DETAILED ACTION
Claim Status
As of the Non-Final Office Action mailed 11/03/2025, claims 1-3, 14, 18-20, 22-24, 27, 29-30, 32, and 36 were pending and claims 32 and 36 were withdrawn for being drawn to nonelected invention.
In Applicant's Response filed on 2/3/2026, claim 1 was amended and claims 2-3, 18, and 30 were canceled.
As such, claims 1, 14, 19-20, 22-24, 27, 29, 32, and 36 are pending and claims 1, 14, 19-20, 22-24, 27, and 29 have been examined herein.
Withdrawn Objections/Rejections
The objections and rejections presented herein represent the full set of objections and rejections currently pending in this application. Any objections or rejections not specifically reiterated are hereby withdrawn.
Claim Objections
Claim 1 is objected to because of the following informalities:
Claim 1 recites “a GEM transcription factor, the input is E2, and the output is a Z3PM transcription factor”. The instant specification states that “GEM” is Gal4 DNA binding domain fused to Estradiol ligand binding domain and Msn2 activation domain (Gal4-E-Msn2), “E2” is estradiol, and “Z3PM” is zinc finger bound to progesterone hormone binding domain and Msn2 activation domain (see para 243). The examiner suggests putting these terms in claim 1 followed by their respective abbreviations in parentheses (i.e., estradiol (E2)).
Claim 1 recites “the input is E2” and “the output is a Z3PM transcription factor.” The examiner suggests amending these limitations to recite “the output of the signal pathway is E2” and “the output of the signaling pathway is a Z3PM transcription factor.”
Appropriate correction is required.
Claim Rejections - 35 USC § 112(d)
The following is a quotation of 35 U.S.C. 112(d):
(d) REFERENCE IN DEPENDENT FORMS.—Subject to subsection (e), a claim in dependent form shall contain a reference to a claim previously set forth and then specify a further limitation of the subject matter claimed. A claim in dependent form shall be construed to incorporate by reference all the limitations of the claim to which it refers.
The following is a quotation of pre-AIA 35 U.S.C. 112, fourth paragraph:
Subject to the following paragraph [i.e., the fifth paragraph of pre-AIA 35 U.S.C. 112], a claim in dependent form shall contain a reference to a claim previously set forth and then specify a further limitation of the subject matter claimed. A claim in dependent form shall be construed to incorporate by reference all the limitations of the claim to which it refers.
Claims 19-20 and 27 are rejected under 35 U.S.C. 112(d) or pre-AIA 35 U.S.C. 112, 4th paragraph, as being of improper dependent form for failing to further limit the subject matter of the claim upon which it depends, or for failing to include all the limitations of the claim upon which it depends. Claim 19 is dependent on currently canceled claim 18. Independent claim 1 specifies that the signaling protein “is a first fusion protein comprising a first leucine zipper fused to a GEM transcription factor.” Claim 20 recites that the signaling protein is “a receptor” and claim 27 states that the receptor is “wherein the receptor is a chimeric antigen receptor or an engineered T cell receptor”. Thus, claims 20 and 21 fail to further limit claim 1. Applicant may cancel the claim(s), amend the claim(s) to place the claim(s) in proper dependent form, rewrite the claim(s) in independent form, or present a sufficient showing that the dependent claim(s) complies with the statutory requirements.
Claim Interpretation
Claim 1 recites “wherein the first and second leucine zippers bind to each other and, when expressed, the switch polypeptide deactivates the signaling protein by localizing the signaling protein to the plasma membrane.”, which recites an intended result of the circuit when used.
Claim Rejections - 35 USC § 103 – Necessitated by 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.
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.
Claim(s) 1, 14, 19-20, 22-24, and 29 is/are rejected under 35 U.S.C. 103 as being unpatentable over Aranda-Diaz et al (ACS Synth Biol. 2017 Mar 17; 6(3):545-554. Epub 27 Dec 2016) in view of Chen et al (ACS Synth. Biol., 9 July 2015; of record).
Aranda-Diaz teaches synthetic modules using transcriptional regulators to achieve concurrent or independent control of the expression of genes or decoupled control of the mean and variance of a single gene (abstract). The reference demonstrates how different arrangements of two simple inducible synthetic Transcriptional Regulators (TRs) can address the challenges of dual gene activation, as well as mean and variance control of gene expression (“wherein the signaling pathway is a synthetic signaling pathway” as in instant claim 24). Specifically, two inducible TRs arranged in parallel can drive the simultaneous and independent expression of two genes, while their arrangement in series can decouple mean and variability of a single gene in a substantial dynamic range (“wherein the signaling protein is a positive or negative regulator of the signaling pathway” as in instant claim 14). The reference teaches an estradiol-responsive TR in which the GAL4-DBD is fused to the hER-LBD and the transcription factor Msn2p activation domain (MSN2-AD) (GEM, Figure 1a, upper). The second TR is a new recombinant protein consisting of the ZIF268-DBD, the hPR-LBD and the MSN2-AD (ZPM, Figure 1a, bottom). Estradiol-bound GEM translocates into the nucleus and binds and activates transcription from promoters that contain Gal4p binding sites, such as pGAL1, which contains four dimeric Gal4 binding sites. Similarly, progesterone-bound ZPM activates transcription from pZ, a modified pGAL1 promoter containing three dimeric Zif268 binding sites but lacking Gal4p binding sites (Figure 1b; “wherein the regulatory sequence operably linked to the nucleic acid sequence encoding the signaling protein is a native promoter of the signaling protein” as in instant claim 23). The reference explored the behavior that would ensure from connecting the GEM and ZPM TRs in series instead of in parallel. In this circuit, the expression of ZPM is under the control of GEM and both are activated by their respective inputs (progesterone and estradiol) (Supplementary Figure S13a; see also Fig. 3a which shows E2 bound to GEM, ZPM as an output of E-GEM bound to pGAL1, and progesterone-bound ZPM driving activation of pZ promoter) (“A molecular feedback circuit, the circuit comprising: a signaling protein that, when activated by an input of a signaling pathway, drives an output of the signaling pathway, wherein the signaling protein is a first fusion protein comprising . . . GEM transcription factor, the input is E2, and the output is a Z3PM transcription factor; and a regulatory sequence responsive to the output and operably linked to a nucleic acid sequence encoding a switch polypeptide, wherein the regulatory sequence is a pZ3 promoter” as in instant claim 1 in-part; “wherein the regulatory sequence comprises a binding site for the transcription factor” as in instant claim 19; “signaling protein is a receptor and the input is a ligand for the receptor” as in instant claim 20). See Fig. 3a, which shows the regulatory sequence operably linked to the signal protein (“wherein the circuit further comprises a regulatory sequence operably linked to a nucleic acid sequence encoding the signaling protein” as in instant claim 22).
The difference between Aranda-Diaz and the instant invention is that it does not teach leucine zipper binding pairs and a plasma membrane-targeting sequestration domain (as in instant claim 1 in-part).
However, Chen teaches novel microscopy-readable two-hybrid fluorescence localization assay using a bait protein and a prey protein and leucine zipper pairs (abstract). The reference teaches that MiCode tags were added to a single pair of newly designed coiled-coil proteins, 100A and 100B, designed to heterodimerize (see “IV Application of MiCoding toward the Screening . . .” para 1). For testing of the interaction between 100A and 100B using the two-hybrid fluorescence localization assay, 100A was designated as the bait and 100B as the prey (same para). The newly designed bait zipper (100A) and prey zipper (100B) were cloned separately and each was incorporated into a DNA segment that included half of the eventual full MiCode (Figure 2B and SI Figure S2). In this single-pair mock assembly (i.e., the identities of the bait and prey were known a priori; however, the bait–prey binding affinity was uncharacterized by other assays), the gene expressing the bait construct was physically linked to the genes expressing the plasma membrane and actin-targeting half of the MiCode (same para). Upon inspection of the fully-MiCoded test strain via microscopy, we observed punctate fluorescence in the RFP channel yielding an interaction score of 1 between bait 100A and prey 100B (same para). The identities of both the bait and prey were verified by identifying the targeted organelles for each of the YFP, CFP, and BFP channels (same para). In the YFP channel, the plasma membrane is visible (same para) (“a plasma membrane-targeting sequestration domain, wherein the first and second leucine zippers bind to each other and, when expressed, the switch polypeptide deactivates the signaling protein localizing the signaling protein to the plasma membrane” as in instant claim 1 in-part). This shows that plasma membrane targeting domains can be utilized in combination with leucine zipper pairs to localize specific proteins to the plasma membrane and can be used for synthetic system designs where hetero-oligomerization without homo-oligomerization is desired and used in genetic circuitry.
Therefore, it would have been obvious prior to the effective filing date of the instantly claimed invention to create a synthetic molecular circuit as taught by Aranda-Diaz, where the circuit includes leucine zipper binding pairs linked to a plasma membrane targeting sequence as taught by Chen, to arrive at the instantly claimed invention. One of ordinary skill would have been motivated to modify the circuit as taught by Aranda-Diaz to include the leucine zipper binding pairs and plasma membrane tag to obtain the predictable result of sequestering proteins of interest to the plasma membrane as taught by the prior art.
Response to Arguments
Applicant's arguments have been fully considered but they are not persuasive. Applicant’s arguments with respect to previously cited Lim reference have been considered but are moot because the new ground of rejection does not rely on that reference applied in the prior rejection of record for any teaching or matter specifically challenged in the argument. Response to arguments will be directed to previously cited reference Chen (applied above).
On p. 7-8 of Remarks, Applicant argues that Chen does not teach or suggest the element of a second leucine zipper linked to a plasma membrane-targeting sequestration domain. Applicant argues that Chen discloses a fluorescent protein linked to various organelle targeting tags, including a plasma-membrane tag, and that while the gene (i.e., the nucleic acid) expressing the bait construct was physically linked to the genes expressing the plasma membrane and actin-targeting half of the MiCode (figure 2B), what is actually expressed are two separate proteins: the bait protein linked to a peroxisome tag, and the MiCode linked to the plasma membrane (or other organelle) tag. Once expressed, the bait protein is imported into the peroxisome, while the MiCode fluorescent protein is targeted to its organelle. There is no disclosure whatsoever of a leucine zipper, bait/prey protein, or coiled-coiled protein pair linked to a plasma membrane tag. Applicant argues that although Chen discusses the recruitment of a fluorescent protein to the plasma membrane, it is silent to the use of plasma membrane sequestration for deactivation of a signaling pathway. Applicant argues that the Chen reference does not teach a first leucine zipper fused to GEM, the input is E2, the output is Z3PM, the regulatory sequence is a pZ3 promoter and other newly added limitations in instant claim 1.
In response, the examiner first notes that newly cited reference Aranda-Diaz teaches synthetic circuits containing GEM, E2, Z3PM, pZ promoter, etc. While it does not teach the use of leucine zipper binding pairs and plasma membrane sequestration, Chen shows how particular protein targets can be recruited to the plasma membrane via leucine binding pairs and plasma membrane tags. Moreover, while Applicant argues that the Chen reference does not show Applicant’s instantly claimed limitations, the examiner notes that Applicant cannot escape the fact that the instant specification states that “useful leucine zipper binding pairs and sequestration domains that may be employed in anchor away strategies employed in the circuits of the present disclosure may include but are not limited to e.g., those described in Chen et al. ACS Synth. Biol., 2015, 4(11):1205-1216, the disclosure of which is incorporated herein by reference in its entirety” (see paragraph 0128). Put simply, the specification states that the product of Chen and the instant application both recite the required limitation of leucine zippers linked to plasma membrane tags. Applicant's argument runs contrary to the clear language in the disclosure. Thus, Applicant’s arguments are not persuasive.
Claim(s) 27 is/are rejected under 35 U.S.C. 103 as being unpatentable over Aranda-Diaz and Chen as applied to claims 1, 14, 19-20, 22-24, and 29 above, and further in view of Roybal et al (Cell, 28 Jan 2016; 164(4):770-9; of record).
The difference between the combined teachings and the invention as instantly claimed is that they do not teach that the signaling protein is a receptor and the input is a ligand for the receptor or that the receptor is synthetic and a chimeric antigen receptor or an engineered T cell receptor.
Roybal teaches precision tumor recognition by T cells using antigen-sensing circuits (title). The reference teaches a class of synthetic Notch (synNotch) receptors (see instant specification para 0086) that use an extracellular recognition domain to recognize a target antigen and ligand engagement leads to cleavage of the receptor and to release of a transcriptional activator domain, which can in turn enter the nucleus and drive expression of user specified target genes (“signaling protein is a receptor and the input is a ligand for the receptor” as in instant claim 20). The reference also teaches the construction of combinatorial antigen recognition T cell circuits in which a synNotch receptor for one antigen drives the inducible expression of a CAR for a second antigen (Introduction para 5). The T cell is engineered to constitutively express a synNotch receptor that recognizes antigen A and the gene for a CAR that recognizes antigen B is also inserted into the cell but is under control of a promoter that requires activation by the synNotch-induced transcription factor (engagement results in receptor cleavage and release of a transcriptional activation domain) (“Design of a two antigen AND-gate circuit” para 1) ( “wherein the synthetic receptor is a chimeric antigen receptor or an engineered T cell receptor” as in instant claim 27). The synNotch receptor was shown to reliably gate CAR expression and was further tested to see if the T cells could be targeted to tumors in vivo (“SynNotch receptors drive tumor-localized CAR expression in vivo” para 1). The synNotch AND-gate T cells displayed remarkably high and reproducible discriminatory action against the two tumors present in the same animal (“Selective combinatorial antigen tumor clearance in vivo” para 2). The reference concludes that the strategy of the sequential AND-gate circuit restricts the expression of the CAR to the tumor microenvironment and (2) has the potential to overcome the problem of off-target cross reaction that can occur with conventional CAR T cells when the target antigen is also present in bystander tissues (Discussion, para 1).
Therefore, it would have been obvious prior to the effective filing date of the instantly claimed invention to create a synthetic molecular circuit as taught by Aranda and Chen in combination, where the circuit contains a ligand input, transcription factor output, regulatory sequence with binding site for the transcription factor and chimeric antigen and T cell receptors as taught by Roybal, to arrive at the instantly claimed invention. Roybal shows the use of a synNotch circuit containing chimeric antigen receptor and T cell receptor. One of ordinary skill would have been motivated to modify the synthetic circuit of Aranda-Diaz and Chen in combination to be in T cells and include the ligand input and receptors of Roybal with a reasonable expectation of advantageously control expression of CAR to only be in tumor microenvironment to treat cancer without off-target effects as taught by the prior art.
Response to Arguments
On p. 8 of Remarks, Applicant argues that the Roybal reference was cited for certain elements of the dependent claims and fails to make up the deficiencies in Lim and Chen.
In response, the examiner notes that, as above, the prior art renders obvious the elements of the instantly claimed circuit. The Roybal reference was cited to render prima facie obvious the limitations of dependent claim 27. Applicant has not provided any arguments challenging the teaching of Roybal with respect to the limitations of claim 27.
Conclusion
No claim is allowed.
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.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to GILLIAN C REGLAS whose telephone number is (571)270-0320. The examiner can normally be reached M-F 9-5.
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/G.R./Examiner, Art Unit 1632
/KARA D JOHNSON/Primary Examiner, Art Unit 1632
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