DETAILED ACTION
Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Application Status
This action is written in response to applicant’s correspondence received 03/11/2024. Claims 55-74 are currently pending and are examined herein.
Information Disclosure Statement
The information disclosure statements (IDS) submitted on 06/21/2023, 03/12/2024, 01/13/2025, and 05/21/2025 are in compliance with the provisions of 37 CFR 1.97. Accordingly, the information disclosure statements are being considered by the examiner. Please note that citation numbers #14 on the IDS of 01/13/2015 and #112 on the IDS of 03/12/2024, as submitted, did not have publication dates listed. The publication dates have been determined and entered into the IDS by the examiner as a courtesy to the applicant. If these dates are in error, the examiner invites correction.
Drawings
The drawings are objected to because the most recent drawings of record (the supplemental drawings received 06/21/2023) include color 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).
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.
Claim Rejections - 35 USC § 102
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 the appropriate paragraphs of 35 U.S.C. 102 that form the basis for the rejections under this section made in this Office action:
A person shall be entitled to a patent unless –
(a)(1) the claimed invention was patented, described in a printed publication, or in public use, on sale, or otherwise available to the public before the effective filing date of the claimed invention.
(a)(2) the claimed invention was described in a patent issued under section 151, or in an application for patent published or deemed published under section 122(b), in which the patent or application, as the case may be, names another inventor and was effectively filed before the effective filing date of the claimed invention.
Claims 55, 58-59, 61, 63-65, and 67-70 are rejected under 35 U.S.C. 102(a)(2) as being anticipated by U.S. PGPUB 20230279351 A1 to Buckner (of record, applicant’s submission, hereinafter ‘Buckner’).
The applied reference has a common applicant and common inventors with the instant application, but names additional inventors not named in the instant application. Based upon the earlier effectively filed date of the reference (06/27/2019), it constitutes prior art under 35 U.S.C. 102(a)(2). This rejection under 35 U.S.C. 102(a)(2) might be overcome by: (1) a showing under 37 CFR 1.130(a) that the subject matter disclosed in the reference was obtained directly or indirectly from the inventor or a joint inventor of this application and is thus not prior art in accordance with 35 U.S.C. 102(b)(2)(A); (2) a showing under 37 CFR 1.130(b) of a prior public disclosure under 35 U.S.C. 102(b)(2)(B) if the same invention is not being claimed; or (3) a statement pursuant to 35 U.S.C. 102(b)(2)(C) establishing that, not later than the effective filing date of the claimed invention, the subject matter disclosed in the reference and the claimed invention were either owned by the same person or subject to an obligation of assignment to the same person or subject to a joint research agreement.
Regarding claim 55, Buckner teaches an engineered Treg comprising first and second polynucleotides in a genome of the cell, comprising promoters (MND) operatively linked to a nucleotide sequence encoding first and second CISC component comprising first and second extracellular binding domains, transmembrane domain, and cytoplasmic domains (FKBP + IL2RG for one CISC and FRB + IL2RB for the other CISC), wherein the first polynucleotide is operably linked to a nucleotide sequence encoding FOXP3 (inserted in a FOXP3 locus), the second polynucleotide is in a TRAC locus and encodes a TCRa/B protein (T1D4 – see FIG. 5), and one wherein dimerization of the first and second CISC components in the presence of a ligand induces a signal transduction event and proliferation (enrichment) of the engineered T cell:
[0238] Details of CISC systems, including structures of first, second and third CISC components and of CISC inducer molecules are described elsewhere herein and in WO/2018/111834 and WO/2019/210078, which are both expressly incorporated by reference in their entireties. Briefly, WO/2018/111834 describes compositions and methods for genetically editing host cells by knock-in (insertion) of genetic constructs encoding a ligand-dimerizable fusion protein chemical-induced signaling complex (CISC). Cellular expression of both fusion protein subunits followed by exposure of the host cells to the chemical ligand permits ligand-induced dimerization of the CISC to transduce a cellular activation signal.
[0067] FIG. 26 depicts schematic diagrams showing exemplary Split IL-2 CISC HDR knock-in constructs for selection of dual-edited cells. In the depicted constructs, CISC (chemically induced signaling complex) is split onto 2 different constructs and each CISC component is co-expressed with a different reporter, in this case either GFP or mCherry. Each construct contains half of a rapamycin-binding complex (either FKBP or FRB domain, with the chimeric endoplasmic reticulum targeting domain fused to one half of an IL-2R signaling complex (IL-2RB or IL-2RG) transmembrane and intracellular domains.
Dual Editing of Human CD4+ T Cells - Examples of Two Loci Approach
[0367] As an alternative dual-editing strategy for generating antigen-specific airT product containing IL-2 CISC components, constructs targeting to the TRAC and FOXP3 loci were developed for two-locus editing. As shown in schematic on FIG. 53, instead of having two constructs targeted to the TRAC locus, one construct is targeted to TRAC locus and the other is targeted to FOXP3 locus.
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[0369] The results of these studies demonstrate that presently disclosed two-loci dual-editing strategies can be used to introduce the IL-2 split-CISC cassette and lead to efficient enrichment of dual-edited cells using Rapalog. Having been successful using this approach for generation and enrichment of dual-edited cells, constructs were designed and cloned for expression of FOXP3 and a pancreatic islet antigen-specific TCR (T1D4) in combination with IL-2 CISC components by targeting the FOXP3 and TRAC loci, respectively. These and a range of other HDR donors are used to generate antigen-specific FOXP3 airTcells (FIG. 66).
Fig. 66:
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Regarding claim 58, Buckner teaches that the cell is a CD4+ cell (see above).
Regarding claim 59, Buckner teaches wherein the second promoter is operably linked to the nucleotides encoding the TCRa/B proteins (see above).
Regarding claim 61, Buckner teaches wherein the first CISC component comprises, in N-to-C terminal order, FKBP and IL2Ry transmembrane and cytoplasmic domains, and the second CISC component comprises, in N-to-C terminal order, FRB and IL2RG transmembrane and cytoplasmic domains (see above).
Regarding claim 63, Buckner teaches that the nucleotide sequence encoding FOXP3 is a FOXP3 cDNA sequence (see above).
Regarding claims 64-65, Buckner teaches that the promoters are constitutive (MND, as shown above, which in e.g. para [0012] Bucker notes is a constitutively active promoter).
Regarding claims 67-69, Buckner teaches methods of making the engineered cell comprising contacting the cell with nucleases targeting the FOXP3 and TRAC loci (i.e., CRISPR/Cas gRNAs and nuclease for the two loci dual editing strategy described above):
[0106] FIG. 60 relates to a strategy for testing two-loci dual-editing of human CD4+ T cells. Panel A depicts a diagram of AAV HDR-donor constructs designed to introduce split IL-2 constructs for selection of dual-edited cells using a two loci dual-editing approach. CISC components are split between 2 constructs and co-expressed with either mCherry or GFP (#3207 and #3251 respectively). Repair templates are flanked by homology arms matched to gRNAs targeting either the TRAC or FOXP3 locus, respectively…Human CD4+ T cells were edited using human TRAC gRNA_4, human FOXP3 gRNA_T9 and #3251 (MND.mCherry.FKBP.IL2RG) and #3207 (MND.GFP.FRB.IL2RB) AAV constructs (two-loci dual editing).
Regarding claim 70, in at least para [0112], describing the constructs shown in FIG. 66 (shown above), Buckner teaches that the FKBP/FRB domains are each half of a rapamycin-binding complex, and , i.e., that the ligand is rapamycin, and in para [0343] Buckner notes that all of the CISC constructs are used to selectively expand engineered cells by contacting them with the rapamycin ligand.
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.
The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
Determining the scope and contents of the prior art.
Ascertaining the differences between the prior art and the claims at issue.
Resolving the level of ordinary skill in the pertinent art.
Considering objective evidence present in the application indicating obviousness or nonobviousness.
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 56-57, 62, 66, and 71-72 are rejected under 35 U.S.C. 103 over U.S. PGPUB 20230279351 A1 to Buckner, as applied to claims 55, 58-59, 61, 63-65, and 67-70.
Buckner teaches the engineered Treg comprising a transgenic TCR of claims 55, 58-59, 61, 63-65, and a method of producing it of claims 67-70, as described above.
Regarding claim 56, Buckner does not teach a specific engineered Treg or construct for engineering it comprising a CAR.
However, Buckner does suggest engineering the Tregs to express CARs:
[0003] Some embodiments provided herein include artificial antigen-specific immunoregulatory T (airT) cells. AirT cells include artificially engineered immune system T lymphocytes stably reprogrammed by gene editing to exhibit certain regulatory T cell (Treg) properties and are also artificially engineered by gene editing, viral vector transduction, transfection or other genetic engineering methodologies to express desired functional T cell antigen receptors (TCR) or other antigen receptors such as chimeric antigen receptors (CAR). In some embodiments, the airT cells are capable of immunosuppressive activity in response to specific antigen recognition by TCR.
Regarding claim 57, Buckner does not teach a specific engineered Treg which is a CD8+ cell, but does suggest alternative embodiments in which the engineered cell is a CD8+ cell, and that the engineered cells suppress proliferation of CD8+ effector T cells (FIG. 94):
[0266] Exemplary T cells that can express TCRs encoded by heterologous genetic material introduced in the cells according to certain embodiments of this disclosure include CD4+ T cells, CD8+ T cells, and related subpopulations thereof (e.g., naive, central memory, stem cell memory, effector memory).
Regarding claim 62, Buckner does not explicitly teach that the specific engineered Treg described above comprised the first CISC-component inserted downstream of the TSDR in the endogenous FOXP3. However, Buckner does teach that the first CISC component was generally inserted into the FOXP3 locus, and teaches that in some embodiments the component is knocked-in downstream of an intronic regulatory T cell (Treg)-specific demethylation region (TSDR) (para [0286]).
Regarding claim 66, Buckner does not explicitly teach that the specific engineered Treg described above comprised in a pharmaceutical composition, but does teach various embodiments of engineered Tregs administered in vivo in pharmaceutically acceptable compositions (see e.g., Examples 6 and 11).
Regarding claims 71-72, Buckner does not explicitly teach a method of treating a disorder in a subject in need thereof comprising the specific engineered Treg described above, but does teach administering various embodiments of the Treg to treat Type 1 diabetes in a mouse model thereof (see Example 6). Buckner also teaches that, “the airT cells are capable of immunosuppressive activity” (para [0003]).
It would have been prima facie obvious to a person having ordinary skill in the art before the effective filing date of the claimed invention to have modified the engineered Treg as taught by Buckner according to the alternative embodiments suggested by Buckner, such as replacing the TCR with a CAR, engineered a CD8+ Treg instead of a CD4+ Treg, inserting the first promoter/CISC component downstream of the TSDR locus, and administering it in vivo in a pharmaceutical composition to treat autoimmune disorders such as diabetes. The ordinary artisan would have had a reasonable expectation of success based on Buckner’s teachings that the Tregs were intended for in vivo use for treatment of autoimmune disease and could be engineered to express TCRs or CARs, and would have been motivated to further insert one of the CISC components downstream of the FOXP3 TSDR locus to produce engineered Tregs capable of stable FOXP3 expression.
Claim 60 is rejected under 35 U.S.C. 103 as being unpatentable over Buckner (cited above), as applied to claims 55, 58-59, 61, 63-65, and 67-70, in view of WIPO Publication 2019084552 A1 to Roth (hereinafter ‘Roth’).
Buckner teaches the engineered Treg of claims 55 and 58, from which claim 60 depends, as discussed above.
Buckner does not teach wherein the second promoter is operably linked to the nucleotide sequence encoding the TCRβ protein and the first portion of the TCRα protein comprising the TCRα variable and joining regions, wherein the nucleotide sequence encoding the first portion of the TCRα protein is in-frame with the endogenous nucleotide sequence encoding the second portion of the TCRα protein.
Roth teaches methods and cells comprising insertion of heterologous TCR under the control of an endogenous TCR promoter:
[0003] The present disclosure is directed to compositions and methods for editing the genome of a human T cell. The inventors have discovered that a heterologous TCR can be inserted into a targeted region in the genome of a T cell, such that the heterologous TCR is under the control of an endogenous TCR promoter.
Roth further teaches that the methods and cells comprising a nucleotide encoding full length TCRβ chain, a variable region of a TCRα chain, a joining region (N-terminus of endogenous TCR alpha subunit) of a TCRα chain, inserted into exon 1 of the TRAC gene (i.e., in-frame with the endogenous sequence):
[0028] Figure la is a schematic depicting insertion of a single non- viral DNA template comprising a nucleic acid sequence encoding, from the N-terminus to the C-terminus, (i) a T2A self-cleaving peptide sequence; (ii) a full-length (i.e., variable region and constant region) of a heterologous TCR-β chain (NYESO-β); (iii) a P2A self -cleaving peptide sequence; (iv) a variable region of a heterologous TCR-a chain (NYESO-a); and (v) a portion of the N-terminus of the endogenous TCR alpha subunit into a T cell via homology directed repair. After insertion of the DNA template in exon 1 of the TRAC gene via homology directed repair, the DNA template was transcribed and translated to produce a full-length NYESO-β chain and a full-length NYESO-a chain that forms an antigen-specific TCR that recognizes the NY-ESO- 1 melanoma neoantigen.
It would have been prima facie obvious to a person having ordinary skill in the art before the effective filing date of the claimed invention to have modified the engineered cell comprising a TCR, as taught by Buckner, to comprise the transgenic single-template TCR as taught by Roth. The ordinary would have been motivated to insert the transgenic TCR into the genome using Roth’s method, based on Roth’s teachings that this would predictably yield a therapeutic T cell which expresses a transgenic TCR under an endogenous promoter while preventing mispairing with endogenous TCR chains.
Claims 55-58 and 61-74 are rejected under 35 U.S.C. 103 as being unpatentable over WIPO Publication 2018/111834 A1 to Scharenberg (published 06/21/2018; hereinafter ‘Scharenberg ‘834’; of record, cited on an IDS) in view of WIPO Publication 2018/080541 A1 to Scharenberg (published 05/03/2018; hereinafter ‘Scharenberg ‘541; of record, cited on an IDS’) and 2019/210280 A1 to Cost (hereinafter ‘Cost’; of record, cited on an IDS).
Regarding claim 1, Scharenberg ‘834 teaches an engineered regulatory T (Treg) cell comprising a first polynucleotide in a genome of the cell, the first polynucleotide comprising a first promoter (MND) operably linked to a nucleotide sequence encoding a first chemically inducible signaling complex (CISC) component comprising first extracellular binding, transmembrane, and cytoplasmic domains (FRB, IL2Rβ), and a second CISC component comprising second extracellular binding, transmembrane, and cytoplasmic domains (FKBP, IL2Rγ), wherein dimerization of the first and second CISC components in the presence of a ligand induces a signal transduction event:
[0008] …a protein sequence encoding a chemical-induced signaling complex (CISC)…the first CISC component comprises a first extracellular binding domain or a portion thereof, a hinge domain, a transmembrane domain, and a signaling domain or a portion thereof…the second CISC component comprises a second extracellular binding domain or portion thereof, a hinge domain, a transmembrane domain, and a signaling domain or portion thereof…first CISC component and the second CISC component are positioned such that when expressed, they dimerize in the presence of a ligand.
[0062] Figure 25 shows a schematic of the Targeted knock-in of an MND promoter and CISC to enrich/expand gene targeted T-cells. The described targeting approach integrates a promoter and both components of an IL2R-CISC V3 into the FOXP3 locus in line with a GFP fusion to the native FOXP3 gene.
[0040] Figure 3 depicts various embodiments of IL2R-CISC architectures. The embodiment shown in Figure 3 shows an architecture for both FRB-IL2RP and for FKBP- IL2Ry, providing schematics for various degrees of flexibility, comprising most flexible (1210 - this embodiment incorporates a short linker sequence the entire first extracellular immunoglobulin superfamily (IgSF) domain of the IL2R and its TM and cytosolic tail regions), medium flexibility (1211 - this embodiment incorporates the entire first extracellular IgSF domain of the IL2R and its TM and cytosolic tail regions), and least flexible (1233 - this embodiment incorporates only the IL2R TM and cytosolic tail regions).
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Scharenberg ‘834 further teaches wherein the first promoter is operatively linked to a nucleotide sequence encoding FOXP3 in the genome to drive ligand-induced enrichment of FOXP3-overexpressing cells:
[0062] The described targeting approach integrates a promoter and both components of an IL2R-CISC V3 into the FOXP3 locus in line with a GFP fusion to the native FOXP3 gene. This architecture is intended to allow for ligand-induced selection of cells which have undergone an accurate gene targeting event.
[0063] Figure 26 depicts a schematic diagram showing an experimental design of targeted knock-in of MND promoter and CISC. This represents an experimental schematic of how a CRISP/Cas9 nuclease is used to induce targeted integration of the cassettes from Figure 25 into the FOXP3 locus, followed by expansion of the gene targeted cells
[0064] Expansion in rapamycin or AP21967 resulted in substantial enrichment of FOXP3 expressing cells, indicating that the IL2R-CISC are able to drive ligand-induced enrichment of gene targeted cell populations, including those in which FOXP3 is overexpressed.
Scharenberg ‘834 further provides a teaching, suggestion or motivation to use the CISC constructs in a CAR T cell. Scharenberg ‘834 introduces the invention in the context of CARs (para [0004]), indicates that in some embodiments the cell comprises a CAR (para [0190]), and notes that there was, “a need for new compositions and methods that allow one to transduce a desired signal through a synthetic complex that cannot be activated through a normal physiological pathway, thus providing a mechanism for activating signaling only within a desired and specifically engineered population of cells” (para[0005]).
Scharenberg ‘541 provides a further teaching, suggestion or motivation to stably express FOXP3 in regulatory T cells under a highly active constitutive promoter (e.g., MDN, as also taught by Scharenberg ‘834) to ensure that the T cells do not revert to a non-suppressive phenotype:
[0055] …methods are provided herein to avoid the risks that may arise from regulatory cells changing into pro-inflammatory cells. As shown in Figure 1, FOXP3 expressed from an iTreg is used as a master regulator of the immune system and is used for tolerance and immune suppression.
[0080] In the alternatives described herein, the cells for therapeutic application are engineered to have stable FOXP3 expression through the use of a gene editing nuclease to modify the regulatory elements of the FOXP3 locus to provide for stable FOXP3 expression. In the exemplary data provided, a constitutive promoter is placed upstream of the FOXP3 coding exons (examples of constitutive promoters include EF1 alpha promoter, the PGK promoter, and/or the MND promoter, among many others) to drive FOXP3 expression, but a variety of approaches are envisioned to modify the regulatory elements so as to allow for stable FOXP3 expression. By several approaches used to modify the endogenous regulatory elements, the claimed therapeutic cell exhibits constitutive expression of the native FOXP3 gene, such that it is no longer susceptible to regulation that could result in FOXP3 gene silencing and reversion to a non-suppressive cell phenotype
Scharenberg ‘541 teaches that the FOXP3 overexpression/stable expression is achieved by insertion of a constitutive promoter:
[0080] In the alternatives described herein, the cells for therapeutic application are engineered to have stable FOXP3 expression through the use of a gene editing nuclease to modify the regulatory elements of the FOXP3 locus to provide for stable FOXP3 expression. In the exemplary data provided, a constitutive promoter is placed upstream of the FOXP3 coding exons (examples of constitutive promoters include EFl alpha promoter, the PGK promoter, and/or the MND promoter, among many others) to drive FOXP3 expression, but a variety of approaches are envisioned to modify the regulatory elements so as to allow for stable FOXP3 expression. By several approaches used to modify the endogenous regulatory elements, the claimed therapeutic cell exhibits constitutive expression of the native FOXP3 gene, such that it is no longer susceptible to regulation that could result in FOXP3 gene silencing and reversion to a non-suppressive cell phenotype. Accordingly, in the alternative methods described herein, the problem of loss of FOXP3 expression due to epigenetic influences on the native regulatory sequences and promoter has been solved.
Scharenberg ‘541 further echoes Scharenberg ‘834’s suggestion that the engineered cell may also comprise a CAR, which would allow the T-cell to target a specific tissue in need for the delivery of cells expressing FOXP3 to a tissue specific site:
[0090] In some alternatives, the cells can also be further engineered to express a chimeric antigen receptor or a TCR (T cell receptor) or other targeting moiety. The cells can be engineered to express a CAR for targeting a specific tissue or cell…Allowing a T-cell to express a CAR or TRC would allow the T-cell to target a specific tissue in need for the delivery of cells expressing FOXP3 to a tissue specific site.
Scharenberg ‘541 further notes that the engineered cell may address flaws with the use of natural regulatory T cells, such as Treg instability, and may be used for ameliorating complications from graft-versus-host disease:
[0085] There are also potential flaws with the use of natural Tregs . For example, autoimmune patients are genetically predisposed to Treg instability. For example, it is plausible for a CAR bearing nTreg to convert to a CAR T effector cell. nTreg also retain the potential for epigenetic regulation of FOXP3, which could lead to the down regulation of FOXP3 induction, which means that the function or an nTreg population may never be fully predictable. Also, natural Tregs may not include the correct TCR (T cell receptor) specificities…Thus the methods provided herein are an improvement over using the transfer of natural Tregs by using engineered cells as there is potential for linking CAR expression to regulatory T cell function to avoid potential engraftment of CAR Tregs that have the potential to convert to pro inflammatory CAR T cells.
[0073] "Graft-versus-host disease" (GVHD) as described herein, refers to a medical complication following the receipt of transplanted tissue from a genetically different person. GvHD is commonly associated with stem cell or bone marrow transplant but the term also applies to other forms of tissue graft. Immune cells in the donated tissue recognize the recipient as foreign and not "self." In some alternatives herein, the methods provided can be used for preventing or ameliorating the complications that can arise from GVHD.
Neither of the above references explicitly teach that the second polynucleotide is in integrated into a TRAC locus in the genome.
However, Cost teaches an analogous CISC system for T cell proliferation/survival, wherein the CISC components comprise extracellular binding domains (FRB and/or FKBD) fused to transmembrane and intracellular signaling domains (ILR2β and/or ILR2γ) (see FIG. 1 and its description, quoted below):
[0157] FIG. 1 shows schematics for donor template constructs # 1 -# 11 , depicting elements present in the donor template constructs (not shown to scale) and their relative positions. 5’ HA: 5’ homology arm; 3’ HA: 3’ homology arm; s pA: synthetic polyA signal; SV40 pA: SV40 polyA signal; pMSCV: murine stem cell virus (MSCV) promoter; CD8 sp: CD8 signal peptide; CD8 tm: CD8 transmembrane domain; CD28: CD28 co-stimulatory domain; 4-1BB: 4-1BB co-stimulatory domain; CD3z: OP)3-z cytoplasmic signaling domain; WPRE3: Woodchuck Hepatitis Virus (WHP) Posttranscriptional Regulatory Element (WPRE) 3; CISCB: CISC subunit with FRB domain and IL2RP domain; tCISOy: CISC subunit with FKBP domain and fragment of IL2Ry domain; b2M CR: P2-microglobulin chimeric receptor; FRB: naked FRB domain polypeptide; P2A, T2A: self-cleaving peptide; ER: endoplasmic reticulum signal sequence; CNb30: mutant calcineurin polypeptide; tLNGFR: truncated low- affinity nerve growth factor receptor.
Cost further teaches both CISC components were integrated into the genome, with one CISC component inserted into the TRAC locus together with a CAR, to disrupt the endogenous TRAC gene so that the cell is unable to express a functional unmodified T cell receptor:
[0003] The present disclosure relates to compositions and methods for controlled plasma cell depletion in an individual. In particular, the compositions include a general architecture for generating physiologically functional synthetic chemical-induced signaling complexes (CISCs) that allow for controlling the survival and/or proliferation of T cells.
[0013] In some embodiments, the b) nucleic acid encoding a CAR that can recognize BCMA is inserted into the region of the endogenous TRA gene encoding the TRAC domain
[0125] In some embodiments, nucleic acid encoding i) the first CISC component is inserted into an endogenous IL2RG gene, and/or nucleic acid encoding ii) the second CISC component is inserted into the region of the endogenous TRA gene encoding the TRAC domain; or nucleic acid encoding i) the first CISC component is inserted into the region of the endogenous TRA gene encoding the TRAC domain, and/or nucleic acid encoding ii) the second CISC component is inserted into the endogenous IL2RG gene.
[0223] In some embodiments, the systems described herein comprise one or more donor templates comprising nucleic acid encoding the following system components: i) an anti plasma cell construct; ii) a first CISC component comprising an IL2RB signaling domain; iii) a polypeptide that confers resistance to rapamycin; iv) a selectable marker; v) a polypeptide that confers resistance to one or more calcineurin inhibitors; and vi) a second CISC component comprising an IL2Ry signaling domain or fragment thereof. In some embodiments, the one or more donor templates comprise a first donor template and a second donor template. In some embodiments, the first donor template is configured to be inserted in a first endogenous gene and the second donor template is configured to be inserted in a second endogenous gene…the first donor template is inserted into the region of the endogenous TRA gene encoding the TRAC domain. In some embodiments, insertion of the first donor template results in a non-functional TRAC domain. The TRAC domain in a cell is non-functional if the cell is unable to express a functional native (unmodified) T cell receptor.
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the engineered regulatory T cell comprising CISC components and a constitutive promoter integrated into the endogenous FOXP3 locus for stable FOXP3 overexpression, as taught by the combination of Scharenberg ‘834 and Scharenberg ‘541, to further comprise a chimeric antigen receptor, as suggested by Scharenberg ‘834 and Scharenberg ‘541. As taught by Cost, the CISC system can successfully be adapted to insert the two components at two different genes in the genome, and the ordinary artisan would have been motivated to choose an endogenous TRAC locus as one of the insertion sites because disruption of the endogenous TRAC locus is required to disrupt the endogenous unmodified T cell receptor, as also taught by Cost. The ordinary artisan would have had a reasonable expectation that adapting the CISC system for insertion of one component at the FOXP3 locus and the other at an endogenous TRAC locus would have been successful because Cost teaches that each of the two CISC components can be inserted at different loci.
Regarding claim 56, Cost teaches the second promoter operably linked to the nucleotide sequence encoding the CAR (see FIG. 1 and para [0157], cited above).
Regarding claims 57 and 58, Cost teaches that the cell is a CD4+ or CD8+ cell:
[0248] In some embodiments, the engineered cells are T cells, or precursor cells capable of differentiating into T cells. In some embodiments, the engineered cells are CD3+, CD8+, and/or CD4+ T lymphocytes.
Example 9: Simultaneous TI into a TRAC gene and an IL2RG gene
[0406] Flow cytometry was performed to illustrate the efficiency of dual targeted integration. CD8+ T cells were stimulated with CD3/CD28 beads for three days, the beads removed, and then one day later the cells were treated with TRAC 1 RNP + BCMA CAR-CISCP AAV and IL2RG GC12 RNP + FRB-tLNGFR-CNb30-CISCy AAV.
Regarding claim 61, Scharenberg ‘834 and Cost teach wherein one of the CISC components comprises, in N-to-C-terminal order, a FKBP domain and IL-2Rγ transmembrane and cytoplasmic domains, and the other CISC component comprises, in N-to-C-terminal order, a FRB domain and IL-2Rβ transmembrane and cytoplasmic domains (see e.g., FIG. 3 of Scharenberg ‘834 and FIG. 1 of Cost).
Regarding claim 62, Scharenberg ‘541 teaches wherein the first promoter is operably linked to a first coding exon of an endogenous FOXP3 gene in the Treg cell, wherein the first promoter is downstream of the TSDR:
[0120] Edited T-cells show fully methylated TSDR's, despite known high level FOXP3 expression, demonstrating that integration of a promoter downstream of the TSDR effectively bypasses the normal epigenetic regulation of the FOXP3 locus (Figure 26).
Please see also Figures 4 and 5 of Scharenberg ‘541, which show insertion of various constructs comprising a constitutive promoter operably linked to the first coding exon of FOXP3 (i.e., exon 2) and inserted downstream of the epigenetic regulation site (i.e., the TSDR).
Regarding claim 63, Scharenberg ‘541 teaches that the FOXP3 sequence is a FOXP3 exon, i.e., a FOXP3 cDNA sequence.
Regarding claims 64 and 65, Scharenberg ‘541, Scharenberg ‘834 and Cost all teach wherein both promoters are constitutive (MND) promoters (see above).
Regarding claim 66, Scharenberg ‘541 (para [0017]) teaches pharmaceutical compositions comprising the cell.
Regarding claim 67, the combination of references cited above teaches the limitations of the engineered cell, as discussed above, and further teaches methods of producing it by contacting the cell with a polynucleotide (expression vector) (see above).
Regarding claim 68, Scharenberg ‘541 teaches nucleases targeting the FOXP3 locus (para [0011] and Cost teaches nucleases targeting the TRAC locus (para [0221]).
Regarding claim 69, Cost teaches editing the cell with a CRISPR/Cas system, i.e., contacting the cell with an RNA-guided Cas nuclease and two gRNAs targeting two loci (para [0221]).
Regarding claim 70, Scharenberg ‘834 (para [0018]) and Cost (para [0241]) both teach the CISC components and methods comprising contacting the cell with the ligand, causing the components to dimerize.
Regarding claims 71-74, while all of the cited references concern engineered T cells for therapeutic use, Scharenberg ‘541 most explicitly teaches methods of treating autoimmune and alloimmune disorders comprising administering the engineered cell to subjects in need thereof. Both Scharenberg ‘834 (para [0140]) and Cost (para [0279]) also teach wherein the engineered T cells may be allogenic or autologous to the subject.
Claim 59 is rejected under 35 U.S.C. 103 as being unpatentable over Scharenberg ‘834, Scharenberg ‘541 and Cost as applied to claims 55-58 and 61-74, further in view of Schober (Orthotopic T-Cell Receptor Replacement—An “Enabler” for TCR-Based Therapies. Cells. 2020 Jun 1;9(6):1367.)
Scharenberg ‘834, Scharenberg ‘541 and Cost render obvious the engineered Treg cell of claims 55 and 58, from which the instantly rejected claim depends, as described above.
Scharenberg ‘834, Scharenberg ‘541 and Cost do not teach wherein the second promoter is operably linked to sequences encoding TCRα and TCRβ proteins.
Schober teaches that transgenic TCRs offer advantages over CAR T cells (p. 2/20):
…current CAR T-cell manufacturing protocols are still lengthy and cumbersome, which can introduce considerable heterogeneity into the “living drug”. In clinical reality, the success rate and the speed of generating cell products are some of the most important bottlenecks of applying CAR T cells. Furthermore, it is questionable how much of the success of CAR T cells can be explained by the nature of the CAR itself…
…In contrast to CAR T cells, TCR engineered T cells are not limited to target surface antigens, and important studies demonstrated the feasibility of using TCR-engineered T cells for therapy of different cancer entities…T cells that react to checkpoint therapy often recognize mutation-derived neo-epitopes…Neo-epitopes also bear the advantage of being very specific to the tumor itself, ameliorating the danger of off-target recognition…Importantly, targeting neo-epitopes can only be achieved via the TCR (and not, for example, with a CAR).
Schober further suggests replacing the endogenous TCR with the transgenic TCR via gene editing, and shows a schematic in Figure 2 depicting orthotopic TCR replacement through CRISPR-mediated insertion of TCR alpha and beta at the TRAC locus with knockout of the endogenous TCR beta chain.
It would have been prima facie obvious to a person having ordinary skill in the art before the effective filing date of the claimed invention to have modified the engineered T cell comprising a CAR, as taught by Scharenberg ‘834, Scharenberg ‘541 and Cost, to comprise a transgenic TCR instead, as taught by Schober. Schober teaches that transgenic TCRs have advantages over CARs, such as a greater range of and specificity to certain antigens, as described above.
Claim 60 is rejected under 35 U.S.C. 103 as being unpatentable over Scharenberg ‘834, Scharenberg ‘541 and Cost as applied to claims 55-58 and 61-74, further in view of Schober (Orthotopic T-Cell Receptor Replacement—An “Enabler” for TCR-Based Therapies. Cells. 2020 Jun 1;9(6):1367.) and WIPO Publication 2019084552 A1 to Roth (hereinafter ‘Roth’).
Scharenberg ‘834, Scharenberg ‘541 and Cost render obvious the engineered Treg cell of claims 55 and 58, from which the instantly rejected claim depends, as described above.
Scharenberg ‘834, Scharenberg ‘541 and Cost do not teach wherein the second promoter is operably linked to sequences encoding TCRα and TCRβ proteins.
Schober teaches that transgenic TCRs offer advantages over CAR T cells, as already discussed above.
Schober further notes that if endogenous TCR is not, “eliminated upon introduction of a transgenic TCR…remaining TCRs suppress surface expression and, therefore, diminish functionality through competition for a limited number of CD3 molecules…Moreover, since the TCR is a heterodimeric receptor, transgenic a- and endogenous B-chains (and vice versa) can also mispair. Such mispaired…TCR variants can lead to novel antigen specificities…Mispairing was shown to lead to allow-reactivity in a human in vitro setting…and to GvHD or xeno-reactivity in a murine in vivo setting” (3/20):
While Schober provides one solution, that of knocking out the endogenous TCR, as already discussed above, Roth provides a further solution to endogenous and transgenic TCR chain mispairing, which is simultaneous knock-in of transgenic TCR-a and TCR-B and knockout of the endogenous genes in a single multiplexed round of editing and preventing the potentially undesirable pairing of an antigen-specific TCRα chain with an endogenous TCRβ (Figure 2):
[0146] Both the TCR-a and TCR-β chains can be knocked in simultaneously in a single multiplexed round of editing (Figure 2). This is similar to the targeting strategy in Figure la and Figure lb, except that at both the TCR-a and TCR-β constant loci, only the variable regions of the desired antigen specific TCR are inserted. This has the benefit of both reducing the total size of the insertions (from one 1.5 kbp insertion to two 500 bp insertions), but also means that any T cell expressing both chains of the desired antigen specific TCR will have both its previously recombined endogenous TCR-a and TCR-β chains knocked out, preventing the potentially undesirable pairing of an inserted antigen specific TCR-a chain with an endogenous TCR-β chain for example.
Roth teaches methods and cells comprising insertion of heterologous TCR under the control of an endogenous TCR promoter:
[0003] The present disclosure is directed to compositions and methods for editing the genome of a human T cell. The inventors have discovered that a heterologous TCR can be inserted into a targeted region in the genome of a T cell, such that the heterologous TCR is under the control of an endogenous TCR promoter.
Roth further teaches that the methods and cells comprising a nucleotide encoding full length TCRβ chain, a variable region of a TCRα chain, a joining region (N-terminus of endogenous TCR alpha subunit) of a TCRα chain, inserted into exon 1 of the TRAC gene:
[0028] Figure la is a schematic depicting insertion of a single non- viral DNA template comprising a nucleic acid sequence encoding, from the N-terminus to the C-terminus, (i) a T2A self-cleaving peptide sequence; (ii) a full-length (i.e., variable region and constant region) of a heterologous TCR-β chain (NYESO-β); (iii) a P2A self -cleaving peptide sequence; (iv) a variable region of a heterologous TCR-a chain (NYESO-a); and (v) a portion of the N-terminus of the endogenous TCR alpha subunit into a T cell via homology directed repair. After insertion of the DNA template in exon 1 of the TRAC gene via homology directed repair, the DNA template was transcribed and translated to produce a full-length NYESO-β chain and a full-length NYESO-a chain that forms an antigen-specific TCR that recognizes the NY-ESO- 1 melanoma neoantigen.
It would have been prima facie obvious to a person having ordinary skill in the art before the effective filing date of the claimed invention to have modified the engineered cell comprising a CAR, as taught by Scharenberg ‘834, Scharenberg ‘541 and Cost, to comprise a transgenic TCR, as taught by Schober and Roth. The ordinary would have been motivated to engineer a transgenic TCR instead of a CAR based on Schober’s teachings regarding the advantages of TCRs in terms of specificity, and would further have been motivated to insert the transgenic TCR into the genome using Roth’s method, based on Roth’s teachings that this would predictably yield a therapeutic T cell which expresses a transgenic TCR under an endogenous promoter while preventing mispairing with endogenous TCR chains.
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 55 and 58 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claims 79, 83 and 85 of copending Application No. 17/596,493. Although the claims at issue are not identical, they are not patentably distinct from each other for the following reasons.
Copending claim 79 recites an engineered CD4+ regulatory T (Treg) cell, which is a species within the genus of instantly claimed Treg cells and thus anticipates that limitation of instant claim 55. Copending claim 79 recites the following characteristics of the Treg which anticipate those recited in instant claim 55:
A first inserted nucleic acid in a FOXP3 gene locus (i.e., in the genome of the cell), encoding a first CISC component with a CISC inducer molecule (ligand) binding domains (i.e., extracellular binding domains selected from FKBP and FRB) linked to an activation signal transduction domain (i.e., a cytoplasmic domain selected from IL-2Rβ and IL-2Rγ)
A second inserted nucleic acid in a TRAC gene locus, encoding a second CISC component with a CISC inducer molecule binding domain linked to an activation signal transduction domain selected from those noted above for the first component, the second nucleic acid further encoding a TCRα and TCRβ variable region
Copending claim 79 differs from instant claim 55 in that it does not recite the transmembrane domain, and recites only the TCR variable regions, not the full TCRs, as claim 55 is interpreted as requiring. However, copending claim 83 depends from copending claim 79 and recites TCRα/β polypeptides encoded by the inserted nucleotides. These polypeptides are interpreted as encompassing the full length polypeptides. Likewise, copending claim 85, which also depends from copending claim 79, recites specific transmembrane domains of the CISC components. In combination, copending claims 79, 83 and 85 anticipate the limitations of instant claim 55.
Regarding instant claim 58, copending claim 79 recites that the cell is a CD4+ Treg.
This is a provisional nonstatutory double patenting rejection because the patentably indistinct claims have not in fact been patented.
Conclusion
No claim is allowed at this time.
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/AMANDA M ZAHORIK/Examiner, Art Unit 1636
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