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 .
Priority
Acknowledgment is made of applicant’s claim for foreign priority under 35 U.S.C. 119 (a)-(d). The certified copy of the foreign Application EP21306680.6, filed on December 01, 2021 been filed.
Status of the Claims
Amendments dated 05/30/2024 are entered.
Claims 1-15 are pending and are examined herein.
Information Disclosure Statement
The information disclosure statement (IDS) submitted on 05/30/2024 is acknowledged and is being considered by the examiner.
Claim Objections
Claim 1 is objected to because of the following informality:
Line 2 should recite “the method comprising the following steps:” instead of “the method comprising the following steps;” with a semicolon
Claim 12 is objected to because of the following informality:
Line 2 recites “(15)” when is should not; for consistency (cf. claim 1).
Appropriate correction is required.
Claim Interpretations
Per paragraph 0002 of the specification, “A recombinant protein is a protein produced by cells whose DNA has been modified by genetic engineering.” Thus, the ribosomes and other proteins produced by cells whose DNA has been modified by genetic engineering are recombinant proteins.
Per paragraph 0026 of the specification, by "first genetically engineered organism or part thereof", it is meant any single living plant, animal, algae, yeast or bacteria which has been genetically
engineered.
Per paragraph 0031 of the specification, an “affinity ligand” is “a molecule which is able to bind to a specific target, the specific target being according to the invention (1) a recombinant protein and/or (2) an oil body protein, as long as the affinity ligand which is either fused to a recombinant protein or an oil body protein allows forming complexes comprising the oil body protein and the recombinant protein.”
Per paragraph 0037 of the specification, "flotation" “[means] that the mix is introduced into a liquid solution, such as a buffer at pH 7, a phosphate-buffered saline solution or the like, and is centrifuged.”
Regarding claim 5, paragraph 0045 of the specification recites the following:
By “at least one inactivated gene" as used herein it is meant a gene which cannot encode RNA or a protein or encodes no functional protein, or which encodes a protein with reduced activity by at least 90 %. Said "inactivated gene" can encode a protein with reduced functionality, or be a fully inactivated gene, encoding no functional protein or not encoding any protein.
Claim Rejections - 35 USC § 112(b)
The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
Claims 1-13 are rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention. All dependent claims are included in these rejections unless they contain a limitation that overcomes the deficiencies of the parent claim from which they depend. The rationale for this determination is explained below.
Regarding the rejection of claims 1-13, the preamble in claim 1 recites a method for “the purification of recombinant proteins produced by a first genetically engineered organism.” However, the claim further recites active steps where the first step of claim 1 recites “providing the first genetically engineered organism expressing the recombinant proteins or a part thereof” such that the first genetically engineered organism could alternatively be expressing only a part of the recombinant proteins. Subsequent steps would fail to accomplish “purification of recombinant proteins” as recited in the preamble. Thus, claims 1-15 are rejected under 35 U.S.C. 112(b) as being incomplete for omitting essential steps, such omission amounting to a gap between the steps. See MPEP § 2172.01.
Claims 1-13 are additionally rejected because the last step of the method of claim 1 is “Recovering the complexes comprising the oil body proteins and the recombinant proteins by flotation of the mix.” The recited active steps do not accomplish “purification of recombinant proteins produced by a first genetically engineered organism” as recited in the preamble even with the alternative where first genetically engineered organism is expressing the recombinant proteins. At the end of the method of claim 1, the “recombinant proteins produced by a first genetically engineered organism” are still in complexes comprising the oil body proteins and the affinity ligand which allows forming complexes and is fused to the recombinant proteins and/or the oil body proteins.
Claim 6 is further rejected on the basis that it contains an indefinite Markush grouping of alternatives. A Markush grouping is a closed group of alternatives, i.e., the selection is made from a group "consisting of" (rather than "comprising" or "including") the alternative members. The transitional term "comprising", which is synonymous with "including," "containing," or "characterized by," is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. The recitation of “The oil body proteins are selected among a list comprising … combinations thereof” is such that the Markush grouping of oil body proteins is not a closed group of alternatives. It is unclear what other alternatives are intended to be encompassed by the claim (emphasis provided). See MPEP § 2111.03 and MPEP § 2173.05(h).
Claim 8 is similarly rejected on the basis that it contains an indefinite Markush grouping of alternatives because it recites “the recombinant proteins are selected among a list comprising insulin, growth factors, therapeutic antibodies, antigens, structural and bioactive peptides or combinations thereof” (emphasis provided).
Claim 12 is further rejected. The phrase "preferably" renders the claim indefinite because it is unclear whether the limitation(s) following the phrase are part of the claimed invention. Description of examples or preferences is properly set forth in the specification rather than the claims. If stated in the claims, examples and preferences may lead to confusion over the intended scope of a claim. See MPEP § 2173.05(d).
Claim Rejections - 35 USC § 103
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. Considering objective evidence present in the application indicating obviousness or nonobviousness.
Claims 1-15 are rejected under 35 U.S.C. 103 as being unpatentable over Moloney (U.S. Patent Application Publication No. US20050039235A1, assigned to SemBioSys Genetics Inc, titled ‘Methods for the production of insulin in plants’, published 2005, Patent publication cited in IDS) in view of Rooijen (U.S. Patent Application Publication No. US5856452A, assigned to SemBioSys Genetics Inc, titled ‘Oil bodies and associated proteins as affinity matrices’, published 1999), Ahmad (Ahmad A, et al. Green biofactories: recombinant protein production in plants. Recent Pat Biotechnol. 2010 Nov;4(3):242-59. PMID: 21171961., published 2010, Cited in IDS), and Ludman (Ludman M, Fátyol K. The virological model plant, Nicotiana benthamiana expresses a single functional RDR6 homeolog. Virology. 2019 Nov;537:143-148. doi: 10.1016/j.virol.2019.08.017. Epub 2019 Aug 29. PMID: 31493652., published 2019).
Regarding claims 1-5, Moloney teaches “recombinant expression of insulin fusion proteins in transformed Arabidopsis thaliana lines” and “a method of purifying insulin from plant” (c.f., claim 1’s pre amble and 1st step: “A method for the purification of recombinant proteins produced by a first genetically engineered organism, the method comprising the following steps; providing the genetically engineered organism expressing the recombinant proteins or a part thereof” and claim 2’s “genetically engineered plant”) (para 0059 and 0166).
Moloney also teaches “a stabilizing polypeptide that permits the association of the insulin polypeptide with an oil body upon breakage of the integrity of the plant cell [where] such a stabilizing polypeptide is a single chain antibody with specificity for an oil body [or a] single chain antibody [that] specifically binds an oleosin” (c.f., claim 1’s “wherein the recombinant proteins and/or the oil body proteins are fused with an affinity ligand which allows forming complexes comprising the oil body proteins and the recombinant proteins”) (emphasis added; para 0122; claims 15, 19, 20 and 13).
Flotation is defined in paragraph 0037 of the Specification of the instant application as meaning “that the mix is introduced into a liquid solution…and is centrifuged.”
Moloney teaches in para 0197 that “transgenic Arabidopsis thaliana seeds were ground with a mortar and pestle in 50 μl of 50 mM Tris-HCl pH 8.0, …a reducing SDS-PAGE sample buffer (6×SDS sample buffer, 0.35 M Tris-HCl pH 6.8, 30% glycerol, 10% SDS, 0.012% bromophenol blue, 5% β-mercaptoethanol) [(i.e., a liquid solution)] was added to the slurry and mixed by briefly vortexing [and then] centrifuged” (i.e., flotation). Moloney next teaches in para 0198 that “the soluble aqueous fraction was removed with a 26 G 5/8 1 ml syringe and the fat pad [(i.e., complexes comprising the oil body proteins and the recombinant proteins)] was re-suspended in 100 μl phosphate buffer supplemented with salt (20 mM Na2HPO4 pH 8.0, 0.5 M NaCl)” (i.e., claim 1’s 3rd step: “Recovering the complexes comprising the oil body proteins and the recombinant proteins by flotation of the mix”) (para 0198).
Moloney teaches “production of a fusion protein comprising insulin in chloroplasts of transgenic tobacco…results in the accumulation of insulin in green tissue, primarily the tobacco leaves” (c.f., claim 3)(para 0009).
Moloney does not explicitly teach:
Mixing the first genetically engineered organism or part thereof with oil body proteins which are not obtained from the first engineered organism,
that the genetically engineered plant expressing the recombinant proteins has been engineered by introducing a nucleic acid construct comprising the coding sequence of the recombinant proteins via agroinfiltration.
the genetically engineered plant or part thereof expressing the recombinant proteins comprises at least one inactivated gene involved in the Transcriptional Gene Silencing (TGS) and/or at least one inactivated gene involved in the Post Transcriptional Gene Silencing (PTGS) mechanism.
Rooijen is in the field of “Oil bodies and associated proteins as affinity matrices” (title) and teaches in example 2 that “Oil bodies were prepared from 5 g of wild type Brassica napus cv Westar seeds” (i.e., claim 1’s “oil body proteins which are not obtained from the first engineered organism”).
Rooijen teaches in example 3, which spans col 16 line 25 to col 18 line 25, that “Soluble protein is extracted through sonication of cells induced to express the ligand-GFP fusion [(i.e., the first genetically engineered organism or part thereof) and] is mixed with 2 ml of oil bodies prepared as described above, from seeds of non-transgenic plants” (col 16 line 25 to col 18 line 25). This reads on claim 1’s 2nd step: “Mixing the first genetically engineered organism or part thereof with oil body proteins which are not obtained from the first engineered organism, wherein the recombinant proteins and/or the oil body proteins are fused with an affinity ligand which allows forming complexes comprising the oil body proteins and the recombinant proteins.”
Ahmad is in the field of “recombinant protein production in plants” (title) and teaches that “to obtain high recombinant protein yields, agro-infiltration in Nicotiana benthamiana leaves, when combined with the co-expression of a suppressor of gene silencing [like p19 and HC-Pro], has widely established itself as the most utilized transient expression system in plants” (c.f., claim 4’s “engineered by introducing a nucleic acid construct comprising the coding sequence of the recombinant proteins via agroinfiltration” (page 14, col 1 and page 3, col 1 ). This also reads on claim 5 as it teaches co-expression of a suppressor of gene silencing (i.e., at least one in activated gene involved in the Transcriptional Gene TGS and/or at least one inactivated gene involved in the PTGS mechanism. Additionally, it is well known in the art that the widely used laboratory strain of Nicotiana benthamiana (often referred to as the LAB or TW16 strain) possesses a naturally occurring, non-functional Rdr1 gene where RDR1 is involved in both the TGS and in the PTGS mechanisms (see Ludman page 144, col 1, 1st full para).
More explicitly, regarding claim 5, Ludman “report[s] that the LAB strain of N. benthamiana expresses a single functional RDR6 homeolog [and] Inactivation of this gene by CRISPR/ Cas9” (i.e., the genetically engineered plant or part thereof expressing the recombinant proteins comprises at least one inactivated gene involved in the TGS and in the PTGS mechanism)(page 144, col 1, 2nd full para).
Therefore, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify the combined recombinant protein, affinity ligand, and oil body protein-mediated purification of the protein teachings of Moloney and Rooijen with the Agroinfiltration and suppressing of gene silencing teachings of Ahmad and Ludman arriving at the claimed invention and with a reasonable expectation of success. One would have been motivated to do this in order to take advantage of “several breakthroughs…allowing for very high accumulation levels, mainly through…transient expression, coupled with…protein fusions [and] quick and simple purification strategies… using oleosin [that] shown promise as efficient and cost-effective methods for non-chromatographic separation” (Ahmed, Abstract).
Regarding claims 6-7, Moloney teaches “stabilizing polypeptide is a single chain antibody with specificity for an oil body [or a] single chain antibody [that] specifically binds an oleosin” (para 0122).
Regarding claim 8, Moloney teaches “Methods for the production of insulin in plants” (title)
Regarding claim 9, Rooijen teaches that “Oleosin-specific ligands may be identified and isolated from a peptide phage display library screened with oleosin protein” (i.e., an artificial affinity ligand derived through phage display)(example 3, para 2).
Regarding claims 10-11, Moloney teaches “a single-chain Fv antibody (scFv) with species-specific affinity against the 18 kDa oleosin from Arabidopsis thaliana denoted D9scFv” (emphasis provided; para 0190).
Regarding claim 12, Rooijen teaches in example 3, which spans col 16 line 25 to col 18 line 25, that “Soluble protein is extracted through sonication of cells induced to express the ligand-GFP fusion [(i.e., the first genetically engineered organism or part thereof) and] is mixed with 2 ml of oil bodies prepared as described above, from seeds of non-transgenic plants [(i.e., oilseed plant comprising oil body proteins)]” (col 16 line 25 to col 18 line 25).
Regarding claim 13, Moloney teaches “a cleavage site may be located upstream of the N-terminus and downstream the C-terminus of the insulin allowing for the insulin polypeptide to be cleaved from the fusion partner, thereby obtaining isolated insulin” (i.e., the affinity ligand fused with the recombinant proteins and/or with the oil body proteins contains a cleavage site)(para 0123).
Regarding claim 14, Rooijen teaches an “aqueous phase containing the purified IgG-GFP fusion protein” and that “if desired, the IgG-affinity tag may be cleaved from GFP using Factor Xa at a concentration of 1U/50 ug of protein [so that] GFP could then be further purified using the oleosin-protein A oil body affinity matrix to remove the IgG affinity tag, (i.e., the method further comprises a step of purifying the recombinant proteins from the complexes) (col 24, lines 24-33).
Regarding claim 15, Moloney teaches in para 0180-0185 “Construction of pSBS4404: PRS-D9scFv-Klip27-MI-KDEL” where KLIP27 is “a Trypsin Cleavable Pro-Peptide” situated between an anti-oleosin scFv and insulin protein (MI) and then in para 0202-0204 teaches “Cleavage and HPLC Analysis of 4404 Expressing Arabidopsis Seed” where “concentrated sample was resuspended in 105 μl of double distilled water [and] then cleaved with trypsin (1:300 trypsin:total protein ratio, in 50 mM Tris-HCl pH 8.0, on ice for 90 min)” such that the insulin was cleaved from the ligand (i.e., purifying the recombinant proteins from the complexes includes modifying the pH to cleave the affinity ligand from the complexes).
Conclusion
No claims allowed.
Contact Information
Any inquiry concerning this communication or earlier communications from the examiner should be directed to YVETTE B TAMUKONG whose telephone number is (571)272-1040. The examiner can normally be reached M-Th 730-5 EST.
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/YVETTE BIH TAMUKONG/ Examiner, Art Unit 1662
/BRATISLAV STANKOVIC/ Supervisory Patent Examiner, Art Units 1661 & 1662
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