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 .
2. Applicant’s response filed on 07/15/2026 is acknowledged.
3. Claims 1-20 are pending.
4. Applicant’s election without traverse of Group I, claims 1-12, in the reply filed on 07/15/2026 is acknowledged. Upon further consideration the Examiner has withdrawn the species election requirement.
5. Claims 13-20 are withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected Group, there being no allowable generic or linking claim. Election was made without traverse in the reply filed on 07/15/2026.
6. Claims 1-12 are under consideration for their full scope.
7. Applicant’s IDS document filed on 07/17/2023 has been considered.
8. 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.
9. 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.
10. Claims 1-12 are rejected under 35 U.S.C. 102(a)(1) as being anticipated by U.S. Patent Application Publication 2020/0163899 (IDS filed on 07/17/2023; Reference 1).
U.S. Patent Application Publication 2020/0163899 teaches in the following paragraphs:
[0036] “Prussian blue particle(s) or nanoparticle(s)” refer to particles containing Prussian blue, preferably in an amount and form suitable for administration to a subject during a photothermal treatment. This definition includes but is not limited to the Prussian blue compositions described by U.S. 2014/0271487 which is incorporated by reference. [0037] These particles may contain components a chemical formula of A.sub.xB.sub.yM.sub.z[M′(CN.sub.6].sub.a.n(H.sub.2O), where A represents at least one of VO″ Ca, V, Cr, Mn, Fe, Co, Ni, Cu, In, Ga, Sr, lr, Nb, Li, Na, K, Rb, Cs, Fr, TI, Mo, Ru, Rh, Pd, Ag, Cd, In, Lu, Ba, Hf, Ta, W, Os, Pt, Hg, La, Eu, Gd, Tb, Dy and Ho, in any oxidation state and any combination thereof; B represents at least one of VO″ Ca, V, Cr, Mn, Fe, Co, Ni, Cu, In, Ga, Sr, lr, Nb, Li, Na, K, Rb, Cs, Fr, TI, Mo, Ru, Rh, Pd, Ag, Cd, In, Lu, Ba, Hf, Ta, W, Os, Pt, Hg, La, Eu, Gd, Tb, Dyand Ho, in any oxidation state and any combination thereof; M represents at least one of VO″ Ca, V, Cr, Mn, Fe, Co, Ni, Cu, In, Ga, Sr, lr, Nb, Li, Na, K, Rb, Cs, Fr, TI, Mo, Ru, Rh, Pd, Ag, Cd, In, Lu, Ba, Hf, Ta, W, Os, Pt, Hg, La, Eu, Gd, Tb, Dyand Ho, in any oxidation state and any combination thereof; M′ represents at least one of VO″ Ca, V, Cr, Mn, Fe, Co, Ni, Cu, In, Ga, Sr, lr, Nb, Li, Na, K, Rb, Cs, Fr, TI, Mo, Ru, Rh, Pd, Ag, Cd, In, Lu, Ba, Hf, Ta, W, Os, Pt, Hg, La, Eu, Gd, Tb, Dy and Ho, in any oxidation state and any combination thereof; x is from 0.1 to about 1; Y is from 0 to about 1; z is from 0.1 to about 4; a is from 0.1 to about 4; and n is from 0.1 to about 24.
[0254] Nanoparticle Synthesis and Bioconjugation. PBNP were synthesized at room temperature as previously described. See Volz H G. Pigments, Inorganic. Encyclopedia of Industrial Chemistry. Weinheim: Wiley-VCH; 2006, incorporated herein by reference in its entirety. Briefly, a solution containing 10.6 mg K.sub.4[Fe(CN).sub.6].3H.sub.2O (2.5×10.sup.−5 mol) in 5 mL of MQ was added to a vigorously stirred solution containing 6.8 mg Fe(Cl).sub.3.6H.sub.2O (2.5×10.sup.−5 mol) in 10 mL of MQ. The resulting blue precipitate containing PBNP was rinsed to remove unreacted components by centrifugation and sonication. PBNP were coated with filtered nonfluorescent- or AlexaFluor 488-conjugated avidin (1 mg/ml, Life Technologies, Grand Island, N.Y.) at a ratio of 0.1 mg avidin per 1 mg PBNP via electrostatic self-assembly. See Jaiswal J K, Mattoussi H, Mauro J M, Simon S M. Long-term multiple color imaging of live cells using quantum dot bioconjugates. Nat Biotechnol. 2003 January; 21(1):47-51, incorporated herein by reference in its entirety. This mixture was protected from light and the fluorophore-conjugated avidin was allowed to coat the PBNP over a period of 3 hours an orbital shaker at 4° C. [0255] Nanoparticle Characterization. Following fabrication, the size and zeta potential of the PBNP or avidin-coated PBNP was determined using a Zetasizer Nano ZS (Malvern Instruments, Malvern, UK). Vis NIR spectra of PBNP and PBNP-Tc constructs in the range of 500-1100 nm were measured on a Genesys 10S spectrophotometer (ThermoFisher Scientific, Waltham, Mass.) using the VISIONlite software. [0256] Cell Sources. Jurkat Cells. Human Jurkat T cells were obtained from ATCC (Jurkat Clone E6-1, ATCC TIB-152) and were used to examine feasibility of cellular backpacking with a general T cell moiety. Jurkat cells were maintained in RPMI1640 with 10% fetal bovine serum and 1% GlutaMax. [0257] Primary Cell PBMC. [0258] Human peripheral blood mononuclear cells (PBMC) were obtained from healthy volunteers upon informed consent or deidentified discarded blood products under Institutional Review Board-approved protocols at Children's National Health System and Baylor College of Medicine. Whole blood was washed with sterile PBS (Cellgro, Manassas, Va.) and PBMC were isolated by ficoll density gradient separation. Red blood cells were lysed (ACK Lysing Buffer) and remaining PBMC were cultured in RPMI 1640 medium supplemented with 2 mmol/L GlutaMAX TM-1 and fetal bovine serum. PBMC were used to generate PHA blasts, lymphoblastoid cell lines (LCL), and antigen-specific T cell lines. [0259] CMV T Cells. [0260] For CMV-specific T cells, PBMC were expanded by stimulation with autologous antigen presenting cells pulsed with an overlapping peptide library spanning the CMV pp65 protein, as previously described. Prior to use in cell proliferation and co-culture, cells were restimulated from a frozen aliquot with irradiated PHA blasts (30 Gy) pulsed with the same overlapping peptide mix in the presence of IL2 (100 U/mL). PHA blasts were generated from PBMC by stimulation with phytohemagglutinin (5 μg/mL) and IL2 (100 U/mL). T cells were maintained in RPMI 1640 supplemented with 45% Click's medium (Irvine Scientific, Santa Ana, Calif.), 2 mmol/L GlutaMAX TM-1, and 5% human AB serum (Valley Biomedical, Winchester Va.)]. [0261] EBV T Cells. [0262] For EBV-specific T cells, PBMC were expanded by stimulation with either autologous antigen presenting cells pulsed with an overlapping peptide library spanning the EBV BZLF1, LMP2, and EBNA1 proteins, as previously described, or autologous irradiated LCL. Prior to use in cell proliferation and co culture, cells were restimulated from a frozen aliquot with irradiated autologous lymphoblastoid cell lines. LCL were first grown by infection with EBV B95-8 virus in the presence of cyclosporine (1 μg/mL) and expanded over 4 weeks. T cells were maintained in RPMI 1640 supplemented with 45% Click's medium (Irvine Scientific, Santa Ana, Calif.), 2 mmol/L GlutaMAX TM-1, and 5% human AB serum (Valley Biomedical, Winchester Va.)]. [0263] Backpacking of PBNP onto T cells. Jurkat Tc, EBV Tc, or CMV Tc were biotinylated by co-incubating with 1 mg/mL biotinylation reagent (EZ-Link™ Sulfo-NHS-LC-Biotin) at 4° C. on an orbital shaker, as previously described (Jaiswal 2003). The sulfo-NHS group of the biotinylation reagent covalently binds to free amines of cell surface proteins. The biotinylated T cells were then added to a solution of fluorescent avidin-coated PBNP (10.sup.−7-10.sup.−8 mg PBNP/T cell). The mixture was protected from light and the avidin-biotin interactions were allowed to occur for 0.5-1 h at 4° C. on an orbital shaker. The cells were then rinsed to remove unbound nanoparticles by centrifugation. Following this, the PBNP were effectively “backpacked” onto T cells and the construct identified as PBNP-Tc. The efficiency of the backpacking was evaluated using confocal microscopy, and flow cytometry. [0264] Prior to imaging, T cells were stained with viability dyes (DAPI, CFSE, Calcein Blue AM or Calcein Red/Orange) as per the manufacturer's specifications. Images were obtained at the Children's National Medical Center/George Washington University Core Microscopy on the Olympus BX61, Zeiss Apotome, and Olympus FV100 Confocal microscopes, and scale bar is indicative of 20 μm. [0265] Flow Cytometry. The phenotype of naked and backpacked T cells was characterized by staining with antibodies against a panel of T cell markers such as CD3 (145-2C11), CD4 (Gk1.5), CD8 (53-6.7), CD19, CD25, CD45RO, CD45RA, and PD-1 (CD279, RMP1-30, Biolegend) conjugated to FITC, PE, PerCP, APC, APC-Cy7, or PE-Cy7 (BD Biosciences). Examination of T cell backpacking was performed after T cells were stained with Calcein Blue viability dye or CD3 antibody, and coupled to Alexa Fluor 488-avidin-coated PBNP, as described above. Gates were set based on unstained cells. Samples were acquired on the FACSCalibur and BD Accuri flow cytometers, and results were analyzed using Flow Jo 7.6.5 (Tree Star Inc., Ashland, Oreg.). [0266] Photothermal Therapy. To evaluate PTT efficacy in vitro, PBNP or PBNP-Tc constructs were co cultured with target cells. The co-cultures were established in a 96 well plate and individual wells were subject to PTT using an NIR laser (808 nm Collimated Diode Laser System, Laserglow Technologies) at 2.5 W/cm.sup.2 for 10 minutes. The temperature of each well during PTT was monitored using a thermocouple or FLIR thermal imaging system (FLIR, Billerca, Mass.). The photothermal conversion efficiencies of each experimental condition was determined using previously described methodology (Roper 2007, Hoffman 2014). This formulaic determination of photothermal conversion efficiency measured the effective photothermal conversion efficiencies of PBNP or PBNP-Tc in the presence of cells and cell culture media components. [0267] Cell Proliferation. To determine the ability of T cells to proliferate, cells were labelled with CFSE according to the manufacturer's protocol, and stimulated with their corresponding targets. Proliferation was measured after 24 hours. [0268] IFNγ Secretion by ELISPOT. Millipore Multi Screen HTS filter plates (Millipore) were coated with IFN g capture antibody (Mabtech) at a concentration of 10 ug/mL for 4 hours or overnight at 4 C. Plates were washed with PBS and blocked for 1 hour at 37 C. Cells were then plated at a concentration of 1×10.sup.6/mL, and exposed to peptide targets containing actin, CMV antigens, or EBV antigens. For development, plates were washed in PBS/.05% Tween 20 (Sigma-Aldrich) and incubated with biotinylated IFN-g detection antibody (0.5 ug/mL; Mabtech) for 2 hours at 37 C, followed by incubation with streptavidin coupled alkaline phosphatase complex (Vectastain; Vector Laboratories) for 1 hour at room temperature and spots were developed by incubation with 3-amino-9-ethylcarbazole substrate (Sigma) solution. Spot-forming cells (SFC) were counted and evaluated by Zellnet Consulting using an automated plate reader system (Karl Zeiss). [0269] Co-Culture Experiments. To assess their efficacy against target cells, PBNP-Tc constructs were added at a 2:1 ratio to target cells (LCL or PHA blasts), and cultured in RPMI medium for 4-8 hours after which point PTT was administered as described above. Target cells were labeled with CFSE/Cell Trace Far Red according to the manufacturer's protocol prior to inclusion into the assay. Following completion of co culture and/or PTT, flow cytometry was used to examine CFSE/Cell Trace Far Red expression on target cells to determine cell viability. Results were compared to a negative control of target cells alone, irradiated target cells, and positive controls of target cells artificially killed by adding DMSO. Experimental groups were as follows: (i) target cells alone, (ii) irradiated target cells (30Gy), (iii) target cells with an irrelevant cell line (Eol or Phx), (iv) target cells with DMSO, (v) target cells with Tc, (vi) target cells with PBNP, (vii) target cells with PBNP-Tc constructs, (viii) target cells with laser, (ix) target cells with Tc and laser, (x) target cells with PBNP and laser, and (xi) target cells with PBNP-Tc constructs and laser. To assess specificity and activation of T cells, EBV Tc (naked or backpacked with PBNP) were stimulated with irradiated LCL cells at a 2:1 ratio, and CMV Tc (naked or backpacked with PBNP) were stimulated with antigen-loaded PHA blasts at a 2:1 ratio for 24 hours and IFNγ ELISpot assay was performed as per manufacturer's protocol (Mabtech, Cincinnati, Ohio).
[0266] Photothermal Therapy. To evaluate PTT efficacy in vitro, PBNP or PBNP-Tc constructs were co cultured with target cells. The co-cultures were established in a 96 well plate and individual wells were subject to PTT using an NIR laser (808 nm Collimated Diode Laser System, Laserglow Technologies) at 2.5 W/cm.sup.2 for 10 minutes. The temperature of each well during PTT was monitored using a thermocouple or FLIR thermal imaging system (FLIR, Billerca, Mass.). The photothermal conversion efficiencies of each experimental condition was determined using previously described methodology (Roper 2007, Hoffman 2014). This formulaic determination of photothermal conversion efficiency measured the effective photothermal conversion efficiencies of PBNP or PBNP-Tc in the presence of cells and cell culture media components. [0267] Cell Proliferation. To determine the ability of T cells to proliferate, cells were labelled with CFSE according to the manufacturer's protocol, and stimulated with their corresponding targets. Proliferation was measured after 24 hours. [0268] IFNγ Secretion by ELISPOT. Millipore Multi Screen HTS filter plates (Millipore) were coated with IFN g capture antibody (Mabtech) at a concentration of 10 ug/mL for 4 hours or overnight at 4 C. Plates were washed with PBS and blocked for 1 hour at 37 C. Cells were then plated at a concentration of 1×10.sup.6/mL, and exposed to peptide targets containing actin, CMV antigens, or EBV antigens. For development, plates were washed in PBS/.05% Tween 20 (Sigma-Aldrich) and incubated with biotinylated IFN-g detection antibody (0.5 ug/mL; Mabtech) for 2 hours at 37 C, followed by incubation with streptavidin coupled alkaline phosphatase complex (Vectastain; Vector Laboratories) for 1 hour at room temperature and spots were developed by incubation with 3-amino-9-ethylcarbazole substrate (Sigma) solution. Spot-forming cells (SFC) were counted and evaluated by Zellnet Consulting using an automated plate reader system (Karl Zeiss). [0269] Co-Culture Experiments. To assess their efficacy against target cells, PBNP-Tc constructs were added at a 2:1 ratio to target cells (LCL or PHA blasts), and cultured in RPMI medium for 4-8 hours after which point PTT was administered as described above. Target cells were labeled with CFSE/Cell Trace Far Red according to the manufacturer's protocol prior to inclusion into the assay. Following completion of co culture and/or PTT, flow cytometry was used to examine CFSE/Cell Trace Far Red expression on target cells to determine cell viability. Results were compared to a negative control of target cells alone, irradiated target cells, and positive controls of target cells artificially killed by adding DMSO. Experimental groups were as follows: (i) target cells alone, (ii) irradiated target cells (30Gy), (iii) target cells with an irrelevant cell line (Eol or Phx), (iv) target cells with DMSO, (v) target cells with Tc, (vi) target cells with PBNP, (vii) target cells with PBNP-Tc constructs, (viii) target cells with laser, (ix) target cells with Tc and laser, (x) target cells with PBNP and laser, and (xi) target cells with PBNP-Tc constructs and laser. To assess specificity and activation of T cells, EBV Tc (naked or backpacked with PBNP) were stimulated with irradiated LCL cells at a 2:1 ratio, and CMV Tc (naked or backpacked with PBNP) were stimulated with antigen-loaded PHA blasts at a 2:1 ratio for 24 hours and IFNγ ELISpot assay was performed as per manufacturer's protocol (Mabtech, Cincinnati, Ohio).
The reference teachings anticipate the claimed invention.
11. No claim is allowed.
12. Any inquiry concerning this communication or earlier communications from the examiner should be directed to NORA MAUREEN ROONEY whose telephone number is (571)272-9937. The examiner can normally be reached on M-F from 8:00am to 4:30pm.
If attempts to reach the examiner by telephone are unsuccessful, the examiner' s supervisor, Misook Yu, can be reached at telephone number (571) 272-0839. The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300.
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September 19, 2026
/Nora M Rooney/
Primary Examiner, Art Unit 1641
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