have been fully considered but are not persuasive. The Applicant argues that the exogenous genes integrated into the chromosome are not lost, where the strains can consistently output a concentration of protein. This argument is not persuasive, as integration of an exogenous gene into the chromosome, and specifically the galK locus, is a known strategy to stably introduce a gene into the chromosome (i.e., it is known that chromosomal integration will establish a gene that is not “lost over time” and that galK is a locus which can safely harbor such integrations of exogenous genes). Furthermore, the use of constitutive promoters to maintain high production of a desired protein is a known strategy as taught by prior art (see above). Furthermore, as discussed in the above 103 rejection, the finding that the presence of an increase in the concentration of disulfide and prolyl isomerases improves the yield of properly folded protein production is not an unexpected finding because the art teaches specifically that such chaperone improve the yield of properly folded proteins (103 rejection, above). The Applicant refers to Figures 13A and 13B, which show an increased yield in IgG antibodies. However, given that it was already known that the expression of such chaperones improves the yield of properly folded proteins, the result is not unpredictable or unexpected, where the prior art teaches such outcomes and therefore a motivation to generate such bacterial cells expressing the recited isomerases. Furthermore, the specific loci recited such as galK do not appear to offer a specific advantage, as the exogenous genes were integrated in operable functionality with a constitutive promoter and therefore do not rely upon the galK promoter, as recited in claim 38 and recited in the specification (e.g., paragraph 167). Furthermore, the specification does not demonstrate that galK and/or xylA are specifically advantageous, where Figure 13A and 13B simply show that the overexpression of dsbC and FkpA (from constitutive promoters) yields an increase in IgG folded protein production, which is to be expected because prior art such as Schlapshcy has already taught this phenomenon (Title and Abstract).
Double Patenting – Maintained/Updated in Response to Amendment
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.
As an initial matter, the following claim interpretation of claim 38 is given for each of the following rejections:
Interpretation of Claim 38
As an initial matter, claims 38 and 59 can broadly be interpreted in the following manner: claims 38 and 59 recite “the bacterial strain comprises native genes encoding native disulfide isomerase and/or prolyl isomerase, and the native genes comprise altered codons to suppress recombination with the exogenous genes,” (step “b”, final lines of the claim). This claim language does not specify or establish a connection between the “native” isomerase genes and the “exogenous” disulfide and prolyl isomerases. In other words, step “b” of claims 38 and 59 do not establish or recite that the isomerase gene must be the same/encode the same protein as the exogenous disulfide and/or prolyl isomerase recited previously as the “exogenous” isomerases recited in step “i” of the claims. The “native” isomerase genes recited in the claims are therefore only structurally defined in that they comprise “altered” codons compared with the exogenous isomerases. Thus, claim 38 broadly reads on E. coli strains which natively comprise different isomerases with respect to exogenously introduced isomerases, which would comprise “altered” codons relative to the exogenous isomerases (see below).
Furthermore, as each individual rejection below renders obvious the use of E. coli, the following inherent characteristics of the E. coli cells recited in the claims of corresponding patent documents and/or prior art is given and applied to each of the individual rejections below:
As evidenced by UniProt, E. coli strain comprises two disulfide isomerases: dsbG and dsbC (see pages 2 and 6 of UniProt). As evidenced by Shao, DsbG is a disulfide isomerase (Title, Abstract) Furthermore, an alignment of E. Coli K12 isomerase DsbG and DsbC is given on page 1 of UniProt, where the region of closest identity is 27% (page 1 of UniProt). Thus, E. Coli strain K12 encodes multiple isomerase genes which encode protein sequences which are significantly different (UniProt pages 1-2 and 6). Thus, the “native” isomerase DsbG is broadly interpreted to comprise “altered codons” relative to the isomerase DsbC. Such differences in the gene encoding DsbG and an exogenously introduced DsbC would inherently “suppress recombination” between the two gene sequences. Thus, the E. coli strains taught by the art inherently comprise a “native” disulfide isomerase gene that has “altered codons” relative to DsbC with suppressed recombination as a result of an “altered codon” sequence (i.e., K12 encodes the disulfide isomerase DsbG).
This above claim interpretation of claim 38 along with the inherent properties of E. coli cells is applied to each of the double patenting rejections below, and for the sake of brevity, is not repeated for each rejection.
Claims 38, 40-45, 47-50, and 52-59 are rejected on the ground of nonstatutory double patenting as being unpatentable over the claims of U.S. Patent No. US 10190145 B2 in view of Kurokawa (Kurokawa Y et al. Appl Environ Microbiol. 2000 Sep;66(9):3960-5), Ow (Ow DS et al. Microb Cell Fact. 2010 Apr 13;9:22), Court (Court D et al. Annu Rev Genet. 2002;36:361-88), Chatterjee (WO 2004/081033 A2), Choi (Choi et al. Chemical Engineering Science, Volume 61, Issue 3, 2006, 876-885), Jonasson (Jonasson P et al. Biotechnol Appl Biochem. 2002 Apr;35(2):91-105), and Madden (WO 2010/085731 A2). The rejection is further evidenced by UniProt (UniProt Accession numbers, sequences, and alignments for UniProt accession numbers of E. coli K12 dsbC and DsbG genes, published 2010) and Shao (Shao F et al. J Biol Chem. 2000 May 5;275(18):13349-52).
Regarding claim 38:
Claims 1-3 of US 10190145 B2 read as follows:
1. A bacterial cell free synthesis system for expressing biologically active proteins comprising: i) a cell free S30 extract of E. coli bacteria having an active oxidative phosphorylation system, containing biologically functioning tRNA, amino acids and ribosomes necessary for cell free protein synthesis and where the bacteria were transformed with genes encoding a disulfide isomerase and a prolyl isomerase wherein the two isomerases are expressed in the bacteria at a total concentration of at least 1 g/liter of extract; and ii) a nucleic acid encoding a protein of interest, wherein the protein of interest comprises a disulfide bond and a proline residue, and where a concentration of the protein of interest is increased compared to the sum of the concentration expressed by bacteria separately transformed with individual genes encoding the disulfide isomerase and the prolyl isomerase, and the incubation conditions are otherwise the same.
2. The system of claim 1, wherein the disulfide isomerase is DsbA, DsbB, DsbC, DsbD, or yeast PDI, and the prolyl isomerase is FkpA, SlyD, trigger factor (tig) or Skp.
3. The system of claim 1, wherein the bacteria from which the extract is prepared expresses at least one of the disulfide isomerase and the prolyl isomerase from a gene operably linked to a constitutive promoter
Regarding claims 38, 40-45, and 47 of the present application, the teachings of the prior art references Kurokowa, Ow, Court, Choi, Chatterjee, and Jonasson are given in the 103 rejection, above and recited here. The claim limitations of claims 1 and 2 of US 10190145 B2 appear to recite the same bacterial cell free synthesis systems and cell-free extracts of the present claims 47 and 52, which require a cell free extract from a bacterial strain. Claim 3 of US 10190145 B2 recites a bacterial strain from which the extract is prepared, wherein the bacterial strain expresses an excess of disulfide isomerase and prolyl isomerase. Claim 3 of US 10190145 B2 recites that the isomerases are operably linked to a constitutive promoter (i.e., the bacteria are expressing a high level of the protein).
Furthermore, Chatterjee teaches that E. coli K12 A19 can be used in cell-free synthesis systems such as those recited in claim 1 of ‘145 (page 39, fourth paragraph of Chatterjee).
Madden is a patent document which teaches the development of recombinant microorganisms for the production of desired pathways (Title, Abstract, and throughout). Madden teaches that it is a known and useful technique to integrate heterologous/exogenous genes into the chromosome of a desired microorganism (page 66, final paragraph). Furthermore, Madden teaches that “Genes cloned into MB5718 can be inserted into the chromosome at the galK locus allowing them to be stably expressed without the requirement for antibiotic selection,” (page 67, first paragraph). Thus, Madden teaches that, when creating recombinant microorganisms, the galK locus is a known safe harbor site for introducing heterologous genes into a bacterial chromosome, where furthermore there is an advantage to doing so because 1) galK is a known site to receive heterologous genes and 2) introducing heterologous genes into the galK locus allows for the stable introduction of heterologous genes into a chromosome (page 67, first paragraph).
It would have been obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention, to combine the teachings of either Kurokowa, Court, and Choi or Ow, Court, and Choi and to integrate either the DsbC construct of Kurokowa or the FkpA construct of Ow directly into the E. coli genome, as taught by Court because such a combination is the simple combination of prior art elements according to a known method to yield predictable results. The results were predictable because Kurokowa, Ow, Court, and Choi are all discussing the genetic engineering of E.coli specifically, and therefore there is a reasonable expectation of success that the genomic integration approach taught by Court would work in the bacterial strains taught by both Kurokowa and Ow. Furthermore, a practitioner would be motivated to constitutively express either the DsbC of Kurokowa or the FkpA of Ow because 1) Kurokawa and Ow teach the benefits of overexpressing these proteins (Abstract, Introduction of both, and see documents) and 2) Choi teaches that constitutive expression systems are not only alternatives to inducible systems such as those taught by Kurokowa and Ow but also have advantages such as the fact that they are cheaper (page 882, left column, first paragraph). Furthermore, a practitioner would have a reasonable expectation of success because Choi teaches that inducible systems are interchangeable with constitutive expression systems and also teaches working examples of constitutive expression systems (page 882, left column, first paragraph and Table 2).
Furthermore, Chatterjee teaches that such cell free synthesis systems can comprise bacterial strains which have ompT proteases (page 39, fourth paragraph). Thus, recitation of ompT proteases is obvious in view of the teachings of Chatterjee, where the combination of references is the simple substitution of one bacterial strain for another with predictable results because Chatterjee and ‘145 both concern the same subject matter and field of endeavor, where Chatterjee teaches that such ompT positive strains can be used for the purposes and systems recited.
With regards to the claim limitation that the intracellular concentration be at least 1 mg/mL, although this specific value is not taught in Kurokowa, Ow, Court, and Choi, a practitioner of ordinary skill in the art could arrive at this value by routine optimization and lab work. Furthermore, a practitioner would be motivated to overexpress the recited proteins especially in light of the fact that Ow teaches that the efficacy of the overexpressed proteins is in direct proportion to a dosage dependence (page 10, left column, second paragraph). A practitioner would therefore be motivated to optimize the expression levels of the beneficial proteins DsbC and FkpA taught by Kurokowa and Ow through routine methods to arrive at the presently claimed invention. Furthermore, MPEP 2144.05 Section II(A) specifically states that differences in concentration fall under routine optimization and will not support patentability unless there is evidence indicating such concentration is critical.
Additionally, the galK locus is already a known safe harbor expression site for bacterial gene integration, as taught by Madden. Thus, the combination including Madden is the simple combination of known prior art elements with predictable success. Furthermore, the practitioner is motivated to integrate heterologous genes into the galK locus because Madden teaches that such integration is useful, as it stabilizes offers stable integration into the chromosome.
Regarding claims 48-50, it would have been obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention, to combine the teachings of Kurokowa, Court, Choi, Chatterjee, and Jonasson, to arrive at a bacterial strain comprising two copies of the dsbC gene integrated into its chromosome because such a combination is simply the combination of prior art elements according to known methods to yield predictable results. Furthermore, a practitioner would have been motivated to create a bacterial strain containing two copies of the dsbC gene because 1) multiple copies of a gene or sequence can improve yield of the protein product, as taught by Jonasson and 2) dsbC as a protein product was known to have value in the cell-free protein synthesis method taught by Chatterjee. A practitioner would have been motivated to produce as much dsbC as possible in order to use in the cocktail described by Chatterjee, and would therefore be motivated to create a bacterial strain capable of producing as much dsbC as possible. Inserting multiple copies of the dsbC gene into the genome of a bacterial strain would be an obvious solution to the need taught by Chatterjee as taught by the combination of Kurokowa, Court, and Jonasson (see above).
Regarding claims 52-56, the claim limitations of claims 1 and 2 of US 10190145 B2 to recite the same bacterial cell free synthesis systems and cell-free extracts of the present claims 47 and 52 with the exception that claims 1 and 2 of US 10190145 B2 recite that the protein of interest contains a proline. However, proline is an amino acid which could be found in a “protein of interest.” Also note that claim 1 recites a cell free extract, and thus reads on the recitation of the present application’s claim 52, which recites “the cell free extract of claim 47”.
Regarding claim 57, claim 18 of US 10190145 B2 reads:
“A method of expressing properly folded, biologically active proteins in a bacterial cell free synthesis system comprising the steps of: i) combining a bacterial extract with a nucleic acid encoding a protein of interest comprising a disulfide bond and a proline residue; and ii) incubating the bacterial extract with the nucleic acid under conditions permitting the expression and proper folding of the protein of interest, wherein the bacterial extract is an S30 extract of E. coli bacteria comprising biologically functioning tRNA, amino acids, ribosomes necessary for cell free protein synthesis, a protein disulfide isomerase and a peptidyl-prolyl cis-trans isomerase, wherein the protein disulfide isomerase and the peptidyl-prolyl cis-trans isomerase are present at a concentration of at least 1 g/liter of extract, and where the expression of the protein of interest is increased to a concentration greater than the sum of the concentration expressed when the bacterial extract comprises one but not both of the protein disulfide isomerase and the peptidyl-prolyl cis-trans isomerase, and the incubation conditions are otherwise the same.”
Thus, claim 18 appears to recite the same limitations of claim 57 of the instant application.
Regarding claim 58, with regards to the claim limitation that an extract prepared from the bacterial strain comprises the isomerases at a concentration of be at least 1 g/L, although this specific value is not taught in Kurokowa, Ow, Court, and Choi, a practitioner of ordinary skill in the art could arrive at this value by routine optimization and lab work. Furthermore, a practitioner would be motivated to overexpress the recited proteins especially in light of the fact that Ow teaches that the efficacy of the overexpressed proteins is in direct proportion to a dosage dependence (page 10, left column, second paragraph). A practitioner would therefore be motivated to optimize the expression levels of the beneficial proteins DsbC and FkpA taught by Kurokowa and Ow through routine methods to arrive at the presently claimed invention. Furthermore, MPEP 2144.05 Section II(A) specifically states that differences in concentration fall under routine optimization and will not support patentability unless there is evidence indicating such concentration is critical.
Regarding claim 59, the limitations of this claim are addressed in the rejection of claim 38, above and in the evidentiary statement at the beginning of the Double Patenting section of this office action.
Claims 38, 40-45, 47-50, and 52-59 are rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1-3, 13, and 26 of U.S. Patent No. 10774354 B2 in view of Kurokawa (Kurokawa Y et al. Appl Environ Microbiol. 2000 Sep;66(9):3960-5), Ow (Ow DS et al. Microb Cell Fact. 2010 Apr 13;9:22), Court (Court D et al. Annu Rev Genet. 2002;36:361-88), ), Choi (Choi et al. Chemical Engineering Science, Volume 61, Issue 3, 2006, 876-885), Chatterjee (WO 2004/081033 A2), Jonasson (Jonasson P et al. Biotechnol Appl Biochem. 2002 Apr;35(2):91-105), and Madden (WO 2010/085731 A2). The rejection is further evidenced by UniProt (UniProt Accession numbers, sequences, and alignments for UniProt accession numbers of E. coli K12 dsbC and DsbG genes, published 2010) and Shao (Shao F et al. J Biol Chem. 2000 May 5;275(18):13349-52).
Claims 1-3 and 13 of US 10774354 B2 recite the following:
1. A bacterial cell free synthesis system for expressing biologically active proteins comprising: i) a cell free S30 extract of E. coli bacteria having an active oxidative phosphorylation system, containing biologically functioning tRNA, amino acids and ribosomes necessary for cell free protein synthesis and wherein an exogenous DsbC disulfide isomerase and an exogenous FkpA prolyl isomerase were expressed in the bacteria at a level of at least 1 g/liter of extract; and ii) a nucleic acid encoding a protein of interest, wherein the protein of interest comprises a disulfide bond and a proline residue, where said bacterial cell free synthesis system expresses the protein of interest to a concentration of at least about 100 mg/L.
2. The system of claim 1, wherein the bacteria from which the extract is prepared was co-transformed with genes encoding the exogenous DsbC disulfide isomerase and the exogenous FkpA prolyl isomerase.
3. The system of claim 1, wherein the bacteria from which the extract is prepared expresses at least one of the exogenous DsbC disulfide isomerase and the exogenous FkpA prolyl isomerase from a gene operably linked to a constitutive promoter.
Claim 13 recites that mutations in ompT or lonP may be present, which means that the bacteria from which the extract was prepared comprise ompT and/or lonP.
Regarding claims 38, 40-45, and 47 of the present application, the teachings of the prior art references Kurokowa, Ow, Court, Choi Chatterjee, and Jonasson are given in the 103 rejection, above. The claim limitations of claims 1 and 2 of US 10774354 B2 to recite the same bacterial cell free synthesis systems and cell-free extracts of the present claims 47 and 52, which require a cell free extract from a bacterial strain. Claim 3 of US 10774354 B2 recites a bacterial strain from which the extract is prepared, wherein the bacterial strain expresses an excess of DsbC and/or FkpA. Claim 3 of US 10774354 B2 recites that DsbC/FkpA are operably linked to a constitutive promoter (i.e., the bacteria are expressing a high level of the protein).
Furthermore, Chatterjee teaches that E. coli K12 A19 can be used in cell-free synthesis systems such as those recited in claim 1 of ‘145 (page 39, fourth paragraph of Chatterjee).
Madden is a patent document which teaches the development of recombinant microorganisms for the production of desired pathways (Title, Abstract, and throughout). Madden teaches that it is a known and useful technique to integrate heterologous/exogenous genes into the chromosome of a desired microorganism (page 66, final paragraph). Furthermore, Madden teaches that “Genes cloned into MB5718 can be inserted into the chromosome at the galK locus allowing them to be stably expressed without the requirement for antibiotic selection,” (page 67, first paragraph). Thus, Madden teaches that, when creating recombinant microorganisms, the galK locus is a known safe harbor site for introducing heterologous genes into a bacterial chromosome, where furthermore there is an advantage to doing so because 1) galK is a known site to receive heterologous genes and 2) introducing heterologous genes into the galK locus allows for the stable introduction of heterologous genes into a chromosome (page 67, first paragraph).
It would have been obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention, to combine the teachings of either Kurokowa, Court, and Choi or Ow, Court, and Choi and to integrate either the DsbC construct of Kurokowa or the FkpA construct of Ow directly into the E. coli genome, as taught by Court because such a combination is the simple combination of prior art elements according to a known method to yield predictable results. The results were predictable because Kurokowa, Ow, Court, and Choi are all discussing the genetic engineering of E.coli specifically, and therefore there is a reasonable expectation of success that the genomic integration approach taught by Court would work in the bacterial strains taught by both Kurokowa and Ow. Furthermore, a practitioner would be motivated to constitutively express either the DsbC of Kurokowa or the FkpA of Ow because 1) Kurokawa and Ow teach the benefits of overexpressing these proteins (Abstract, Introduction of both, and see documents) and 2) Choi teaches that constitutive expression systems are not only alternatives to inducible systems such as those taught by Kurokowa and Ow but also have advantages such as the fact that they are cheaper (page 882, left column, first paragraph). Furthermore, a practitioner would have a reasonable expectation of success because Choi teaches that inducible systems are interchangeable with constitutive expression systems and also teaches working examples of constitutive expression systems (page 882, left column, first paragraph and Table 2).
Furthermore, Chatterjee teaches that such cell free synthesis systems can comprise bacterial strains which have ompT proteases (page 39, fourth paragraph). Thus, recitation of ompT proteases is obvious in view of the teachings of Chatterjee, where the combination of references is the simple substitution of one bacterial strain for another with predictable results because Chatterjee and ‘354 both concern the same subject matter and field of endeavor, where Chatterjee teaches that such ompT positive strains can be used for the same purposes and systems recited in ‘354.
With regards to the claim limitation that the intracellular concentration be at least 1 mg/mL, although this specific value is not taught in Kurokowa, Ow, Court, and Choi, a practitioner of ordinary skill in the art could arrive at this value by routine optimization and lab work. Furthermore, a practitioner would be motivated to overexpress the recited proteins especially in light of the fact that Ow teaches that the efficacy of the overexpressed proteins is in direct proportion to a dosage dependence (page 10, left column, second paragraph). A practitioner would therefore be motivated to optimize the expression levels of the beneficial proteins DsbC and FkpA taught by Kurokowa and Ow through routine methods to arrive at the presently claimed invention. Furthermore, MPEP 2144.05 Section II(A) specifically states that differences in concentration fall under routine optimization and will not support patentability unless there is evidence indicating such concentration is critical.
Additionally, the galK locus is already a known safe harbor expression site for bacterial gene integration, as taught by Madden. Thus, the combination including Madden is the simple combination of known prior art elements with predictable success. Furthermore, the practitioner is motivated to integrate heterologous genes into the galK locus because Madden teaches that such integration is useful, as it stabilizes offers stable integration into the chromosome.
Regarding claims 48-50, it would have been obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to combine the teachings of Kurokowa, Court, Choi, Chatterjee, and Jonasson, to arrive at a bacterial strain comprising two copies of the dsbC gene integrated into its chromosome because such a combination is simply the combination of prior art elements according to known methods to yield predictable results. Furthermore, a practitioner would have been motivated to create a bacterial strain containing two copies of the dsbC gene because 1) multiple copies of a gene or sequence can improve yield of the protein product, as taught by Jonasson (throughout) and 2) dsbC as a protein product was known to have value in the cell-free protein synthesis method taught by Chatterjee (throughout). A practitioner would have been motivated to produce as much dsbC as possible in order to use in the cocktail described by Chatterjee, and would therefore be motivated to create a bacterial strain capable of producing as much dsbC as possible. Inserting multiple copies of the dsbC gene into the genome of a bacterial strain would be an obvious solution to the need taught by Chatterjee as taught by the combination of Kurokowa, Court, and Jonasson (see above).
Regarding claims 52-56, the claim limitations of claims 1-3 of US 10774354 B2 recite the same bacterial cell free synthesis systems and cell-free extracts of the present claims 47 and 52 with the exception that claims 1-3 of US 10774354 B2 recite that the protein of interest contains a proline. However, proline is an amino acid which could be found in a “protein of interest.” Also note that claim 1 recites a cell free extract, and thus reads on the recitation of the present application’s claim 52, which recites “the cell free extract of claim 47”.
Regarding claim 57, claim 26 of US 10774354 B2 recites:
26. A method of expressing properly folded, biologically active proteins in a bacterial cell free synthesis system comprising the steps of: i) incubating the bacterial cell free synthesis system of claim 1 under conditions permitting the expression and proper folding of the protein of interest.
Thus, claim 26 recites the same limitations of claim 57 of the instant application.
Regarding claim 58, with regards to the claim limitation that an extract prepared from the bacterial strain comprises the isomerases at a concentration of be at least 1 g/L, although this specific value is not taught in Kurokowa, Ow, Court, and Choi, a practitioner of ordinary skill in the art could arrive at this value by routine optimization and lab work. Furthermore, a practitioner would be motivated to overexpress the recited proteins especially in light of the fact that Ow teaches that the efficacy of the overexpressed proteins is in direct proportion to a dosage dependence (page 10, left column, second paragraph). A practitioner would therefore be motivated to optimize the expression levels of the beneficial proteins DsbC and FkpA taught by Kurokowa and Ow through routine methods to arrive at the presently claimed invention. Furthermore, MPEP 2144.05 Section II(A) specifically states that differences in concentration fall under routine optimization and will not support patentability unless there is evidence indicating such concentration is critical.
Regarding claim 59, these claim limitations are addressed in the rejection of claim 38, above, and in the evidentiary statement provided at the beginning of the Double Patenting rejection section of this office action.
Claims 38, 40, 45, and 47-48, and 59 are rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1-13 of U.S. Patent No. 9,650,621 B2 (‘621) in view of Chatterjee (WO 2004/081033 A2, of record), Anderson (US 2011/0136169), Kurokawa (Kurokawa Y et al. Appl Environ Microbiol. 2000 Sep;66(9):3960-5), Ow (Ow DS et al. Microb Cell Fact. 2010 Apr 13;9:22), Chatterjee (WO 2004/081033 A2) Choi (Choi et al. Chemical Engineering Science, Volume 61, Issue 3, 2006, 876-885), and Madden (WO 2010/085731 A2). The rejection is further evidenced by UniProt (UniProt Accession numbers, sequences, and alignments for UniProt accession numbers of E. coli K12 dsbC and DsbG genes, published 2010) and Shao (Shao F et al. J Biol Chem. 2000 May 5;275(18):13349-52).
The teachings of Chatterjee, Anderson, Kurokowa, Ow, Choi, Church and Jonasson from the 103 rejection are reiterated herein.
Regarding the claims of ‘621, claims 1-3 of ‘621 recite an OmpT1-sensistive RF1 protein. Furthermore, claims 6-7 and 11-13 recite bacterial cell-free synthesis systems, methods of preparing bacterial cell-free synthesis systems, and a method of expressing a protein of interest in a bacterial cell-free synthesis system comprising the combination of a nucleic acid template encoding a protein of interest with bacterial cell free synthesis extract to produce a bacterial cell-free synthesis system and expressing a protein of interest. Claim 8 recites a bacterial cell comprising the OmpT1-sensitive RF1 protein. Claim 8 also recites that nucleic acids are incorporated into the genome of the bacterial cell. Claim 11 furthermore recites that the bacterial strain is ompT positive. Claim 12 recites that the OmpT1 positive bacteria is an E. coli. Furthermore, claim 13 recites a method of cell-free synthesis where bacterial cells express wildtype ompT. Thus, the claims of the ‘621 patent are generally drawn to methods of protein expression using cell-free synthesis, bacterial extracts, and cells comprising components for cell-free synthesis and their extracts, where furthermore ompT positive E. coli cells are recited in claims 11 and 12.
Regarding claim 45, ‘621 recites that the cells are E. coli (claim 12)
Regarding claim 47, ‘621 recites a cell-free extract prepared from bacterial strains (claim 11).
Regarding claim 48, ‘621 recites a method of culturing a bacterial strain that permit the expression of exogenous proteins (claim 13).
With regards to claims 38, 40, 45, and 47-48, ‘621 does not recite the claim limitations of an exogenous disulfide isomerase or exogenous prolyl isomerase linked to a constitutive promoter and expressed in the cell at a concentration of 1 mg/mL, where the disulfide isomerase is DsbC and the prolyl isomerase is FkpA.
The teachings of Kurokowa, Ow, Chatterjee, Choi, are given inthe103 rejection above and included here.
Regarding claim 38, Kurokawa is a research article which focuses on the overexpression of the protein disulfide isomerase DsbC on a plasmid cassette system in E. coli (Abstract, Title, and see document). Kurokawa therefore teaches a bacterial strain (E. coli) comprising an expression cassette that expresses a high level of the disulfide isomerase DsbC (Abstract, Title, and see document). The DsbC gene was expressed in a non-native, arabinose-inducible expression construct (Materials and Methods, sections “Bacterial Strains and Plasmids” and “Culture Conditions and Protein Expression”), and thus the DsbC gene was an “exogenous” disulfide isomerase following the claim language interpretation outlined in the above “Claim Interpretation” section. Note also that the title of Kurokawa identifies DsbC as a “disulfide isomerase.” The Abstract of Kurokawa states that the DsbC proteins in their study were “overexpressed.” Furthermore, Kurokawa teaches that the overexpression of DsbC increased the expression of a protein of interest severalfold (Abstract), and further teaches that such overexpression is essential in stabilizing and solubilizing the expression of recombinant proteins of interest (Abstract and page 3960 in its entirety to 3961, left column, first paragraph). Kurokawa therefore also teaches a motivation to overexpress DsbC in E. coli (Abstract and page 3960 in its entirety to 3961, left column, first paragraph).
Ow teaches the overexpression of FkpA from an arabinose-inducible, non-native cassette in E. coli cells (Abstract Materials and Methods section entitled “Cloning and bacterial strains”), wherein the FkpA gene is under the control of an inducible promoter (i.e., it is overexpressed). Ow teaches that FkpA is a prolyl isomerase (background, second paragraph). Ow therefore teaches a bacterial strain (E. coli) comprising an expression cassette that expresses an exogenous (see “Claim Interpretation” section, above) prolyl isomerase. Furthermore, Ow teaches that the overexpression of FkpA is known to alleviate the stress response of E. coli during the accumulation of misfolded proteins, suppresses the formation of inclusion bodies, promotes proper protein folding, and significantly improves the solubility and functional expression of recombinant protein expression such as the expression of recombinant antibodies (scFv) (page 2, left column, second paragraph). Furthermore, Ow teaches and reduced to practice the expression of FkpA to increase scFv solubility and cell viability in a dose dependent manner (page 10, left column, second paragraph). Thus, Ow teaches a motivation to overexpress FkpA, as the benefits of FkpA in helping cell viability and proper protein folding are taught by Ow to be dose-dependent (page 10, left column, second paragraph).
Chatterjee is a patent document which focuses on the synthesis of proteins by cell-free protein expression (Abstract and throughout). Chatterjee teaches that the addition of molecular chaperones or foldases can be applied to their cell-free synthesis methods/systems, including the addition of both the disulfide isomerase DsbC and the prolyl isomerase FkpA proteins as part of a protein cocktail (page 7, third paragraph, page 24-25, beginning with the final paragraph of page 24, page 45 first paragraph, and claims 43 and 44). Chatterjee teaches that the addition of the proteins DsbC and FkpA isomerases to the cell-free synthesis systems that they teach are useful because they improve expression of proteins (bottom of page 24 into page 25). Chatterjee teaches cell-free systems for protein synthesis, that such systems have commercial advantages, and furthermore that the addition of the chaperone/folding proteins DsbC and FkpA to their systems offers advantages because they improve protein production (Background, first paragraph, and bottom of page 24 into page 25). Chatterjee therefore teaches a motivation to produce DsbC and FkpA proteins, so that they can be added to their cell-free synthesis systems.
Choi is a review article that teaches the production of recombinant proteins in E. coli (Title, Abstract, and see document). Choi teaches that E. coli is widely used for the production of recombinant proteins for industrial purposes (Abstract). Choi teaches a number of constitutively expressed systems in industrial E. coli (Table 2). Choi teaches that inducible expression systems such as those taught by Kurokawa and Ow can be expensive, and that constitutive expression systems are a ready alternative (page 882, left column, first paragraph). Choi therefore teaches that the use of constitutive promoters to express proteins in E. coli is a known method in the industry that can also have advantages over inducible systems for proteins that are intended to be overexpressed (page 882, left column, first paragraph).
Madden is a patent document which teaches the development of recombinant microorganisms for the production of desired pathways (Title, Abstract, and throughout). Madden teaches that it is a known and useful technique to integrate heterologous/exogenous genes into the chromosome of a desired microorganism (page 66, final paragraph). Furthermore, Madden teaches that “Genes cloned into MB5718 can be inserted into the chromosome at the galK locus allowing them to be stably expressed without the requirement for antibiotic selection,” (page 67, first paragraph). Thus, Madden teaches that, when creating recombinant microorganisms, the galK locus is a known safe harbor site for introducing heterologous genes into a bacterial chromosome, where furthermore there is an advantage to doing so because 1) galK is a known site to receive heterologous genes and 2) introducing heterologous genes into the galK locus allows for the stable introduction of heterologous genes into a chromosome (page 67, first paragraph).
It would have been obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to combine the claims of the ‘621 patent with either Kurokawa, Chatterjee, and Choi or Ow, Chatterjee, and Choi, and to include disulfide and/or prolyl isomerases such as DsbC and FkpA as taught by Kurokawa/Ow/Chatterjee because Kurokawa/Ow and Chatterjee teach that overexpression of such isomerases has great benefit in the expression and stabilization of proteins of interest. Furthermore, Chatterjee teaches a motivation to produce isomerases such as those taught by Kurokawa and Ow because they are useful in cell-free synthesis systems such those recited in the claims of ‘621 (Kurokawa Abstract and page 3960 in its entirety to 3961, left column, first paragraph, and Ow page 2, left column, second paragraph, and Chatterjee Background, first paragraph, and bottom of page 24 into page 25). Furthermore, ‘621, Kurokawa, Ow, Chatterjee, and Choi all concern the expression of recombinant proteins of interest and recombinant protein expression; a practitioner would therefore be motivated to combine the beneficial teachings of Kurokawa and Ow with Chatterjee and the claims of the ‘621 patent with a reasonable expectation of success because each of these documents is in the same field of endeavor. Furthermore, a practitioner would be motivated to constitutively express either the DsbC of Kurokawa or the FkpA of Ow because Kurokawa and Ow teach the benefits of overexpressing these proteins (Abstract, Introduction of both, and see documents). Additionally, Choi teaches that constitutive expression systems are not only alternatives to inducible systems such as those taught by Kurokawa and Ow but also have advantages such as the fact that they are cheaper (page 882, left column, first paragraph). Also, Chatterjee teaches that these proteins are useful in cell-free protein synthesis and therefore provides motivation to combine the teachings with ‘621 (Background, first paragraph, and bottom of page 24 into page 25). Furthermore, a practitioner would have a reasonable expectation of success because Choi teaches that inducible systems are interchangeable with constitutive expression systems and also teaches working examples of constitutive expression systems (page 882, left column, first paragraph and Table 2).
With regards to the claim limitation that the intracellular concentration be at least 1 mg/mL, although this specific value is not taught in Kurokawa, Ow, or Choi, a practitioner of ordinary skill in the art could arrive at this value by routine optimization and lab work. Furthermore, a practitioner would be motivated to overexpress the recited proteins especially in light of the fact that Ow teaches that the efficacy of the overexpressed proteins is in direct proportion to a dosage dependence (page 10, left column, second paragraph). A practitioner would therefore be motivated to optimize the expression levels of the beneficial proteins DsbC and FkpA taught by Kurokawa and Ow through routine methods to arrive at the presently claimed invention. Furthermore, MPEP 2144.05 Section II(A) specifically states that differences in concentration fall under routine optimization and will not support patentability unless there is evidence indicating such concentration is critical.
Additionally, the galK locus is already a known safe harbor expression site for bacterial gene integration, as taught by Madden. Thus, the combination including Madden is the simple combination of known prior art elements with predictable success. Furthermore, the practitioner is motivated to integrate heterologous genes into the galK locus because Madden teaches that such integration is useful, as it stabilizes offers stable integration into the chromosome.
Regarding claim 40, ‘621 does not recite that the exogenous disulfide isomerase is DsbC and the exogenous prolyl isomerase is FkpA.
Kurokawa and Ow teach the DsbC and FkpA genes, respectively (see rejection of claim 38). Kurokawa teaches that there are advantages to overexpressing DsbC including the stabilization of expressed recombinant proteins(Abstract and page 3960 in its entirety to 3961, left column, first paragraph).
Ow teaches that the overexpression of FkpA is known to alleviate the stress response of E. coli during the accumulation of misfolded proteins, suppresses the formation of inclusion bodies, promotes proper protein folding, and significantly improves the solubility and functional expression of recombinant protein expression such as the expression of recombinant antibodies (scFv) (page 2, left column, second paragraph).
It would have been obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to combine the claims of the ‘621 patent with either Kurokawa, Chatterjee, and Choi or Ow, Chatterjee, and Choi, and to include disulfide and/or prolyl isomerases such as DsbC and FkpA as taught by Kurokawa/Ow/Chatterjee because Kurokawa/Ow and Chatterjee teach that overexpression of such isomerases has great benefit in the expression and stabilization of proteins of interest.
Regarding claim 59, these claim limitations are addressed in the rejection of claim 38 above, and in the evidentiary statement discussed at the beginning of the Double Patenting section of this office action.
Claims 41-44, 49-50, and 52-58 are rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1-13 of U.S. Patent No. 9,650,621 B2 (‘621) in view of Chatterjee (WO 2004/081033 A2, of record), Anderson (US 2011/0136169) Kurokawa (Kurokawa Y et al. Appl Environ Microbiol. 2000 Sep;66(9):3960-5), Ow (Ow DS et al. Microb Cell Fact. 2010 Apr 13;9:22), Chatterjee (WO 2004/081033 A2), and Choi (Choi et al. Chemical Engineering Science, Volume 61, Issue 3, 2006, 876-885), and Madden (WO 2010/085731 A2) as applied to claims 38, 40, 45, 47-48, above, and further in view of Jonasson (Jonasson P et al. Biotechnol Appl Biochem. 2002 Apr;35(2):91-105).
The teachings of Chatterjee, Anderson, Kurokowa, Ow, Choi, Church and Jonasson from the 103 rejection are reiterated herein.
The claim recitations of ‘621 are discussed above. Regarding claim 41, the rejection of claims 38 and 40 are discussed above.
Chatterjee teaches a strong motivation to generate isomerases such as DsbC and FkpA because such proteins have beneficial effects in cell-free synthesis systems and methods such as those recited in the claims of ‘621 (Chatterjee Background, first paragraph, and bottom of page 24 into page 25, claims of ‘621).
Regarding claim 41, ‘621 does not recite the dsbC gene. None of ‘621, Kurokawa, Ow, Chatterjee, or Choi teach or suggest that their bacterial strains comprise two copies of the dsbC gene.
Jonasson is a review article focused on the facilitated production and recovery of recombinant proteins from E. coli (Title, Abstract, and throughout). Jonasson teaches the strategy of gene multimerization for increased protein yields in recombinant E. coli (page 100, left column, second paragraph). Jonasson teaches that using multiple copies of a gene can increase the yield of a target protein (page 100, left column, second paragraph).
It would have been obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to combine the claims of ‘621 and the teachings of Kurokawa, Ow, Choi, Chatterjee, and Jonasson, to arrive at a bacterial strain comprising two copies of the dsbC gene integrated into its chromosome because such a combination is simply the combination of prior art elements according to known methods to yield predictable results. Furthermore, a practitioner would be motivated to create a bacterial strain containing two copies of the dsbC gene because 1) multiple copies of a gene or sequence can improve yield of the protein product, as taught by Jonasson (page 100, left column, second paragraph) and 2) dsbC as a protein product was known to have value in the cell-free protein synthesis method taught by Chatterjee and ‘621 (Background, first paragraph, and bottom of page 24 into page 25, claim of ‘621). A practitioner would be motivated to produce as much dsbC as possible in order to use in the cocktail described by Chatterjee, and would therefore be motivated to create a bacterial strain capable of producing as much dsbC as possible. Inserting multiple copies of the dsbC gene into the genome of a bacterial strain would be an obvious solution to the need taught by Chatterjee as taught by the combination of ‘621, Kurokawa, Choi, and Jonasson.
Regarding claim 42, ‘621 does not recite the FkpA gene.
Chatterjee also teaches that FkpA is an important gene product in the cell-free synthesis cocktails that they teach (Background, first paragraph, and bottom of page 24 into page 25). Kurokawa, Ow, Choi, Chatterjee, and Jonasson do not teach the strain according to claim 41 further comprising a plasmid with two copies of the FkpA gene linked to a promoter.
It would have been obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to further modify the strains of E. coli rendered obvious by the claims of ‘621 and the teachings of Kurokawa, Ow, Choi, Chatterjee, and Jonasson to create bacterial cell strain capable of producing an abundance of FkpA as presently recited. A practitioner would be motivated to combine the teachings of ‘621, Kurokawa, Ow, Choi, Chatterjee, and Jonasson to include two copies of FkpA within the cell recited in claim 41, because FkpA enhances protein expression in the methods taught by Chatterjee, and FkpA is therefore a valuable protein to produce (Background, first paragraph, and bottom of page 24 into page 25). A practitioner would therefore be motivated to produce as much FkpA as possible, so that it could be used in a cell-free synthesis system taught by ‘621 and Chatterjee. To accomplish the production of FkpA, a practitioner could use the overexpression system of Ow and the suggestion to use multiple copies of the gene as suggested by Jonasson (page 100, left column, second paragraph).
Regarding claim 43, ‘621 claims integration of constructs into the chromosome (claim 8), and the advantages of multiple copies of FkpA in a bacterial strain are rendered obvious by the combination of Ow, Chatterjee, and Jonasson.
Regarding claim 44, ‘621 claims integrating gene copies into a bacteria’s chromosome (claim 8). Jonasson teaches that including multiple copies of a target protein can increase yield (Jonasson page 100, left column, second paragraph). Chatterjee teaches that dsbC and FkpA are important proteins in their cell-free synthesis systems such as those recited in the claims of ‘621 (Background, first paragraph, and bottom of page 24 into page 25). A practitioner would therefore be motivated to create a bacterial strain which overexpressed dsbC and FkpA, so that these proteins could be used in the systems taught by Chatterjee. Claim 44 is therefore the simple combination of known prior art elements.
Regarding claims 49 and 50, these claim limitations are addressed in the rejections of claims 41-44. As discussed above, a bacterial strain including two copies of DsbC and FkpA, wherein the gene copies are either in the chromosome or on plasmids, is the simple combination of prior art elements to yield predictable results. The prior art elements (DsbC, FkpA, two copies of each gene, plasmid and/or chromosome integration), are discussed in the above rejections of claims 38, 40-45 and 47-48.
Regarding claim 52, ‘621 recites bacterial cell-free synthesis systems, methods of preparing bacterial cell-free synthesis systems, and a method of expressing a protein of interest in a bacterial cell-free synthesis system comprising the combination of a nucleic acid template encoding a protein of interest with bacterial cell free synthesis extract to produce a bacterial cell-free synthesis system and expressing a protein of interest (claims 6-7 and 11-13).
‘621 does not recite an active oxidation phosphorylation system, tRNA, amino acids and ribosomes for cell-free protein synthesis, or that the exogenous prolyl isomerase or disulfide isomerase were expressed at a level of at least 1 gm/liter of extract. ‘621 does not recite that the protein of interest is expressed at a level of 100 mg/L.
Both Kurokawa and Ow teach the generation of cell free extracts (Kurokawa “Fractionation and Analysis of Proteins,” page 3961, left column, third paragraph, and Ow, page 11, right column, third paragraph).
Chatterjee also teaches that cell free extracts are used in their cell-free synthesis systems and methods (e.g., page 33, second paragraph). Chatterjee further teaches that their cell-free synthesis systems comprise one or more energy sources providing chemical energy for protein or biological macromolecule synthesis (claim 1 of Chatterjee), wherein the one of more energy sources generates or regenerates high-energy triphosphate compounds (claim 2 of Chatterjee, i.e., Chatterjee teaches an energy source for active oxidative phosphorylation), tRNAs (e.g., claim 11 and page 8, third paragraph), amino acids and ribosomes (page 8, third paragraph), a DNA template for protein synthesis (page 8, first paragraph).
It would have been obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to combine the teachings of ‘621 with Court, Kurokawa, Ow, Chatterjee, Choi, and Jonasson because such a combination is the simple combination of known prior art elements to yield predictable results. Furthermore, the claims of ‘621 recite methods of cell-free synthesis; a practitioner would therefore be motivated to incorporate the components of cell-free synthesis systems taught by Chatterjee with ‘621 simply so that the systems of ‘621 would be functional (claims 3-15 of ‘621). With regards to the claim limitations that the protein chaperone be expressed in the bacterial strain at a level of at least 1 gm/liter or extract, and that the protein of interest be expressed at a concentration of at least 100 mg/L, Chatterjee teaches that “the skilled artisan will recognize that concentrations of the various components of the incubation medium can be adjusted as is known in the art while still maintaining the synthetic function”, (page 33, second paragraph). Thus, the exact concentration of the protein chaperone and its expression could be determined through routine experimentation and optimization. Similarly, the concentration of the production of a protein of interest could be determined through routine optimization and experimentation of the methods of Chatterjee.
Regarding claim 53, ‘621 also recites cell free extracts to be used in cell-free synthesis systems (claim 13). A bacterial cell comprising two copies of the dsbC gene is discussed in the rejection of claim 41. Furthermore, Chatterjee teaches the use of cell extracts to be used in their cell-free synthesis systems (page 28, final paragraph).
Regarding claim 54, ‘621 also recites cell free extracts to be used in cell-free synthesis systems (claim 13). The claim limitations of claim 54 are addressed above in the rejection of claim 42. Furthermore, Chatterjee teaches the use of cell extracts to be used in their cell-free synthesis systems (page 28, final paragraph).
Regarding claim 55, ‘621 also recites cell free extracts to be used in cell-free synthesis systems (claim 13). The claim limitations of claim 55 are addressed above in the rejection of claim 43. Furthermore, Chatterjee teaches the use of cell extracts to be used in their cell-free synthesis systems (page 28, final paragraph).
Regarding claim 56, ‘621 also recites cell free extracts to be used in cell-free synthesis systems (claim 13). The claim limitations of claim 56 are addressed above in the rejection of claim 44. Furthermore, Chatterjee teaches the use of cell extracts to be used in their cell-free synthesis systems (page 28, final paragraph).
Regarding claim 57, ‘621 recites a method of expressing a recombinant protein of interest in a cell free synthesis system.
Regarding claim 58, ‘621 and the prior art references cited do not explicitly teach that the isomerase is produced with a concentration of at least 1g/L.
Chatterjee teaches the importance of producing the isomerases DsbC and FkpA and therefore provides a motivation for the production of these proteins, which can be used in their commercially relevant cell-free synthesis systems (bottom of page 24 and into page 25, and see rejection of claim 41).
It would have been obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to arrive at the presently recited claim limitation where the isomerase is present at a concentration of at least 1 gm/liter, because such a concentration could be arrived at through routine experimentation given that Chatterjee has already taught that the production of the recited isomerases has commercial value (see rejection of claim 41). A practitioner would therefore be motivated to optimize the expression of the recited isomerases in the extracts of the strains, and the recited concentration range could be arrived at through routine experimentation. See MPEP 2144.05, section II(A) for a discussion on routine optimization.
Claims 38, 40, 45, and 47-48 , and 59 are rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1-13 of U.S. Patent No. 10,450,353 B2 (‘353) in view of Chatterjee (WO 2004/081033 A2, of record), Anderson (US 2011/0136169)Kurokawa (Kurokawa Y et al. Appl Environ Microbiol. 2000 Sep;66(9):3960-5), Ow (Ow DS et al. Microb Cell Fact. 2010 Apr 13;9:22), Chatterjee (WO 2004/081033 A2) and Choi (Choi et al. Chemical Engineering Science, Volume 61, Issue 3, 2006, 876-885) and Madden (WO 2010/085731 A2). The rejection is further evidenced by UniProt (UniProt Accession numbers, sequences, and alignments for UniProt accession numbers of E. coli K12 dsbC and DsbG genes, published 2010) and Shao (Shao F et al. J Biol Chem. 2000 May 5;275(18):13349-52).
The teachings of Chatterjee, Anderson, Kurokowa, Ow, Choi, Church and Jonasson from the 103 rejection are reiterated herein.
Regarding the claims of ‘353, claims 1-3 of ‘353 recite an OmpT1-sensistive RF1 protein and a bacterial cell comprising the OmpT1 sensitive RF1 protein. Claim 3 recites that the bacterial strain is positive for OmpT1. Claim 5 recites the incorporation of nucleic acid constructs into the genome of a cell. Furthermore, claims 6-7 and 11-12 recite bacterial cell-free synthesis systems, methods of preparing bacterial cell-free synthesis systems, and a method of expressing a protein of interest in a bacterial cell-free synthesis system comprising the combination of a nucleic acid template encoding a protein of interest with bacterial cell free synthesis extract to produce a bacterial cell-free synthesis system and expressing a protein of interest. Claim 11 further recites that the strain has a wild-type ompT. Claim 12 also recites that the cultured bacteria is ompT positive. Claim 13 recites that the OmpT1 positive bacteria is an E. coli. Thus, ‘353 is generally drawn to methods of protein expression using cell-free synthesis, bacterial extracts, and cells comprising components for cell-free synthesis and their extracts.
Regarding claim 45, ‘353 recites that the bacteria is an E. coli (claim 13).
Regarding claim 47,’353 recites a cell free synthesis extract (claim 12).
Regarding claim 48, ‘353 recites a method comprising culturing a bacterial strain to permit the overexpression of a protein of interest (claim 12).
With regards to claims 38, 40, 45, and 47-48, ‘353 does not recite the claim limitations of an exogenous disulfide isomerase or exogenous prolyl isomerase linked to a constitutive promoter and expressed in the cell at a concentration of 1 mg/mL, where the disulfide isomerase is DsbC and the prolyl isomerase is FkpA.
Regarding claim 38, Kurokawa is a research article which focuses on the overexpression of the protein disulfide isomerase DsbC on a plasmid cassette system in E. coli (Abstract, Title, and see document). Kurokawa therefore teaches a bacterial strain (E. coli) comprising an expression cassette that expresses a high level of the disulfide isomerase DsbC (Abstract, Title, and see document). The DsbC gene was expressed in a non-native, arabinose-inducible expression construct (Materials and Methods, sections “Bacterial Strains and Plasmids” and “Culture Conditions and Protein Expression”), and thus the DsbC gene was an “exogenous” disulfide isomerase following the claim language interpretation outlined in the above “Claim Interpretation” section. Note also that the title of Kurokawa identifies DsbC as a “disulfide isomerase.” The Abstract of Kurokawa states that the DsbC proteins in their study were “overexpressed.” Furthermore, Kurokawa teaches that the overexpression of DsbC increased the expression of a protein of interest severalfold (Abstract), and further teaches that such overexpression is essential in stabilizing and solubilizing the expression of recombinant proteins of interest (Abstract and page 3960 in its entirety to 3961, left column, first paragraph). Kurokawa therefore also teaches a motivation to overexpress DsbC in E. coli (Abstract and page 3960 in its entirety to 3961, left column, first paragraph).
Ow teaches the overexpression of FkpA from an arabinose-inducible, non-native cassette in E. coli cells (Abstract Materials and Methods section entitled “Cloning and bacterial strains”), wherein the FkpA gene is under the control of an inducible promoter (i.e., it is overexpressed). Ow teaches that FkpA is a prolyl isomerase (background, second paragraph). Ow therefore teaches a bacterial strain (E. coli) comprising an expression cassette that expresses an exogenous (see “Claim Interpretation” section, above) prolyl isomerase. Furthermore, Ow teaches that the overexpression of FkpA is known to alleviate the stress response of E. coli during the accumulation of misfolded proteins, suppresses the formation of inclusion bodies, promotes proper protein folding, and significantly improves the solubility and functional expression of recombinant protein expression such as the expression of recombinant antibodies (scFv) (page 2, left column, second paragraph). Furthermore, Ow teaches and reduced to practice the expression of FkpA to increase scFv solubility and cell viability in a dose dependent manner (page 10, left column, second paragraph). Thus, Ow teaches a motivation to overexpress FkpA, as the benefits of FkpA in helping cell viability and proper protein folding are taught by Ow to be dose-dependent (page 10, left column, second paragraph).
Chatterjee is a patent document which focuses on the synthesis of proteins by cell-free protein expression (Abstract and throughout). Chatterjee teaches that the addition of molecular chaperones or foldases can be applied to their cell-free synthesis methods/systems, including the addition of both the disulfide isomerase DsbC and the prolyl isomerase FkpA proteins as part of a protein cocktail (page 7, third paragraph, page 24-25, beginning with the final paragraph of page 24, page 45 first paragraph, and claims 43 and 44). Chatterjee teaches that the addition of the proteins DsbC and FkpA isomerases to the cell-free synthesis systems that they teach are useful because they improve expression of proteins (bottom of page 24 into page 25). Chatterjee teaches cell-free systems for protein synthesis, that such systems have commercial advantages, and furthermore that the addition of the chaperone/folding proteins DsbC and FkpA to their systems offers advantages because they improve protein production (Background, first paragraph, and bottom of page 24 into page 25). Chatterjee therefore teaches a motivation to produce DsbC and FkpA proteins, so that they can be added to their cell-free synthesis systems.
Choi is a review article that teaches the production of recombinant proteins in E. coli (Title, Abstract, and see document). Choi teaches that E. coli is widely used for the production of recombinant proteins for industrial purposes (Abstract). Choi teaches a number of constitutively expressed systems in industrial E. coli (Table 2). Choi teaches that inducible expression systems such as those taught by Kurokawa and Ow can be expensive, and that constitutive expression systems are a ready alternative (page 882, left column, first paragraph). Choi therefore teaches that the use of constitutive promoters to express proteins in E. coli is a known method in the industry that can also have advantages over inducible systems for proteins that are intended to be overexpressed (page 882, left column, first paragraph).
Madden is a patent document which teaches the development of recombinant microorganisms for the production of desired pathways (Title, Abstract, and throughout). Madden teaches that it is a known and useful technique to integrate heterologous/exogenous genes into the chromosome of a desired microorganism (page 66, final paragraph). Furthermore, Madden teaches that “Genes cloned into MB5718 can be inserted into the chromosome at the galK locus allowing them to be stably expressed without the requirement for antibiotic selection,” (page 67, first paragraph). Thus, Madden teaches that, when creating recombinant microorganisms, the galK locus is a known safe harbor site for introducing heterologous genes into a bacterial chromosome, where furthermore there is an advantage to doing so because 1) galK is a known site to receive heterologous genes and 2) introducing heterologous genes into the galK locus allows for the stable introduction of heterologous genes into a chromosome (page 67, first paragraph).
It would have been obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to combine the teachings ‘353 with either Kurokawa, Chatterjee, and Choi or Ow, Chatterjee, and Choi, and to include disulfide and/or prolyl isomerases such as DsbC and FkpA as taught by Kerokan/Ow/Chatterjee because Kurokawa/Ow and Chatterjee teach that overexpression of such isomerases has great benefit in the expression and stabilization of proteins of interest. Furthermore Chatterjee teaches a motivation to produce isomerases such as those taught by Kurokawa and Ow because they are useful in cell-free synthesis systems such those recited in the claims of ‘353 (Kurokawa Abstract and page 3960 in its entirety to 3961, left column, first paragraph, and Ow page 2, left column, second paragraph, and Chatterjee Background, first paragraph, and bottom of page 24 into page 25). Furthermore, ‘353, Kurokawa, Ow, Chatterjee, and Choi all concern the expression of recombinant proteins of interest and recombinant protein expression; a practitioner would therefore be motivated to combine the beneficial teachings of Kurokawa and Ow with Chatterjee and ‘353 with a reasonable expectation of success because each of these documents is in the same field of endeavor. Furthermore, a practitioner would be motivated to constitutively express either the DsbC of Kurokawa or the FkpA of Ow because Kurokawa and Ow teach the benefits of overexpressing these proteins (Abstract, Introduction of both, and see documents). Furthermore Choi teaches that constitutive expression systems are not only alternatives to inducible systems such as those taught by Kurokawa and Ow but also have advantages such as the fact that they are cheaper (page 882, left column, first paragraph) Also, Chatterjee teaches that these proteins are useful in cell-free protein synthesis and therefore provides motivation to combine the teachings with ‘353 (Background, first paragraph, and bottom of page 24 into page 25). A practitioner would have a reasonable expectation of success because Choi teaches that inducible systems are interchangeable with constitutive expression systems and also teaches working examples of constitutive expression systems (page 882, left column, first paragraph and Table 2).
With regards to the claim limitation that the intracellular concentration be at least 1 mg/mL, although this specific value is not taught in Kurokawa, Ow, and Choi, a practitioner of ordinary skill in the art could arrive at this value by routine optimization and lab work. Furthermore, a practitioner would be motivated to overexpress the recited proteins especially in light of the fact that Ow teaches that the efficacy of the overexpressed proteins is in direct proportion to a dosage dependence (page 10, left column, second paragraph). A practitioner would therefore be motivated to optimize the expression levels of the beneficial proteins DsbC and FkpA taught by Kurokawa and Ow through routine methods to arrive at the presently claimed invention. Furthermore, MPEP 2144.05 Section II(A) specifically states that differences in concentration fall under routine optimization and will not support patentability unless there is evidence indicating such concentration is critical.
Additionally, the galK locus is already a known safe harbor expression site for bacterial gene integration, as taught by Madden. Thus, the combination including Madden is the simple combination of known prior art elements with predictable success. Furthermore, the practitioner is motivated to integrate heterologous genes into the galK locus because Madden teaches that such integration is useful, as it stabilizes offers stable integration into the chromosome.
Regarding claim 40, ‘353 does not recite that the exogenous disulfide isomerase is DsbC and the exogenous prolyl isomerase is FkpA.
Kurokawa and Ow teach the DsbC and FkpA genes, respectively (see rejection of claim 38). Kurokawa teaches that there are advantages to overexpressing DsbC including the stabilization of expressed recombinant proteins(Abstract and page 3960 in its entirety to 3961, left column, first paragraph).
Ow teaches that the overexpression of FkpA is known to alleviate the stress response of E. coli during the accumulation of misfolded proteins, suppresses the formation of inclusion bodies, promotes proper protein folding, and significantly improves the solubility and functional expression of recombinant protein expression such as the expression of recombinant antibodies (scFv) (page 2, left column, second paragraph).
It would have been obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to combine the claims of the ‘353 patent with either Kurokawa, Chatterjee, and Choi or Ow, Chatterjee, and Choi, and to include disulfide and/or prolyl isomerases such as DsbC and FkpA as taught by Kurokawa/Ow/Chatterjee because Kurokawa/Ow and Chatterjee teach that overexpression of such isomerases has great benefit in the expression and stabilization of proteins of interest.
Regarding claim 59, these claim limitations are addressed in the rejection of claim 38 and in the evidentiary statement provided at the top of the Double Patenting section of this office action.
Claims 41-44, 49-50, and 52-58 are rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1-13 of U.S. Patent No. 10,450,353 B2 (‘353) in view of Kurokawa (Kurokawa Y et al. Appl Environ Microbiol. 2000 Sep;66(9):3960-5), Ow (Ow DS et al. Microb Cell Fact. 2010 Apr 13;9:22), Chatterjee (WO 2004/081033 A2), and Choi (Choi et al. Chemical Engineering Science, Volume 61, Issue 3, 2006, 876-885) and Madden (WO 2010/085731 A2) as applied to claims 38, 40, 45, 47-48, above, and further in view of Jonasson (Jonasson P et al. Biotechnol Appl Biochem. 2002 Apr;35(2):91-105).
The teachings of Chatterjee, Anderson, Kurokowa, Ow, Choi, Church and Jonasson from the 103 rejection are reiterated herein.
A discussion of the recitation of claim limitations of ‘353 is given above. Regarding claim 41, the rejection of claims 38 and 40 are discussed above.
Regarding claim 41, Chatterjee teaches a strong motivation to generate isomerases such as DsbC and FkpA because such proteins have beneficial effects in cell-free synthesis systems and methods such as those recited in the claims of ‘353 (Chatterjee Background, first paragraph, and bottom of page 24 into page 25, claims of ‘353).
Regarding claim 41, ‘353 does not recite the dsbC gene. None of ‘353, Kurokawa, Ow, Chatterjee, or Choi teach or suggest that their bacterial strains comprise two copies of the dsbC gene.
Jonasson is a review article focused on the facilitated production and recovery of recombinant proteins from E. coli (Title, Abstract, and throughout). Jonasson teaches the strategy of gene multimerization for increased protein yields in recombinant E. coli (page 100, left column, second paragraph). Jonasson teaches that using multiple copies of a gene can increase the yield of a target protein (page 100, left column, second paragraph).
Madden is a patent document which teaches the development of recombinant microorganisms for the production of desired pathways (Title, Abstract, and throughout). Madden teaches that it is a known and useful technique to integrate heterologous/exogenous genes into the chromosome of a desired microorganism (page 66, final paragraph). Furthermore, Madden teaches that “Genes cloned into MB5718 can be inserted into the chromosome at the galK locus allowing them to be stably expressed without the requirement for antibiotic selection,” (page 67, first paragraph). Thus, Madden teaches that, when creating recombinant microorganisms, the galK locus is a known safe harbor site for introducing heterologous genes into a bacterial chromosome, where furthermore there is an advantage to doing so because 1) galK is a known site to receive heterologous genes and 2) introducing heterologous genes into the galK locus allows for the stable introduction of heterologous genes into a chromosome (page 67, first paragraph).
It would have been obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to combine the teachings of ‘353, Kurokawa, Ow, Choi, Chatterjee, and Jonasson, to arrive at a bacterial strain comprising two copies of the dsbC gene integrated into its chromosome because such a combination is simply the combination of prior art elements according to known methods to yield predictable results. Furthermore, a practitioner would be motivated to create a bacterial strain containing two copies of the dsbC gene because 1) multiple copies of a gene or sequence can improve yield of the protein product, as taught by Jonasson (page 100, left column, second paragraph) and 2) dsbC as a protein product was known to have value in the cell-free protein synthesis method taught by Chatterjee and ‘353 (Background, first paragraph, and bottom of page 24 into page 25, claims of ‘353). A practitioner would be motivated to produce as much dsbC as possible in order to use in the cocktail described by Chatterjee, and would therefore be motivated to create a bacterial strain capable of producing as much dsbC as possible. Inserting multiple copies of the dsbC gene into the genome of a bacterial strain would be an obvious solution to the need taught by Chatterjee as taught by the combination of ‘353, Kurokawa, Choi, and Jonasson.
Additionally, the galK locus is already a known safe harbor expression site for bacterial gene integration, as taught by Madden. Thus, the combination including Madden is the simple combination of known prior art elements with predictable success. Furthermore, the practitioner is motivated to integrate heterologous genes into the galK locus because Madden teaches that such integration is useful, as it stabilizes offers stable integration into the chromosome.
Regarding claim 42, ‘353 does not recite the FkpA gene.
Chatterjee also teaches that FkpA is an important gene product in the cell-free synthesis cocktails that they teach (Background, first paragraph, and bottom of page 24 into page 25). Kurokawa, Ow, Choi, Chatterjee, and Jonasson do not teach the strain according to claim 41 further comprising a plasmid with two copies of the FkpA gene linked to a promoter.
It would have been obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to further modify the strains of E. coli rendered obvious by ‘353, Kurokawa, Ow, Choi, Chatterjee, and Jonasson to create
a bacterial cell strain capable of producing an abundance of FkpA as presently recited. A practitioner would be motivated to combine the teachings of ‘353, Kurokawa, Ow, Choi, Chatterjee, and Jonasson to include two copies of FkpA within the cell recited in claim 41, because FkpA enhances protein expression in the methods taught by Chatterjee, and FkpA is therefore a valuable protein to produce (Background, first paragraph, and bottom of page 24 into page 25). A practitioner would therefore be motivated to produce as much FkpA as possible, so that it could be used in a cell-free synthesis system taught by ‘353 and Chatterjee. To accomplish the production of FkpA, a practitioner could use the overexpression system of Ow and the suggestion to use multiple copies of the gene as suggested by Jonasson (page 100, left column, second paragraph).
Regarding claim 43, ‘353 teaches integration of constructs into the chromosome (claim 5), and the advantages of multiple copies of FkpA in a bacterial strain are rendered obvious by the combination of Ow, Chatterjee, and Jonasson.
Regarding claim 44, ‘353 teaches integrating gene copies into a bacteria’s chromosome (claim 5). Jonasson teaches that including multiple copies of a target protein can increase yield (Jonasson page 100, left column, second paragraph). Chatterjee teaches that dsbC and FkpA are important proteins in their cell-free synthesis systems such as those recited in the claims of ‘353 (Background, first paragraph, and bottom of page 24 into page 25, claims 6-13 of ‘353). A practitioner would therefore be motivated to create a bacterial strain which overexpressed dsbC and FkpA, so that these proteins could be used in the systems taught by Chatterjee. Claim 44 is therefore the simple combination of known prior art elements.
Regarding claims 49 and 50, these claim limitations are addressed in the rejections of claims 41-44. As discussed above, a bacterial strain including two copies of DsbC and FkpA, wherein the gene copies are either in the chromosome or on plasmids, is the simple combination of prior art elements to yield predictable results. The prior art elements (DsbC, FkpA, two copies of each gene, plasmid and/or chromosome integration), are discussed in the above rejections of claims 38, 40-45 and 47-48.
Regarding claim 52, ‘353 recites bacterial cell-free synthesis systems, methods of preparing bacterial cell-free synthesis systems, and a method of expressing a protein of interest in a bacterial cell-free synthesis system comprising the combination of a nucleic acid template encoding a protein of interest with bacterial cell free synthesis extract to produce a bacterial cell-free synthesis system and expressing a protein of interest (claims 6-7 and 11-12).
‘353 does not recite an active oxidation phosphorylation system, tRNA, amino acids and ribosomes for cell-free protein synthesis, or that the exogenous prolyl isomerase or disulfide isomerase were expressed at a level of at least 1 gm/liter of extract. ‘353 does not recite that the protein of interest is expressed at a level of 100 mg/L.
Both Kurokawa and Ow teach the generation of cell free extracts (Kurokawa “Fractionation and Analysis of Proteins,” page 3961, left column, third paragraph, and Ow, page 11, right column, third paragraph).
Chatterjee also teaches that cell free extracts are used in their cell-free synthesis systems and methods (e.g., page 33, second paragraph). Chatterjee further teaches that their cell-free synthesis systems comprise one or more energy sources providing chemical energy for protein or biological macromolecule synthesis (claim 1 of Chatterjee), wherein the one of more energy sources generates or regenerates high-energy triphosphate compounds (claim 2 of Chatterjee, i.e., Chatterjee teaches an energy source for active oxidative phosphorylation), tRNAs (e.g., claim 11 and page 8, third paragraph), amino acids and ribosomes (page 8, third paragraph), a DNA template for protein synthesis (page 8, first paragraph).
It would have been obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to combine the teachings of ‘353 with Court, Kurokawa, Ow, Chatterjee, Choi, and Jonasson because such a combination is the simple combination of known prior art elements to yield predictable results. Furthermore, the claims of ‘353 recite methods of cell-free synthesis; a practitioner would therefore be motivated to incorporate the components of cell-free synthesis systems taught by Chatterjee with ‘353 simply so that the systems of ‘353 would be functional (claims of ‘353). With regards to the claim limitations that the protein chaperone be expressed in the bacterial strain at a level of at least 1 gm/liter or extract, and that the protein of interest be expressed at a concentration of at least 100 mg/L, Chatterjee teaches that “the skilled artisan will recognize that concentrations of the various components of the incubation medium can be adjusted as is known in the art while still maintaining the synthetic function”, (page 33, second paragraph). Thus, the exact concentration of the protein chaperone and its expression could be determined through routine experimentation and optimization. Similarly, the concentration of the production of a protein of interest could be determined through routine optimization and experimentation of the methods of Chatterjee.
Regarding claim 53, ‘353 also recites cell free-extracts to be used in cell-free synthesis systems (claims 11-13). A bacterial cell comprising two copies of the dsbC gene is discussed in the rejection of claim 41. Furthermore, Chatterjee teaches the use of cell extracts to be used in their cell-free synthesis systems (page 28, final paragraph).
Regarding claim 54, ‘353 also recites cell free extracts to be used in cell-free synthesis systems (claims 11-13). The claim limitations of claim 54 are addressed above in the rejection of claim 42. Furthermore, Chatterjee teaches the use of cell extracts to be used in their cell-free synthesis systems (page 28, final paragraph).
Regarding claim 55, ‘353 also recites cell free extracts to be used in cell-free synthesis systems (claims 11-13). The claim limitations of claim 55 are addressed above in the rejection of claim 43. Furthermore, Chatterjee teaches the use of cell extracts to be used in their cell-free synthesis systems (page 28, final paragraph).
Regarding claim 56, ‘353 also recites cell free extracts to be used in cell-free synthesis systems (claims 11-13). The claim limitations of claim 56 are addressed above in the rejection of claim 44. Furthermore, Chatterjee teaches the use of cell extracts to be used in their cell-free synthesis systems (page 28, final paragraph).
Regarding claim 57, ‘353 recites a method for expressing a properly folded protein of interest in a bacterial cell-free synthesis system comprising incubating a cell-free synthesis system under conditions which permit the expression of a protein of interest (claim 11).
Regarding claim 58, ‘353 and the prior art references cited do not explicitly teach that the isomerase is produced with a concentration of at least 1g/L.
Chatterjee teaches the importance of producing the isomerases DsbC and FkpA and therefore provides a motivation for the production of these proteins, which can be used in their commercially relevant cell-free synthesis systems (bottom of page 24 and into page 25, and see rejection of claim 41).
It would have been obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to arrive at the presently recited claim limitation where the isomerase is present at a concentration of at least 1 gm/liter, because such a concentration could be arrived at through routine experimentation given that Chatterjee has already taught that the production of the recited isomerases has commercial value (see rejection of claim 41). A practitioner would therefore be motivated to optimize the expression of the recited isomerases in the extracts of the strains, and the recited concentration range could be arrived at through routine experimentation. See MPEP 2144.05, section II(A) for a discussion on routine optimization.
Claims 38, 40, 45, and 47-48 and 59 are rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1-13 of U.S. Patent No. 11,261,219 B2 (‘219) in view of Kurokawa (Kurokawa Y et al. Appl Environ Microbiol. 2000 Sep;66(9):3960-5), Ow (Ow DS et al. Microb Cell Fact. 2010 Apr 13;9:22), Chatterjee (WO 2004/081033 A2), Court (Court D et al. Annu Rev Genet. 2002;36:361-88), Choi (Choi et al. Chemical Engineering Science, Volume 61, Issue 3, 2006, 876-885), and Jonasson (Jonasson P et al. Biotechnol Appl Biochem. 2002 Apr;35(2):91-105) and Madden (WO 2010/085731 A2). The rejection is further evidenced by UniProt (UniProt Accession numbers, sequences, and alignments for UniProt accession numbers of E. coli K12 dsbC and DsbG genes, published 2010) and Shao (Shao F et al. J Biol Chem. 2000 May 5;275(18):13349-52).
The teachings of Chatterjee, Kurokowa, Ow, Choi, Church and Jonasson from the 103 rejection are reiterated herein.
Regarding the claims of ‘219, claims 1-5 of ‘219 recite an OmpT1-sensistive RF1 protein and a bacterial cell (E. coli, claims 2 and 8) comprising the OmpT1 sensitive RF1 protein, where claim 1 further recites that the cell is positive for ompT. Furthermore, claims 3-15 recite bacterial cell-free synthesis systems, methods of preparing bacterial cell-free synthesis systems, and a method of expressing a protein of interest in a bacterial cell-free synthesis system comprising the combination of a nucleic acid template encoding a protein of interest with bacterial cell free synthesis extract to produce a bacterial cell-free synthesis system and expressing a protein of interest. Claim 3 further specifies that the bacterial cell is ompT positive. Thus, ‘219 is generally drawn to methods of protein expression using cell-free synthesis, bacterial extracts, and cells comprising components for cell-free synthesis and their extracts, where the bacterial strains are positive for ompT.
Regarding claim 45, ‘219 recites E. coli (claim 2).
Regarding claim 47, ‘219 recites a cell free extract (claim 3).
Regarding claim 48, ‘219 recites a method of producing an exogenous protein of interest by culturing a bacterial strain under conditions which permit the expression of the exogenous protein (claim 7).
With regards to claims 38, 40, 45, and 47-48, ‘219 does not recite the claim limitations of an exogenous disulfide isomerase or exogenous prolyl isomerase linked to a constitutive promoter and expressed in the cell at a concentration of 1 mg/mL, where the disulfide isomerase is DsbC and the prolyl isomerase is FkpA. ‘219 also does not recite that the expression of their constructs are integrated genomically.
Regarding claim 38, Kurokawa is a research article which focuses on the overexpression of the protein disulfide isomerase DsbC on a plasmid cassette system in E. coli (Abstract, Title, and see document). Kurokawa therefore teaches a bacterial strain (E. coli) comprising an expression cassette that expresses a high level of the disulfide isomerase DsbC (Abstract, Title, and see document). The DsbC gene was expressed in a non-native, arabinose-inducible expression construct (Materials and Methods, sections “Bacterial Strains and Plasmids” and “Culture Conditions and Protein Expression”), and thus the DsbC gene was an “exogenous” disulfide isomerase following the claim language interpretation outlined in the above “Claim Interpretation” section. Note also that the title of Kurokawa identifies DsbC as a “disulfide isomerase.” The Abstract of Kurokawa states that the DsbC proteins in their study were “overexpressed.” Furthermore, Kurokawa teaches that the overexpression of DsbC increased the expression of a protein of interest severalfold (Abstract), and further teaches that such overexpression is essential in stabilizing and solubilizing the expression of recombinant proteins of interest (Abstract and page 3960 in its entirety to 3961, left column, first paragraph). Kurokawa therefore also teaches a motivation to overexpress DsbC in E. coli (Abstract and page 3960 in its entirety to 3961, left column, first paragraph).
Ow teaches the overexpression of FkpA from an arabinose-inducible, non-native cassette in E. coli cells (Abstract Materials and Methods section entitled “Cloning and bacterial strains”), wherein the FkpA gene is under the control of an inducible promoter (i.e., it is overexpressed). Ow teaches that FkpA is a prolyl isomerase (background, second paragraph). Ow therefore teaches a bacterial strain (E. coli) comprising an expression cassette that expresses an exogenous (see “Claim Interpretation” section, above) prolyl isomerase. Furthermore, Ow teaches that the overexpression of FkpA is known to alleviate the stress response of E. coli during the accumulation of misfolded proteins, suppresses the formation of inclusion bodies, promotes proper protein folding, and significantly improves the solubility and functional expression of recombinant protein expression such as the expression of recombinant antibodies (scFv) (page 2, left column, second paragraph). Furthermore, Ow teaches and reduced to practice the expression of FkpA to increase scFv solubility and cell viability in a dose dependent manner (page 10, left column, second paragraph). Thus, Ow teaches a motivation to overexpress FkpA, as the benefits of FkpA in helping cell viability and proper protein folding are taught by Ow to be dose-dependent (page 10, left column, second paragraph).
Chatterjee is a patent document which focuses on the synthesis of proteins by cell-free protein expression (Abstract and throughout). Chatterjee teaches that the addition of molecular chaperones or foldases can be applied to their cell-free synthesis methods/systems, including the addition of both the disulfide isomerase DsbC and the prolyl isomerase FkpA proteins as part of a protein cocktail (page 7, third paragraph, page 24-25, beginning with the final paragraph of page 24, page 45 first paragraph, and claims 43 and 44). Chatterjee teaches that the addition of the proteins DsbC and FkpA isomerases to the cell-free synthesis systems that they teach are useful because they improve expression of proteins (bottom of page 24 into page 25). Chatterjee teaches cell-free systems for protein synthesis, that such systems have commercial advantages, and furthermore that the addition of the chaperone/folding proteins DsbC and FkpA to their systems offers advantages because they improve protein production (Background, first paragraph, and bottom of page 24 into page 25). Chatterjee therefore teaches a motivation to produce DsbC and FkpA proteins, so that they can be added to their cell-free synthesis systems.
Court is a review article that focuses on genetic engineering strategies in E. coli (throughout). Court teaches that “in vivo technologies have emerged that, due to
their efficiency and simplicity, may one day replace standard genetic engineering techniques. Constructs can be made on plasmids or directly on the Escherichia coli chromosome from PCR products or synthetic oligonucleotides by homologous recombination”, (Abstract). Court therefore teaches that, when manipulating E. coli strains, constructs can exist either on plasmids or as genomic integrations for the purposes of genetic engineering, and also that such techniques are efficient and simple (Abstract).
Choi is a review article that teaches the production of recombinant proteins in E. coli (Title, Abstract, and see document). Choi teaches that E. coli is widely used for the production of recombinant proteins for industrial purposes (Abstract). Choi teaches a number of constitutively expressed systems in industrial E. coli (Table 2). Choi teaches that inducible expression systems such as those taught by Kurokawa and Ow can be expensive, and that constitutive expression systems are a ready alternative (page 882, left column, first paragraph). Choi therefore teaches that the use of constitutive promoters to express proteins in E. coli is a known method in the industry that can also have advantages over inducible systems for proteins that are intended to be overexpressed (page 882, left column, first paragraph).
Madden is a patent document which teaches the development of recombinant microorganisms for the production of desired pathways (Title, Abstract, and throughout). Madden teaches that it is a known and useful technique to integrate heterologous/exogenous genes into the chromosome of a desired microorganism (page 66, final paragraph). Furthermore, Madden teaches that “Genes cloned into MB5718 can be inserted into the chromosome at the galK locus allowing them to be stably expressed without the requirement for antibiotic selection,” (page 67, first paragraph). Thus, Madden teaches that, when creating recombinant microorganisms, the galK locus is a known safe harbor site for introducing heterologous genes into a bacterial chromosome, where furthermore there is an advantage to doing so because 1) galK is a known site to receive heterologous genes and 2) introducing heterologous genes into the galK locus allows for the stable introduction of heterologous genes into a chromosome (page 67, first paragraph).
It would have been obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to combine the teachings ‘219 with either Kurokawa, Chatterjee, Court, and Choi or Ow, Chatterjee, Court, and Choi, and to include disulfide and/or prolyl isomerases such as DsbC and FkpA as taught by Kerokan/Ow/Chatterjee because Kurokawa/Ow and Chatterjee teach that overexpression of such isomerases has great benefit in the expression and stabilization of proteins of interest. Furthermore Chatterjee teaches a motivation to produce isomerases such as those taught by Kurokawa and Ow because they are useful in cell-free synthesis systems such those recited in the claims of ‘219 (Kurokawa Abstract and page 3960 in its entirety to 3961, left column, first paragraph, and Ow page 2, left column, second paragraph, and Chatterjee Background, first paragraph, and bottom of page 24 into page 25). Furthermore, ‘219, Kurokawa, Ow, Chatterjee, Court, and Choi all concern the expression of recombinant proteins of interest and recombinant protein expression; a practitioner would therefore be motivated to combine the beneficial teachings of Kurokawa and Ow with Chatterjee and ‘219 with a reasonable expectation of success because each of these documents is in the same field of endeavor. Furthermore, a practitioner would be motivated to constitutively express either the DsbC of Kurokawa or the FkpA of Ow because Kurokawa and Ow teach the benefits of overexpressing these proteins (Abstract, Introduction of both, and see documents). Also, Choi teaches that constitutive expression systems are not only alternatives to inducible systems such as those taught by Kurokawa and Ow but also have advantages such as the fact that they are cheaper (page 882, left column, first paragraph). Additionally, Chatterjee teaches that these proteins are useful in cell-free protein synthesis and therefore provides motivation to combine the teachings with ‘219 (Background, first paragraph, and bottom of page 24 into page 25). Furthermore, a practitioner would have a reasonable expectation of success because Choi teaches that inducible systems are interchangeable with constitutive expression systems and also teaches working examples of constitutive expression systems (page 882, left column, first paragraph and Table 2). Additionally, given that Court teaches that genomic integration of constructs into E. coli is efficient and simply a practitioner would be motivated to use this genetic engineering tool with the teachings of ‘219, Kurokawa, Ow, Chatterjee, and Choi (Abstract, Court).
With regards to the claim limitation that the intracellular concentration be at least 1 mg/mL, although this specific value is not taught in Kurokawa, Ow, Court, Chatterjee, and Choi, a practitioner of ordinary skill in the art could arrive at this value by routine optimization and lab work. Furthermore, a practitioner would be motivated to overexpress the recited proteins especially in light of the fact that Ow teaches that the efficacy of the overexpressed proteins is in direct proportion to a dosage dependence (page 10, left column, second paragraph). A practitioner would therefore be motivated to optimize the expression levels of the beneficial proteins DsbC and FkpA taught by Kurokawa and Ow through routine methods to arrive at the presently claimed invention. Furthermore, MPEP 2144.05 Section II(A) specifically states that differences in concentration fall under routine optimization and will not support patentability unless there is evidence indicating such concentration is critical.
Additionally, the galK locus is already a known safe harbor expression site for bacterial gene integration, as taught by Madden. Thus, the combination including Madden is the simple combination of known prior art elements with predictable success. Furthermore, the practitioner is motivated to integrate heterologous genes into the galK locus because Madden teaches that such integration is useful, as it stabilizes offers stable integration into the chromosome.
Regarding claim 40, ‘219 does not recite that the exogenous disulfide isomerase is DsbC and the exogenous prolyl isomerase is FkpA.
Kurokawa and Ow teach the DsbC and FkpA genes, respectively (see rejection of claim 38). Kurokawa teaches that there are advantages to overexpressing DsbC including the stabilization of expressed recombinant proteins(Abstract and page 3960 in its entirety to 3961, left column, first paragraph).
Ow teaches that the overexpression of FkpA is known to alleviate the stress response of E. coli during the accumulation of misfolded proteins, suppresses the formation of inclusion bodies, promotes proper protein folding, and significantly improves the solubility and functional expression of recombinant protein expression such as the expression of recombinant antibodies (scFv) (page 2, left column, second paragraph).
It would have been obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to combine the claims of the ‘219 patent with either Kurokawa, Chatterjee, and Choi or Ow, Chatterjee, and Choi, and to include disulfide and/or prolyl isomerases such as DsbC and FkpA as taught by Kurokawa/Ow/Chatterjee because Kurokawa/Ow and Chatterjee teach that overexpression of such isomerases has great benefit in the expression and stabilization of proteins of interest.
Regarding claim 59, these claim limitations are discussed in the rejection of claim 38, above, and in the evidentiary statement at the beginning of the Double Patenting section of this office action.
Claims 41-44, 49-50, and 52-58 are rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1-13 of U.S. Patent No. 11,261,219 B2 (‘219) in view of Kurokawa (Kurokawa Y et al. Appl Environ Microbiol. 2000 Sep;66(9):3960-5), Ow (Ow DS et al. Microb Cell Fact. 2010 Apr 13;9:22), Chatterjee (WO 2004/081033 A2), Court (Court D et al. Annu Rev Genet. 2002;36:361-88), and Choi (Choi et al. Chemical Engineering Science, Volume 61, Issue 3, 2006, 876-885) and Madden (WO 2010/085731 A2) as applied to claims 38, 40, 45, 47-48, above, and further in view of Jonasson (Jonasson P et al. Biotechnol Appl Biochem. 2002 Apr;35(2):91-105).
A discussion of recitation of the claims of ‘219 is given above.
Regarding claim 41, the rejection of claims 38 and 40 are discussed above.
Chatterjee teaches a strong motivation to generate isomerases such as DsbC and FkpA because such proteins have beneficial effects in cell-free synthesis systems and methods such as those recited in the claims of ‘219 (Chatterjee Background, first paragraph, and bottom of page 24 into page 25, claims of ‘219).
Regarding claim 41, ‘219 does not recite the dsbC gene. None of ‘219, Kurokawa, Ow, Chatterjee, Court, or Choi teach or suggest that their bacterial strains comprise two copies of the dsbC gene.
Jonasson is a review article focused on the facilitated production and recovery of recombinant proteins from E. coli (Title, Abstract, and throughout). Jonasson teaches the strategy of gene multimerization for increased protein yields in recombinant E. coli (page 100, left column, second paragraph). Jonasson teaches that using multiple copies of a gene can increase the yield of a target protein (page 100, left column, second paragraph).
Madden is a patent document which teaches the development of recombinant microorganisms for the production of desired pathways (Title, Abstract, and throughout). Madden teaches that it is a known and useful technique to integrate heterologous/exogenous genes into the chromosome of a desired microorganism (page 66, final paragraph). Furthermore, Madden teaches that “Genes cloned into MB5718 can be inserted into the chromosome at the galK locus allowing them to be stably expressed without the requirement for antibiotic selection,” (page 67, first paragraph). Thus, Madden teaches that, when creating recombinant microorganisms, the galK locus is a known safe harbor site for introducing heterologous genes into a bacterial chromosome, where furthermore there is an advantage to doing so because 1) galK is a known site to receive heterologous genes and 2) introducing heterologous genes into the galK locus allows for the stable introduction of heterologous genes into a chromosome (page 67, first paragraph).
It would have been obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to combine the teachings of ‘219, Kurokawa, Ow, Court, Choi, Chatterjee, and Jonasson, to arrive at a bacterial strain comprising two copies of the dsbC gene integrated into its chromosome because such a combination is simply the combination of prior art elements according to known methods to yield predictable results. Furthermore, a practitioner would be motivated to create a bacterial strain containing two copies of the dsbC gene because 1) multiple copies of a gene or sequence can improve yield of the protein product, as taught by Jonasson (page 100, left column, second paragraph) and 2) dsbC as a protein product was known to have value in the cell-free protein synthesis method taught by Chatterjee and ‘219 (Background, first paragraph, and bottom of page 24 into page 25, claims of ‘219). A practitioner would be motivated to produce as much dsbC as possible in order to use in the cocktail described by Chatterjee, and would therefore be motivated to create a bacterial strain capable of producing as much dsbC as possible. Inserting multiple copies of the dsbC gene into the genome of a bacterial strain would be an obvious solution to the need taught by Chatterjee as taught by the combination of ‘219, Kurokawa, Choi, and Jonasson.
Regarding claim 42, ‘219 does not recite the FkpA gene.
Chatterjee also teaches that FkpA is an important gene product in the cell-free synthesis cocktails that they teach (Background, first paragraph, and bottom of page 24 into page 25). Kurokawa, Ow, Choi, Court, Chatterjee, and Jonasson do not teach the strain according to claim 41 further comprising a plasmid with two copies of the FkpA gene linked to a promoter.
It would have been obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to further modify the strains of E. coli rendered obvious by ‘219, Kurokawa, Ow, Court, Choi, Chatterjee, and Jonasson to create a bacterial cell strain capable of producing an abundance of FkpA as presently recited. A practitioner would be motivated to combine the teachings of ‘219, Kurokawa, Ow, Court, Choi, Chatterjee, and Jonasson to include two copies of FkpA within the cell recited in claim 41, because FkpA enhances protein expression in the methods taught by Chatterjee, and FkpA is therefore a valuable protein to produce (Background, first paragraph, and bottom of page 24 into page 25). A practitioner would therefore be motivated to produce as much FkpA as possible, so that it could be used in a cell-free synthesis system taught by ‘219 and Chatterjee. To accomplish the production of FkpA, a practitioner could use the overexpression system of Ow and the suggestion to use multiple copies of the gene as suggested by Jonasson (page 100, left column, second paragraph).
Regarding claims 43-44, ‘219 does not recite integrating multiple copies of either the dsbC or FkpA genes into the chromosome.
Court teaches that, when manipulating E. coli strains, constructs can exist either on plasmids or as genomic integrations for the purposes of genetic engineering, and also that such techniques are efficient and simple (Abstract).
The advantages of multiple copies of FkpA in a bacterial strain are rendered obvious by the combination of ‘219, Ow, Chatterjee, and Jonasson as discussed above
Jonasson teaches that including multiple copies of a target protein can increase yield (Jonasson page 100, left column, second paragraph). Chatterjee teaches that dsbC and FkpA are important proteins in their cell-free synthesis systems such as those recited in the claims of ‘219 (Background, first paragraph, and bottom of page 24 into page 25, claims 3-15 ‘219).
It would have been obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to combine the teachings of ‘219 with Court, Ow, Chatterjee, and Jonasson to arrive at the claimed invention because such a combination is the simple combination of prior art elements to yield predictable results. A practitioner would be motivated to create a bacterial strain which overexpressed dsbC and FkpA by integrating multiple copies into the chromosome because such integration techniques are known to increase yield (Jonasson, page 100, left column, second paragraph). Furthermore, dsbC and FkpA are known to be important proteins in cell-free synthesis systems such as those recited by ‘219 as taught by Chatterjee (Background, first paragraph, and bottom of page 24 into page 25, claims 3-15 of ‘219).
Regarding claims 49 and 50, these claim limitations are addressed in the rejections of claims 41-44. As discussed above in the rejection of claims 43-44, a bacterial strain including two copies of DsbC and FkpA, wherein the gene copies are either in the chromosome or on plasmids, is the simple combination of prior art elements to yield predictable results. The prior art elements (DsbC, FkpA, two copies of each gene, plasmid and/or chromosome integration), are discussed in the above rejections of claims 38, 40-45 and 47-48.
Regarding claim 52, ‘219 recites cell-free synthesis methods and systems (claims 3-15), and therefore recites methods and systems which would use components of cell-free synthesis systems (claims 3-15). ‘219 recites a nucleic acid encoding a protein of interest (claim 3). ‘219 recites an active phosphorylation system (claim 5).
‘219 does not recite a tRNA, amino acids and ribosomes for cell-free protein synthesis, or that the exogenous prolyl isomerase or disulfide isomerase were expressed at a level of at least 1 gm/liter of extract. ‘219 does not recite that the protein of interest is expressed at a level of 100 mg/L.
Both Kurokawa and Ow teach the generation of cell free extracts (Kurokawa “Fractionation and Analysis of Proteins,” page 3961, left column, third paragraph, and Ow, page 11, right column, third paragraph).
Chatterjee also teaches that cell free extracts are used in their cell-free synthesis systems and methods (e.g., page 33, second paragraph). Chatterjee further teaches that their cell-free synthesis systems comprise one or more energy sources providing chemical energy for protein or biological macromolecule synthesis (claim 1 of Chatterjee), wherein the one of more energy sources generates or regenerates high-energy triphosphate compounds (claim 2 of Chatterjee, i.e., Chatterjee teaches an energy source for active oxidative phosphorylation), tRNAs (e.g., claim 11 and page 8, third paragraph), amino acids and ribosomes (page 8, third paragraph), a DNA template for protein synthesis (page 8, first paragraph).
It would have been obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to combine the teachings of ‘219 with Court, Kurokawa, Ow, Chatterjee, Choi, and Jonasson because such a combination is the simple combination of known prior art elements to yield predictable results. Furthermore, the claims of ‘219 recite methods of cell-free synthesis; a practitioner would therefore be motivated to incorporate the components of cell-free synthesis systems taught by Chatterjee with ‘219 simply so that the systems of ‘219 would be functional (claims 3-15 of ‘219). With regards to the claim limitations that the protein chaperone be expressed in the bacterial strain at a level of at least 1 gm/liter or extract, and that the protein of interest be expressed at a concentration of at least 100 mg/L, Chatterjee teaches that “the skilled artisan will recognize that concentrations of the various components of the incubation medium can be adjusted as is known in the art while still maintaining the synthetic function”, (page 33, second paragraph). Thus, the exact concentration of the protein chaperone and its expression could be determined through routine experimentation and optimization. Similarly, the concentration of the production of a protein of interest could be determined through routine optimization and experimentation of the methods of Chatterjee.
Regarding claim 53, ‘219 recites cell free extracts to be used in cell-free synthesis systems (claims 3-15). A bacterial cell comprising two copies of the dsbC gene is discussed in the rejection of claim 41. Furthermore, Chatterjee teaches the use of cell extracts to be used in their cell-free synthesis systems (page 28, final paragraph).
Regarding claim 54, ‘219 also recites cell free extracts to be used in cell-free synthesis systems (claims 3-15). The claim limitations of 54 are addressed above in the rejection of claim 42. Furthermore, Chatterjee teaches the use of cell extracts to be used in their cell-free synthesis systems (page 28, final paragraph).
Regarding claim 55, ‘219 also recites cell free extracts to be used in cell-free synthesis systems (claims 3-15). The claim limitations of claim 55 are addressed above in the rejection of claim 43. Furthermore, Chatterjee teaches the use of cell extracts to be used in their cell-free synthesis systems (page 28, final paragraph).
Regarding claim 56, ‘219 also recites cell free extracts to be used in cell-free synthesis systems (claims 3-15). The claim limitations of claim 56 are addressed above in the rejection of claim 44. Furthermore, Chatterjee teaches the use of cell extracts to be used in their cell-free synthesis systems (page 28, final paragraph).
Regarding claim 57, ‘219 recites a method of expressing a properly folded biologically active protein of interest in a bacterial cell-free synthesis system comprising culturing a bacterial cell-free synthesis system under conditions allowing for proper folding of the protein of interest (claims 3-15).
Regarding claim 58, ‘219 and the prior art references cited do not explicitly teach that the isomerase is produced with a concentration of at least 1g/L.
Chatterjee teaches the importance of producing the isomerases DsbC and FkpA and therefore provides a motivation for the production of these proteins, which can be used in their commercially relevant cell-free synthesis systems (bottom of page 24 and into page 25, and see rejection of claim 41).
It would have been obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to arrive at the presently recited claim limitation where the isomerase is present at a concentration of at least 1 gm/liter, because such a concentration could be arrived at through routine experimentation given that Chatterjee has already taught that the production of the recited isomerases has commercial value (see rejection of claim 41). A practitioner would therefore be motivated to optimize the expression of the recited isomerases in the extracts of the strains, and the recited concentration range could be arrived at through routine experimentation. See MPEP 2144.05, section II(A) for a discussion on routine optimization.
Response to Arguments/Amendments
The Applicant’s arguments filed 6/1/2026 have been considered but are not persuasive. The Applicant argues that their amendments are sufficient to overcome the Double Patentign rejection, and requests withdrawal of the Double Patenting rejection. However, as discussed above in both the 103 and Double Patenting rejections, the Applicant’s amendments are not sufficient to overcome the rejections in view of previously recited prior art in addition to Madden (see rejection, above). The Applicant does not offer an additional argument. Thus, the Double Patenting rejections are maintained.
Conclusion
Applicant's amendment necessitated the new ground(s) of rejection presented in this Office action. Accordingly, THIS ACTION IS MADE FINAL. See MPEP § 706.07(a). Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action. In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any nonprovisional extension fee (37 CFR 1.17(a)) pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action. In no event, however, will the statutory period for reply expire later than SIX MONTHS from the mailing date of this final action.
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/D.C.R./Examiner, Art Unit 1635
/RAM R SHUKLA/Supervisory Patent Examiner, Art Unit 1635
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