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 .
DETAILED ACTION
Applicant’s response and claim amendments filed on 6/17/2026 are received and entered.
In response to the most recent action, claims 1, 6, 10, and 15 have been amended. Claims 8-9 have been canceled without prejudice. Claims 19 and 20 were withdrawn in a previous action.
Claims 1-7 and 10-18 are currently pending in the instant application.
Any rejections/objections NOT repeated/presented here are withdrawn.
Maintained Rejections
Drawings
Color photographs and color drawings are not accepted in utility applications unless a petition filed under 37 CFR 1.84(a)(2) is granted. Any such petition must be accompanied by the appropriate fee set forth in 37 CFR 1.17(h), one set of color drawings or color photographs, as appropriate, if submitted via the USPTO patent electronic filing system or three sets of color drawings or color photographs, as appropriate, if not submitted via the via USPTO patent electronic filing system, and, unless already present, an amendment to include the following language as the first paragraph of the brief description of the drawings section of the specification:
The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
Color photographs will be accepted if the conditions for accepting color drawings and black and white photographs have been satisfied. See 37 CFR 1.84(b)(2).
Response to Arguments
Examiner notes that the petition for colored drawings has been filed on 6/17/2026. The decision is currently pending, and no decision has been made at the time of this action. Pending an acceptance for the petition of colored drawings, this objection will be maintained.
New Rejections
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.
Claims 1-7, 10-14, and 16-18 are rejected under 35 U.S.C. 103 as being unpatentable over Wang et al. (US 2020/0108145 A1, published 4/9/2020) in view of Chakraborty et al. (A robust strategy for negative selection of Cre-LoxP recombination-based excision of transgenes in induced pluripotent stem cells, PLoS One, Volume 8, Issue 5, all pages, published 2013), Schnutgen et al. (A directional strategy for monitoring Cre-mediated recombination at the cellular level in the mouse, Nature Biotechnology, Volume 21, pgs. 562-656, published 2003), and Abedi et al (US 2021/0324389 A1, filed on 04/15/2021).
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.
Regarding claim 1, Wang teaches a method for remotely controlling and non-invasively manipulating a nucleic acid expression in a cell, or an immune cell, wherein the immune cell can be a macrophage (see paragraph 0008). Want also teaches the method comprises providing a cell, an immune cell or a plurality of cells or immune cells a mechanism of remotely-controlling and non-invasively upregulating expression of a nucleic acid, wherein the nucleic acid can be a CAR, a single chain antibody, or an antibody fragment consisting of a single monomeric variable antibody domain, thereby adding a new specificity, function or target to a cell, an immune cell or a T cell (see claims 1 and 2).
Furthermore, Wang teaches a mammalian or human promoter or transcriptional activator activated by increased temperature, where in a recombinase gene can be controlled. Fig. 18A-C of Wang schematically illustrates that Cre recombinase is under control of the HSP promoter (see paragraph 0122). Fig. 18A shows that upon heat stimulation, heat-activated Cre will act on the two Lox sites to cause recombination, leading to the removal of the stop codon and constitutive expression of the reporter gene (see paragraph 0223).
Regarding claim 2, Wang teaches where upon heat stimulation, Cre will act on the two Lox sites, leading to the removal of the stop codon. This reads where Lox are recombinase are target sites for the recombinase, which flanks a sequence (stop codon) (paragraph 0223).
Regarding claim 5, Wang teaches where in Fig 18A (see paragraph 0223), the target reporter gene is downstream of two lox sites flanking a stop codon. Figure 18A illustrates the genetic orientation as PGK promoter-LoxP-STOP-LoxP-Reporter gene. This reads on where there is a stop cassette (stop codon) situated between the second promoter (PGK) and the payload gene (reporter gene).
Regarding claim 6, Wang teaches HSP promoter gene activation in response to heat shock stimulation in primary human T cells. The heat-sensitive GTM containing HSP-Driven eGFP fused with the PGK-driven mCherry was introduced into primary human T cells by lentiviral infection (see paragraph 0197) and where Fig.14A graphically illustrates HSP-driven eGFP fused with the constitutive PGK promoter-drive mCherry (see paragraph 0200). Therefore, Wang teaches a cargo or payload gene encoding a payload protein, that being mCherry, linked to a PGK promoter, further linked to a recombinase linked to a HSP promoter.
Regarding claim 7, Wang discloses that to convert transient expression of target genes to a permanent one, Cre-Lox recombination system was used (see paragraph 0222). This reads on claim 7 where the chosen recombinase is Cre. Furthermore, Fig. 18A schematically illustrates that the Cre recombinase is under the control of the HSP promoter (see paragraph 0122).
Regarding claim 11, Wang teaches that the nucleic acid or target gene is linked to a promoter activated by increase temperature, optionally linked to a heat shock protein (HSP), optionally a 70B HSP, which can be activated at 43°C. This is exemplified in paragraph [0180] where HEK cells were co-transfected with heat-sensitive GTM (HSP promoter driving the reporter eGFP) and a constitutively expressed mCherry. The heat shock stimulation was applied by incubating cells in a 43°C incubator (see paragraph 0180).
Regarding claim 12, Wang teaches the HSP promoter driven eGFP in Jurkat lymphocytes, wherein the percentage of cells expressing eGFP increased from 0.8% to 30.6% 13 hours after heat shock as compared to the control (see paragraph 0186).
Regarding claim 13, Wang discloses wherein the gene of interest is linked to a mammalian or human promoter, activated by increase temperature, or the nucleic gene is optionally operably linked to a heat shock protein (HSP) (see claim 1).
Regarding claim 16, Wang teaches an engineered cell, or immune cell, or plurality of cells or immune cells, as engineered for the use as a medicament in remotely-controlled and non-invasive manipulation of physiologic and/or a genetic process in a cell, or immune cell, or for the addition of a function or a target specificity to the cell, or immune cell, or plurality of cells or immune cells, or for the manipulation or correction of a pathological process, optionally, for eradicating a tumor or cancer in an individual in vivo (see claim 9). The eradication of a tumor or cancer in an individual indicates where in the engineered cell can express a payload protein that is capable of remodeling a tumor microenvironment.
Regarding claim 17, Wang teaches where Fig. 5 shows proof-of-concept that Gene Transducing Module or GTMs (which comprise of a nucleic acid linked to a promoter, activated by heat, and a thermo-responsive protein-expression nucleic acid linked to an inducible or constitutive promoter) (see paragraph 0033), can be engineered and integrated into the endogenous molecular network for the sensing of ultrasound stimulation to guide gene activations (see paragraph 0152). GTMs are vectors that are engineered into a cell. The cells having the GTMs are not described to comprise a “targeting moiety” binding to a target site of a subject.
Regarding claim 18, Wang recites where “peripheral blood mononuclear cells (PBMCs) containing the designed GTMs are used at different stages to test the feasibility and efficacy of embodiments as provided herein” (see paragraph 0172). It is very well known, to one skilled in the art, that PBMCs, which are derived from the blood, can be differentiated into macrophages. This is further evidenced by Rozner et al. (Generation of macrophages from peripheral blood monocytes in the rhesus monkey, J. Immunol. Methods, Volume 351, Issues 1-2, pgs. 36-40, published 2009).
Regarding claim 1, Wang does not teach wherein the population of heat-inducible macrophages have an exclusive state-switch architecture, wherein in the event of recombination, a second promoter drives the expression of a second payload gene, but in the event there is no recombination, the second promoter is operably linked to a third polynucleotide expressing a pro-death gene capable of halting cell growth and/or inducing cell death in the presence of the pro-death agent. Wang also does not teach that when the second promoter and second polynucleotide are operably linked, the second promoter and third polynucleotide are no longer linked. Regarding claim 3, Wang does not teach where the second polynucleotide of claim 1 is flanked by recombinase target sites, such as loxP sites. Wang only teaches that a stop codon is flanked by LoxP sites which is excised during recombination (see Fig. 18A).
Regarding claim 4, Wang does not teach that prior to the recombination event, the sequence of the payload gene is inverted relative to the promoter.
Regarding claim 10, Wang does not describe the state-switch architecture of claim 1 and does not further teach a pro-death protein and pro-death agent pair.
Regarding claim 14, Wang does not teach where the first inducible promoter comprises a nucleotide sequence at least 80% identical
Regarding claim 1, Schnutgen teaches FLEX switch, which is a mechanism of monitoring Cre-mediated recombination in mice (see abstract). Schnutgen teaches where the Cre-dependent one-way genetic switch is illustrated by plasmid pFLExEGFP, which contains one pair of WT LoxP sites and one pair of lox511 sites, with an alternate organization and a head-to-head orientation within each pair of sites (see Fig. 1). Both loxP and lox551 sites are recognized by Cre, however, lox551 sites recombine efficiently with themselves but not LoxP sites. Furthermore, the plasmid contains a promoter (SV40) driving eGFP as well as a promoterless lacZ gene in the antisense (inverted) orientation. Cred-mediated recombination may first induce inversion of the intervening DNA at either the loxP or lox511 sites (Fig. 1C), thus yielding a direct repeat of either two lox511 or two loxP sites. A further Cre-mediated excision will then remove the DNA located between the two loxP or the two lox511 sites. In the resulting plasmid, a single loxP and lox511 sites are left, making further inversion of the intervening DNA impossible (see Fig. 1D). The SV40 promoter now drives expression of LacZ instead of eGFP (see main). In this instance, in the event of Cre-recombination, the second promoter and second polynucleotide are linked (such as LacZ in the example of Schnutgen). In the event where recombination is not present, the promoter is still linked to eGFP, which could represent a pro-death protein).
Regarding claim 1, Chakraborty teaches a robust strategy for negative selection of Cre-LoxP recombination-based excision of transgenes in induced pluripotent stem cells (see abstract). Chakraborty teaches a proof-of-concept study to insert a therapeutic transgene (Factor IX) in the iPSCs. Cre recombinase was used to excise the antibiotic cassette followed by HSV-tk/ganciclovir negative selection of cells. Fig. 6A (shown below) provides a schematic diagram. In the unexcised form (when recombination doesn’t occur), HSV-tk and blasticidin resistance protein are expressed from the PGK promoter. The addition of ganciclovir leads to halting cell growth. In the event of recombination, the HSV-tk cassette is excised (the second promoter and third polynucleotide are no longer operably linked), and the remaining cells have the promoter linked to the second gene, such as a payload protein or FactorIX.
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Regarding claim 3, Schnutgen teaches Fig. 1, which discloses a SV40 promoter and eGFP and lacZ flanked by LoxP sites. This is also evidenced in Chakraborty Fig. 6A (shown above), where a cassette, which could be a string of polynucleotides representing a second polynucleotide, flanked by two loxP sites.
Regarding claim 4, Schnutgen teaches Fig. 1, which shows the plasmid contains a promoter (SV40) driving eGFP as well as a promoterless lacZ gene in the antisense (inverted) orientation.
Regarding claim 10, Chakraborty Fig. 6A (shown above), teaches the use of a pro-death gene (HSV-TK) with a pro-death agent (Ganciclovir) for the purpose of negative selection.
Regarding claim 14, Abedi teaches a first inducible promoter comprising a nucleic acid selected from the group consisting of SEQ ID NOs: 1-14 capable of inducing transcription of the payload gene to generate a payload transcript upon thermal stimulation (see claim 1). All SEQ ID NOs: 1-14 of Abedi have 100% identity to SEQ ID NOs: 1-14 of the instant application in corresponding order).
It would have been obvious to one with ordinary skill and creativity in the art, before the effective filing date, to combine the teachings of Wang, Schnutgen, Chakraborty, and Abedi to arrive at a population of heat-inducible macrophages comprising of a first promoter linked to a recombinase and a second promoter linked either to a payload gene or a pro-death gene, and where one of the payload gene or pro-death gene is expressed, depending on whether recombination occurs.
One would expect a reasonable chance of success as Wang teaches that a heat inducible Cre-lox system can be used in macrophages to manipulate an immune cell, such as a macrophage (see paragraph 0008 and 0009 of Wang), to express endogenous or exogenous nucleic acid in said cell. While the promoter nucleotide sequence is not explicitly disclosed by Wang, Wang does disclose the use of a mammalian promoter capable of being activated by heat which reads on the promoter taught by Abedi.
Though Wang does not teach an architect where the expression of two genes can be expressed, the claimed switch system is well known in the art. This is exemplified in Schnutgen Fig.1 where two genes, one inverted, are encompassed by loxP sites wherein a recombination event allows for the inversion of the two genes (see Fig. 1A and 1B of Schnutgen). While Schnutgen discloses Cre recombinase is driven by a Rarg promoter, Wang already discloses the use of Cre-recombinase with a heat activated promoter, such as HSP70.
Furthermore, the use of a pro-death gene for the purpose of negative selection in conjunction with the Cre-lox FLEx system is also known. Chakraborty exemplifies this structure in Fig. 6A as a promoter drives a cassette and a transgene of interest, Factor IX. Chakraborty states the unexcised form, the cassette comprises HSV-TK expressed from a PGK promoter, wherein the addition of ganciclovir allows HSV-Tk to halt cell growth. When Cre recombinase is expressed, the HSV-TK cassette is excised and the resulting cell expresses the transgene of interest. The addition of ganciclovir ensures that the resulting cells properly express the transgene of interest (see results of Chakraborty).
Wang already indicates the use of the Cre recombinase to express a payload protein or transgene is art recognized in macrophages to modify said macrophages to better eradicate tumors (see claim 9 of Wang). Furthermore, while Chakraborty indicates a second PGK promoter driving the expression of HSV-Tk is removed or excised with the gene during recombination, it is very well characterized that the inversion or excision of a DNA fragment is dependent on LoxP site orientation. In one of the original papers documenting Cre recombinase activity, Ambreski et al. (Bacteriophage P1 Site-specific recombination, The Journal of Biological Chemistry, Volume 259, No. 3, pgs. 1509-1514, published 1984) teaches “for intramolecular recombination, if the two loxP sites are oriented in opposite directions, then the DNA between the sites is inverted. If the two loxP sites are oriented in the same direction, then the DNA between the sites is excised by recombination (see section titled Recombination with Purified Cre Protein). Fig. 6A of Chakraborty shows loxP sites inserted in the same orientation, leading to excision of HSV-TK or the pro-death gene. It would have been routine, for one skilled in the art, to modify or experiment with the loxP site orientation to either excise or invert the pro-death gene. This inversion mechanism is seen in Schnutgen Fig. 1 where eGFP and LacZ are inverted after recombination and how the promoter now drives the expression of the originally inverted gene (see section titled Main). This further exemplifies that the Cre-lox system can be experimented with and modified to express a gene of interest in a population of cells.
One would be motivated to combine the teachings of Wang, Schnutgen, and Chakraborty for the purpose of providing negative selection to a population of macrophages, ensuring that macrophages that do not undergo recombination to express transgene are eliminated, thereby providing location specific activation of macrophages.
Wang teaches that there are major challenges to CAR-based immunotherapies as non-specific targeting of the CAR-T cells against normal/nonmalignant tissues (on target but off-tumor toxicities) can be life-threatening (see paragraph 0005 of Wang) and where researchers are actively seeking control over the timing and location of the activation of the perfused CAR T cells. Wang also directs the teachings of the invention to immune cells including macrophages (see paragraph 0009 and claim 11). Wang attempts to address this issue by expressing targeting antigens in immune cells driven by location specific ultrasound, providing heat, therefore activating Cre recombinase to express provide targeting capability. Since the FLEX switch system was known in the art to be able to express a first or second gene, depending if recombination occurs, driven by a single promoter, evidenced by Schnutgen and where a pro-death gene, such as HSV-TK, can be used with Cre-lox recombination to negatively select for a population of cells, as evidenced by Chakraborty, one skilled in the art would envision that by modifying the system of Wang to include the FLEX switch system with a pro-death gene and agent, one could further enhance the targeting specificity of macrophages. Under MPEP § 2143, the use of a known technique to improve similar devices (methods, or products in the same way, is a motivation to combine. Here, the prior art teaches that Cre-lox is very well known to express a second (payload) or third (pro-death) gene of interest, dependent on whether recombination occurs, and Chakraborty teaches where negative selection with a pro-death gene can be used to ensure that the remaining population of cells express the transgene of interest. Therefore, this system would be viewed as an improvement on the teachings of Wang to further improve specificity of immune cells which can have deleterious off target effects.
In view of the foregoing, claims 1-7, 10-14, and 16-18 are rejected under 35 U.S.C. 103 as being prima facie obvious, before the effective filing date.
Claims 15 is rejected under 35 U.S.C. 103 as being unpatentable over Wang et al. (US 2020/0108145 A1, published 4/9/2020) in view of Chakraborty et al. (A robust strategy for negative selection of Cre-LoxP recombination-based excision of transgenes in induced pluripotent stem cells, PLoS One, Volume 8, Issue 5, all pages, published 2013), Schnutgen et al. (A directional strategy for monitoring Cre-mediated recombination at the cellular level in the mouse, Nature Biotechnology, Volume 21, pgs. 562-656, published 2003) and Abedi et al (US 2021/0324389 A1, filed on 04/15/2021) in further view Naddafi et al. (The Epigenetic Regulation of Blinatumomab Gene Expression: Tumor Cell-dependent T cell Response against Lymphoma Cells and Cytotoxic Activity, Int. J. Mol. Cell. Med., 05/25/2019).
Regarding claim 1, the combined teachings of Wang, Chakraborty, and Schnutgen would allow one skilled in the art to arrive a population of heat inducible macrophages where a first heat inducible promoter drives a recombinase, and where a second promoter is operably linked to either a second (payload) gene or a third (pro-death) gene, depending on whether a recombination event occurs.
Regarding claim 15, the combined teachings of Wang, Chakraborty, and Schnutgen does not teach where the payload gene is a bispecific T cell engager (BiTE).
Regarding claim 15, Naddafi teaches where BiTEs antibodies (BsAb), specifically Blinatumomab, can form a transient cytolytic synapse between T cells and the tumor target cells (see introduction). BsAbs leads to a discharge of T cells contents and induces tumor cell death. Blinatumomab can redirect T cells toward malignant B cells and induce cancer cell lysis (see introduction).
It would have been obvious to one with ordinary skill in the art, before the effective filing date of the invention, to combine the teachings of Wang, Chakraborty, Schnutgen, and Naddafi, to arrive at a population of heat inducible macrophages, wherein the payload gene of interest is a BiTE.
One would expect a reasonable chance of success as Naddafi teaches expression vector can induce BsAb protein production in cells (see discussion) and that BsAb induces cytotoxicity of T-cells against CD19+ cell line, in vitro (see discussion). Therefore, BiTE is an antibody protein that directs immune cell activity towards a tumor. The invention of Wang recites where the upregulated nucleic acid (or the payload gene) resulting from thermal stimulation expresses a CAR, a single chain antibody, or a single domain antibody, or an antibody fragment consisting of a single monomeric variable antibody domain, to add a new specificity, function, or target cell to the immune cell. Therefore, one skilled in the art would envision that replacing the nucleic acid expressing CAR or an antibody in Wang with a gene expressing a BiTE instead, would functionally result in a similar, if not identical, population of heat-inducible macrophages.
One would be motivated to do so to impart targeting specificity to the immune cell, such as a macrophage, to reduce off target effects, as disclosed in Wang (see paragraph 0005).
In view of the foregoing, claim 15 is rejected under 35 U.S.C. 103 as being prima facie obvious, before the effective filing date.
Double Patenting
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.
Claims 1-18 are rejected on the ground of nonstatutory double patenting as being unpatentable over claim 1, 3, 9, 10, 13, and 17 of U.S. Patent No. 12,359,208 B2 in view of in view of Chakraborty et al. (A robust strategy for negative selection of Cre-LoxP recombination-based excision of transgenes in induced pluripotent stem cells, PLoS One, Volume 8, Issue 5, all pages, published 2013), Schnutgen et al. (A directional strategy for monitoring Cre-mediated recombination at the cellular level in the mouse, Nature Biotechnology, Volume 21, pgs. 562-656, published 2003).
US Patent 12,359,208 B2 and the instant application claim a first inducible promoter selected from a group consisting of SEQ ID NOs: 1-14. Both the instant application and the US Patent have 100% identity between SEQ ID NOs: 1-14. Furthermore, both claim that the first polynucleotide is operably linked to the first promoter. Both also claim a second promoter linked to a second polynucleotide comprising of a payload gene encoding a payload protein comprising of a chimeric antigen receptor (CAR), T-cell receptor (TCR), or a cytokine. Both also claim that upon thermal stimulation, transcription is increased by at least 1.1-fold and that both the nucleic acid composition and the population of heat-inducible macrophages are capable of remodeling the tumor microenvironment.
Regarding claims 1-18 of the instant application and claims 1, 3, 9, 10, 13, and 17 of the issued patent, the issued patent does not recite in claim 1, or any dependent claims, where a second promoter is operably linked to a third polynucleotide, encoding a pro-death gene, in the event recombination does not occur.
Regarding claim 1, Wang teaches where a polynucleotide encoding a gene is expressed when a Cre recombinase under the control of a HSP promoter results in the removal of a stop codon flanked by LoxP sites (see paragraph 0122 and Fig. 18A), which describes the structure disclosed in claim 1 and 10 of the issued patent.
Regarding claim 1, Schnutgen teaches FLEX switch, which is a mechanism of monitoring Cre-mediated recombination in mice (see abstract). Schnutgen teaches where the Cre-dependent one-way genetic switch is illustrated by plasmid pFLExEGFP, which contains one pair of WT LoxP sites and one pair of lox511 sites, with an alternate organization and a head-to-head orientation within each pair of sites (see Fig. 1). Both loxP and lox551 sites are recognized by Cre, however, lox551 sites recombine efficiently with themselves but not LoxP sites. Furthermore, the plasmid contains a promoter (SV40) driving eGFP as well as a promoterless lacZ gene in the antisense (inverted) orientation. Cred-mediated recombination may first induce inversion of the intervening DNA at either the loxP or lox511 sites (Fig. 1C), thus yielding a direct repeat of either two lox511 or two loxP sites. A further Cre-mediated excision will then remove the DNA located between the two loxP or the two lox511 sites. In the resulting plasmid, a single loxP and lox511 sites are left, making further inversion of the intervening DNA impossible (see Fig. 1D). The SV40 promoter now drives expression of LacZ instead of eGFP (see main). In this instance, in the event of Cre-recombination, the second promoter and second polynucleotide are linked (such as LacZ in the example of Schnutgen). In the event where recombination is not present, the promoter is still linked to eGFP, which could represent a pro-death protein).
Regarding claim 1, Chakraborty teaches a robust strategy for negative selection of Cre-LoxP recombination-based excision of transgenes in induced pluripotent stem cells (see abstract). Chakraborty teaches a proof-of-concept study to insert a therapeutic transgene (Factor IX) in the iPSCs. Cre recombinase was used to excise the antibiotic cassette followed by HSV-tk/ganciclovir negative selection of cells. Fig. 6A (shown below) provides a schematic diagram. In the unexcised form (when recombination doesn’t occur), HSV-tk and blasticidin resistance protein are expressed from the PGK promoter. The addition of ganciclovir leads to halting cell growth. In the event of recombination, the HSV-tk cassette is excised (the second promoter and third polynucleotide are no longer operably linked), and the remaining cells have the promoter linked to the second gene, such as a payload protein or FactorIX.
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Regarding claim 15 of the instant application, Naddafi teaches where BiTEs antibodies (BsAb), specifically Blinatumomab, can form a transient cytolytic synapse between T cells and the tumor target cells (see introduction). BsAbs leads to a discharge of T cells contents and induces tumor cell death. Blinatumomab can redirect T cells toward malignant B cells and induce cancer cell lysis (see introduction).
It would have been obvious to modify the claims of US Patent 12,359,208 B2, that being a composition encoding a nucleic acid that encodes a tumor antibody, driven by an inducible promoter responsive to heat, to further comprise the negative selection characteristic of the instant application, in view of Wang, Schnutgen, and, Chakraborty, to arrive at the claims of the instant application.
One would expect a reasonable chance of success as Wang teaches that a heat inducible Cre-lox system can be used in macrophages to manipulate an immune cell, such as a macrophage (see paragraph 0008 and 0009 of Wang), to express endogenous or exogenous nucleic acid in said cell. While the promoter nucleotide sequence is not explicitly disclosed by Wang, Wang does disclose the use of a mammalian promoter capable of being activated by heat which reads on the promoter taught by Abedi.
Though Wang does not teach an architect where the expression of two genes can be expressed, the claimed switch system is well known in the art. This is exemplified in Schnutgen Fig.1 where two genes, one inverted, are encompassed by loxP sites wherein a recombination event allows for the inversion of the two genes (see Fig. 1A and 1B of Schnutgen). While Schnutgen discloses Cre recombinase is driven by a Rarg promoter, Wang already discloses the use of Cre-recombinase with a heat activated promoter, such as HSP70.
Furthermore, the use of a pro-death gene for the purpose of negative selection in conjunction with the Cre-lox FLEx system is also known. Chakraborty exemplifies this structure in Fig. 6A as a promoter drives a cassette and a transgene of interest, Factor IX. Chakraborty states the unexcised form, the cassette comprises HSV-TK expressed from a PGK promoter, wherein the addition of ganciclovir allows HSV-Tk to halt cell growth. When Cre recombinase is expressed, the HSV-TK cassette is excised and the resulting cell expresses the transgene of interest. The addition of ganciclovir ensures that the resulting cells properly express the transgene of interest (see results of Chakraborty).
Wang already indicates the use of the Cre recombinase to express a payload protein or transgene is art recognized in macrophages to modify said macrophages to better eradicate tumors (see claim 9 of Wang). Furthermore, while Chakraborty indicates a second PGK promoter driving the expression of HSV-Tk is removed or excised with the gene during recombination, it is very well characterized that the inversion or excision of a DNA fragment is dependent on LoxP site orientation. In one of the original papers documenting Cre recombinase activity, Ambreski et al. (Bacteriophage P1 Site-specific recombination, The Journal of Biological Chemistry, Volume 259, No. 3, pgs. 1509-1514, published 1984) teaches “for intramolecular recombination, if the two loxP sites are oriented in opposite directions, then the DNA between the sites is inverted. If the two loxP sites are oriented in the same direction, then the DNA between the sites is excised by recombination (see section titled Recombination with Purified Cre Protein). Fig. 6A of Chakraborty shows loxP sites inserted in the same orientation, leading to excision of HSV-TK or the pro-death gene. It would have been routine, for one skilled in the art, to modify or experiment with the loxP site orientation to either excise or invert the pro-death gene. This inversion mechanism is seen in Schnutgen Fig. 1 where eGFP and LacZ are inverted after recombination and how the promoter now drives the expression of the originally inverted gene (see section titled Main). This further exemplifies that the Cre-lox system can be experimented with and modified to express a gene of interest in a population of cells.
Therefore, one would have modified with a reasonable expectation of success, the composition of US Patent 12,359,208 B2 with a Cre recombinase promoter and expect successful transgene expression in a macrophage. One would be motivated to specifically to insert the nucleic acid composition, such as a payload encoding a BiTE antibody, into a tumor-associated macrophage as Wang teaches that off target effects of immune cells pose a serious burden and the combined teachings of Chakraborty and Schnutgen would provide an enhanced method of negative screening genes that do not undergo recombination with location specific heat, in order to reduce the off target effects of macrophages.
Response to Arguments
Applicant argues that, in view of amendments, the teachings of Wang does not disclose the structural architect of claim 1, that being an exclusive state switch system to regulate the expression of either a payload gene or pro-death gene, in the presence or absence of Cre-driven recombination. Wang also does not teach where the payload gene is flanked by LoxP sites, instead teaching where a stop codon is flanked by Cre recognition sites. As the teachings of Xu does not cure the teachings of Wang, the obviousness rejection is therefore moot.
It is noted that examiner has set forth a new rejection that relies on the teachings of Chakraborty and Schnutgen to cure the deficiencies of Wang. Schnutgen teaches the FLEx switch Cre-recombinase system, wherein two genes, eGFP and an inverted LacZ, are flanked by loxP sites. A promoter, SV40, drives the expression of eGFP. In the event of Cre-mediated recombination, eGFP and LacZ are inverted, therefore resulting in an inverted eGFP gene and allowing the SV40 promoter to drive the expression of LacZ instead. This established that it was known in the art that Cre-recombinase can be used in a manner that inverts genes, allowing the expression of a gene to switch between two genes of interest.
Furthermore, the teachings of Wang and Chakraborty provide motivation as to why one would implement this system in macrophages to express a payload protein and a pro-death gene. Wang clearly discloses in paragraph [0005] that off target effects of genetically engineered immune cells can be detrimental to these cells and proposes a mechanism to introduce targeting capability to macrophages. Chakraborty teaches where the Cre-recombinase system can be used to implement a negative selection mechanism into cells, therefore, ensuring that cells that do not recombine still express the pro-death gene. The addition of an agent corresponding with the gene would therefore eliminate any cell population wherein recombination was not successful. One skilled in the art would envision that by combining these well-known elements of the Cre-recombinase system, one would create a population of cells, specifically macrophages, that expresses a payload gene for targeted specificity and a pro-death gene for assurance in the event recombination doesn’t occur, ensuring that the macrophages have a specific, on-target effect.
Conclusion
No claims are allowed.
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/D.T.Y./Examiner, Art Unit 1635
/RAM R SHUKLA/Supervisory Patent Examiner, Art Unit 1635
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