DETAILED ACTION
Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Continued Examination Under 37 CFR 1.114
A request for continued examination under 37 CFR 1.114, including the fee set forth in 37 CFR 1.17(e), was filed in this application after final rejection. Since this application is eligible for continued examination under 37 CFR 1.114, and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114. Applicant's submission filed on September 29, 2025 has been entered.
Application Status and Withdrawn Claims
Applicant’s amendment filed September 29, 2025, amending claims 1, 8 and 20-21 is acknowledged. Claims 1, 4-5, 8-12, 14-15, 17, 20-22 and 24-25 are pending.
Claims 20-22 and 24-25 remain withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected invention, there being no allowable generic or linking claim.
Claims 1, 4-5, 8-12, 14-15, and 17 are under examination.
The amendment to claim 1 requiring the production of the first and second polypeptide in stoichiometric amounts overcomes the §103 rejection over Onishi in view of Lopez.
Any other 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.
Drawings
The drawings are objected to because FIGs 3, 5 and 7, which appears on several sheets, have not been identified by the same number followed by a capital letter. 37 C.F.R. 1.84(u)(1) requires that “partial views intended to form one complete view, on one or several sheets, must be identified by the same number followed by a capital letter.” See MPEP 608.02. Sheets 3, 4 and 5 should be labeled FIG. 3A, FIG. 3B and FIG 3C, respectively. Sheets 7 and 8 should be labeled FIG. 5A and FIG. 5B, respectively. Sheets 10, 11, 12 and 13 should be labeled FIG. 7A, FIG. 7B, FIG. 7C and FIG 7D, respectively.
Corrected drawing sheets in compliance with 37 CFR 1.121(d) are required in reply to the Office action to avoid abandonment of the application. Any amended replacement drawing sheet should include all of the figures appearing on the immediate prior version of the sheet, even if only one figure is being amended. The figure or figure number of an amended drawing should not be labeled as “amended.” If a drawing figure is to be canceled, the appropriate figure must be removed from the replacement sheet, and where necessary, the remaining figures must be renumbered and appropriate changes made to the brief description of the several views of the drawings for consistency. Additional replacement sheets may be necessary to show the renumbering of the remaining figures. Each drawing sheet submitted after the filing date of an application must be labeled in the top margin as either “Replacement Sheet” or “New Sheet” pursuant to 37 CFR 1.121(d). If the changes are not accepted by the examiner, the applicant will be notified and informed of any required corrective action in the next Office action. The objection to the drawings will not be held in abeyance.
Specification
The disclosure is objected to because it contains an embedded hyperlink and/or other form of browser-executable code on page 25. Applicant is required to delete the embedded hyperlink and/or other form of browser-executable code; references to websites should be limited to the top-level domain name without any prefix such as http:// or other browser-executable code. See MPEP § 608.01.
Claim Objections
Claims 11 and 14 are objected to because of the following informalities:
Claim 11 recites “the vector according to claim 10, wherein the first and second subunits are codon optimized for expression in microalgal cells.” In claim 10 “the first and second subunits” refer to parts of a protein. Proteins are not codon optimized. Instead, the polynucleotides that encode proteins are codon optimized. To be more precise, claim 11 should be amended: “wherein the first and second polynucleotides are codon optimized for expression in microalgal cells”.
Claim 14 recites “…and the third expression cassette comprises the bleomycin resistance gene, the HSP70A/RBCS2 chimeric promoter and the 3’ UTR of the RBCS2 gene as cis regulatory regions.” Claim 1 already requires the third expression cassette to comprise the HSP70A/RBCS2 chimeric promoter and the 3’ UTR of the RBCS2 gene as cis regulatory regions, and is therefore redundant in claim 14. It is suggested that “the HSP70A/RBCS2 ... as cis regulatory regions” be deleted.
Additionally, because claim 1 now recites a first and second protein of interest, it is suggested that claim 14 recite “wherein the first protein of interest comprises a light chain of an antibody” and “wherein the second protein of interest comprises a heavy chain of an antibody”.
Appropriate correction is required.
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 8-9 and 17 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.
Claim 8 recites two “further comprising” clauses. The second “further comprising” clause recites “further comprising a nucleotide sequence encoding a signal peptide in the same open reading frame at position 5’ respect to the nucleotide sequence encoding the protein of interest or the protein encoding by the selection gene.” This clause renders claim 8 indefinite for two reasons. First, it is not clear what “the same open reading frame” is referring to. There are three cassettes (i.e., open reading frames) recited in claim 1. Additionally, the two “further comprising” clauses are joined by and/or, so the first clause is not required. It is not clear if the signal peptide must be in the same cassette as the intron, if present, or if “the same open reading frame” is referring to something else. Finally, “the protein of interest” lacks clear antecedent basis since there are two proteins of interest recited and it is not clear which protein of interest is being referred to.
To remedy the indefiniteness, it is suggested to claim each of the vector structures in a separate claim, such as “The vector according to claim 1, further comprising an intro within a region encoding the first protein of interest, and optionally, a nucleotide sequence encoding a signal peptide placed 5’ of the sequence encoding the first protein of interest”. Then add new claims requiring an intron and optionally a signal peptide for the second protein of interest and/or the selection marker gene.
Claim 9 is rejected for depending from claim 8 and not remedying the indefiniteness.
Claim 17 recites “wherein the microalga is from genus Chlamydomonas, preferably the microalga is C. reinhardtii.” Use of “preferably” renders the claim indefinite because it is unclear whether the limitation(s) following the phrase are part of the claimed invention. See MPEP § 2173.05(d).
To remedy the indefiniteness, remove “preferably” from the claim. Alternatively, a new claim can be added that further limits the Chlamydomonas genus by reciting C. reinhardtii.
Claim Rejections - 35 USC § 103
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
This application currently names joint inventors. In considering patentability of the claims the examiner presumes that the subject matter of the various claims was commonly owned as of the effective filing date of the claimed invention(s) absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and effective filing dates of each claim that was not commonly owned as of the effective filing date of the later invention in order for the examiner to consider the applicability of 35 U.S.C. 102(b)(2)(C) for any potential 35 U.S.C. 102(a)(2) prior art against the later invention.
Claims 1, 4-5, 8, 15 and 17 are rejected under 35 U.S.C. 103 as being unpatentable over Jia (Jia et al., Algal Research (2016), 20: 53-56), as evidenced by Lumbreras (Lumbreras et al., Plant Journal (1998), 14: 441-447), and in view of Kumar (Kumar et al., Algal Research (2013), 2: 321-332) and Lopez (Lopez-Paz, Plant Journal (2017), 92: 1232-1244; of record).
Regarding claims 1, 4-5, 15 and 17, Jia teaches a vector for multi-gene expression in C. reinhardtii (i.e., a microalgal cell) (Abstract). Jia teaches a vector comprising gene expression cassette A (GEC A) (i.e., a first expression cassette), gene expression cassette B (GEC B) (i.e., a second expression cassette), and ble resistance (i.e., a third expression cassette comprising the bleomycin selection gene) (Section 2.2; Fig 1). Jia teaches both GEC A and GEC B comprise the HSP70A-RBCS2 chimeric promoter and the RBCS2 3’ UTR terminator (Section 3.1, ¶1). Jia teaches cloning the sequence encoding GFP as GEC A (i.e., a first protein of interest) and cloning the sequence encoding luciferase as GEC B (i.e., a second protein of interest) (Section 2.2, ¶2). Jia teaches the ble resistance gene was amplified from pSP124, which was disclosed in Lumbreras et al., (Section 2.2, ¶1). Lumbreras teaches pSP124 comprises the coding sequence for the ble resistance coding sequence driven by the RBCS2 promoter and terminated with the RBCS2 3’ UTR (Fig 1). Thus, Jia’s multigene expression vector comprises 1) a first expression cassette encoding a first protein of interest regulated by the HSP70A/RBCS2 chimeric promoter and RBCS2 3’ UTR, 2) a second expression cassette encoding a second protein of interest regulated by the HSP70A/RBCS2 chimeric promoter and RBCS2 3’ UTR, and 3) a third expression cassette encoding the bleomycin resistance gene regulated by the RBCS2 promoter and the RBCS2 3’ UTR. Jia teaches “the expression level of GFP was in general correlated with that of luciferase” (i.e., the first and second proteins of interest were produced in stoichiometric amounts) (Section 3.2). Jia also teaches that the expression level of GFP and luciferase varied among transformants (Section 3.2).
Jia does not teach using the RPL23 gene promoter and 3’ UTR or the FDX gene promoter and 3’ UTR. Jia does not teach using the HSP70A/RBCS2 chimeric promoter to drive expression of the bleomycin selection gene.
Kumar teaches evaluating seven different nuclear promoter/3’UTR combinations for expression of proteins of interest in C. reinhardtii (Abstract). Kumar teaches an expression vector comprising the first expression cassette encoding Luciferase (Luc) and a second expression cassette comprising a selection marker coding sequence regulated by the Hsp70A/Rbcs2 chimeric promoter and the Rbcs2 terminator (Fig 1). Kumar teaches that there was a high level of transgene silencing in the strain that used the same Hsp70A/Rbcs2 promoter for expression of both Luciferase and the selection marker (page 330, ¶3). Kumar teaches the gene silencing is likely due to homology dependent transcriptional gene silencing, based on a previous study that showed that the presence of more than one copy of a single promoter driving transgene expression can lead to homology-dependent transcriptional gene silencing (page 330, ¶3). Kumar recommends that expression of the selectable marker and the gene-of-interest should be driven by different promoters (page 330, ¶3).
Lopez teaches the need for discovering additional promoter and 3’ UTR regulatory regions for consistent high transgene expression from the nucleus in C. reinhardtii (¶ spanning pages 1232-1233). Lopez teaches the expression cassette RPL23:Luc:RPL23, which comprises the Luc coding sequence regulated by the RPL23 gene promoter and 3’ UTR (Fig 3). Lopez teaches the expression cassette FDX1:Luc:FDX1, which comprises the Luc coding sequence regulated by the FDX1 gene promoter and 3’ UTR (Fig 3). Lopez teaches the expression cassette AR:Luc:RBCS2, which comprises the Luc coding sequence regulated by the HSP70A/RBCS2 chimeric promoter and RBCS2 3’ UTR, which was previously known in the art (Fig 3). Lopez teaches comparing Luc expression from each of the expression cassettes (Fig 4). Lopez teaches the RPL23:Luc:RPL23 and FDX1:Luc:FDX1 expression cassettes produced similarly high expression of luciferase, and both cassettes produced higher expression than the known expression cassette having the HSP70A/RBCS2 chimeric promoter and RBCS2 3’ UTR (Fig 4a; page 1233, ¶4). Lopez teaches that the RPL23:Luc:RPL23 and FDX1:Luc:FDX1 expression cassettes exhibited very low silencing at 7 and 13 weeks (Fig 4b), which is a desirable feature a transgene expression cassette (¶ spanning pages 1235-1236).
Regarding claims 1, 4-5, 15 and 17, it would have been obvious to one skilled in the art before the effective filing date of the claimed invention to have 1) used the RPL23 promoter/3’UTR and FDX1 promoter/3’UTR regulatory sequences for the expression the first and second genes of interest, and 2) replaced the RBCS2 promoter with the chimeric HSP70A/RBCS2 promoter for expression of bleomycin in Jia’s muti-cassette expression vector. It would have amounted to substituting one known promoter/3’UTR sequences for another by known means to yield predictable results. The skilled artisan would have predicted that the RPL23 promoter/3’UTR and FDX1 promoter/3’UTR regulatory sequences could be used in Jia’s vector for transgene expression in C. reinhardtii because Lopez demonstrates their use for driving high expression of luciferase in C. reinhardtii using a vector that also has a selection marker driven by a second promoter. It also would have been predictable that the HSP70A/RBCS2 promoter could be used to drive expression of bleomycin because both Kumar and Lopez demonstrate driving expression of a selection marker using the chimeric promoter. One would have been motivated to use different promoters for the expression for GFP, Luciferase, and the selection marker because Kumar teaches that using the same promoter to drive transgene expression can lead to transgene silencing. The skilled artisan would have specifically been motivated to choose the RPL23 and FDX1 promoter/3’UTR regulatory regions for expression of the proteins of interest (GEC A and GEC B cassettes) based on their ability to drive exceptionally high transgene expression levels. Based on the transgene expression levels driven by the RPL23 and FDX1 promoter/3’UTR regulatory regions in C. reinhardtii demonstrated in Lopez, the skilled artisan would have predicted that GFP and Luciferase would still be produced at approximately the same levels in a C. reinhardtii cell (i.e., in stoichiometric amounts) using the RPL23 and FDX1 regulatory regions as compared to when the same promoter was used.
Claim 8 is indefinite for the reasons explained above in paragraph 18. For the purposes of examination, claim 8 is interpreted as requiring an intron in either the first, second or third expression cassette and/or a sequence encoding a signal peptide in the first, second or third expression cassette.
Regarding claim 8, Kumar teaches the known Hsp70A/Rbcs2 promoter harbors an intron in the 5’ UTR that is known to enhance transgene expression (page 324, ¶ 4). Thus, the multi-cassette vector with the Hsp70A/Rbcs2 promoter driving expression of bleomycin rendered obvious above for claim 1 already comprised an intron within a region encoding the selection gene.
Claims 10-12 and 14 are rejected under 35 U.S.C. 103 as being unpatentable over Jia (Jia et al., Algal Research (2016), 20: 53-56), Kumar (Kumar et al., Algal Research (2013), 2: 321-332) and Lopez (Lopez-Paz, Plant Journal (2017), 92: 1232-1244, of record), as applied to claims 1, 4-5, 8, 15 and 17 above, and further in view of Tran (Tran et al., Biotechnology and Bioengineering (2009), 104: 663-673).
The teachings of Jia, Kumar and Lopez are recited above and applied as for claims 1, 4-5, 8, 15 and 17.
Jia also teaches that microalgal species can be used for production of human nutrients (page 53, ¶1). Regarding claim 11, Jia teaches codon optimization for the expression of Luciferase and GFP in Chlamydomonas (Section 2.2).
Kumar also teaches Chlamydomonas can be used for the production of biopharmaceuticals (page 321, ¶1). Kumar teaches that as of 2013, most recombinant protein production had used chloroplast expression (page 321, ¶2). Kumar teaches drawbacks of chloroplast-based expression including 1) chloroplasts lack enzymes to carry out important post-translational modifications, and 2) protein processing and targeting machinery necessary for secreting recombinant proteins into the culture media is only operational for nuclear encoded proteins (page 321, ¶2). Kumar teaches nuclear expression of recombinant proteins can overcome the limitations of chloroplast-based expression systems. Regarding claim 11, Kumar teaches codon optimization for the expression of Luciferase in Chlamydomonas (section 2.2).
Lopez also teaches Chlamydomonas is used for algal-based biotechnological applications including production of recombinant protein and other high-value products (page 1232, ¶1). Lopez teaches that Chlamydomonas can secrete recombinant protein into the external media (page 1232, ¶1). Regarding claim 11, Lopez teaches codon optimization for the expression of Luciferase in Chlamydomonas (page 1235, ¶3).
Jia, Kumar and Lopez do not specifically teach using Chlamydomonas for producing heavy and light chains of antibodies.
Tran teaches using Chlamydomonas reinhardtii to synthesize and assemble full length IgG monoclonal antibodies (Abstract). Tran teaches that monoclonal antibodies are composed of two copies of a heavy chain polypeptide and two copies of a light chain polypeptide (i.e., two different subunits of a heterodimeric protein) (page 664, ¶2). Tran teaches integration of two expression cassettes, each encoding either the heavy chain polypeptide or the light chain polypeptide, in the chloroplast genome of C. reinhardtii (Fig 1). Tran teaches accumulation of the human antibody light chain and heavy chain polypeptides in the chloroplast (Fig 2). Tran teaches that human antibody heavy chain antibodies were partially degraded, likely due to a protease in the chloroplast (page 672, ¶3). Tran also teaches that antibodies produced in chloroplast are un-glycosylated, unlike when produced in animal cells (page 672, ¶1).
Regarding claims 10-12 and 14, it would have been obvious to one skilled in the art before the effective filing date of the claimed invention to have provided the multi-cassette expression vector rendered obvious above for claim 1, and cloned in the coding sequence for antibody light chain and heavy chain polypeptides for the nuclear production of antibody heavy and light chains in C. reinhardtii. It would have amounted to cloning known coding sequences by known means to yield predictable results. The skilled artisan would have predicted that the coding sequence for antibody heavy and light chains could be cloned into Jia’s multi-cassette expression vector because 1) Tran teaches cloning various IgG sequences into vectors and 2) various cloning methods are well-known in the art as evidenced by the cited references. The skilled artisan would have been motivated to include the IgG coding sequences in the obvious vector for the purpose of expressing the heavy and light chains from the nucleus to avoid the drawbacks of chloroplast-based expression, such as lack of protein glycosylation and extracellular secretion, indicated by Kumar and Tran.
Claim 9 is rejected under 35 U.S.C. 103 as being unpatentable over Jia (Jia et al., Algal Research (2016), 20: 53-56), Kumar (Kumar et al., Algal Research (2013), 2: 321-332), Lopez (Lopez-Paz, Plant Journal (2017), 92: 1232-1244, of record) and Tran (Tran et al., Biotechnology and Bioengineering (2009), 104: 663-673), as applied to claims 1, 4-5, 8, 10-12, 14-15 and 17 above, and further in view of Hempel (Hempel et al., PloS One (2011), 6(12): e28424).
Claim 9 is indefinite for the reasons explained above in paragraphs 18 and 20. For the purposes of examination, claim 9 is interpreted as requiring a murine signal peptide in either the first, second or third expression cassette.
The teachings of Jia, Kumar, Lopez and Tran are recited above and applied as for claims 1, 4-5, 8, 10-12, 14-15 and 17. Tran also teaches expressing mouse IgG1 in C. reinhardtii chloroplasts (Fig 9).
Jia, Kumar, Lopez and Tran do not expressly teach including a mouse signal peptide in the coding sequence of the proteins of interest or the selection gene.
Hempel teaches antibodies can be produced in algae and cites the Tran reference, of record herein (page 1, ¶2). Hempel teaches the production of a human monoclonal IgG1 antibody in the algae P. tricornutum expressed from the nucleus, as evidenced by accumulation of the heavy and light chains in the endoplasmic reticulum (ER) when fused to an ER-retention signal (page 2, ¶4). Hempel teaches the endogenous signal peptides of the human antibody heavy chain and light chain polypeptides were sufficient to traffic the human protein to the ER in the algal cell (Fig 1, legend).
It would have been obvious to one skilled in the art before the effective filing date of the claimed invention to have used the multi-cassette expression construct rendered obvious above for claim 1, and cloned in the coding sequence for the mouse antibody light chain and heavy chain polypeptides, including the endogenous mouse signal peptide on the IgG gene, for the nuclear production of mouse antibody heavy and light chains in C. reinhardtii. It would have amounted to cloning in known coding sequences by known means to yield predictable results. The skilled artisan would have predicted that the coding sequence for the mouse antibody heavy and light chains could be cloned into Jia’s multi-cassette expression vector because Tran teaches 1) expressing the mouse IgG1 sequences in C. reinhardtii and 2) various cloning methods are well-known in the art as evidenced by the cited references. The skilled artisan would have been motivated to retain the endogenous murine signal peptide from the IgG1 polypeptides for nuclear expression for the purpose of expressing the heavy and light chains from the nucleus, thereby avoiding the drawbacks of chloroplast-based expression. It would have been entirely predictable that the native murine signal polypeptide would function in algal cells because Hempel teaches that other mammalian signal peptides from IgG antibodies are sufficient for ER-trafficking in algal cells.
Response to Arguments
Applicant’s arguments with respect to the §103 rejections of the pending claims have been considered but are moot because the new ground of rejection does not rely on any reference applied in the prior rejection of record for any teaching or matter specifically challenged in the argument.
Conclusion
No claims are allowable.
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/CATHERINE KONOPKA/Examiner, Art Unit 1635