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 .
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.
Status of Claims
Claims 1-20 are pending. Claims 19-20 are withdrawn. Claims 1-18 are under examination.
Withdrawn Objections/Rejections
In light of the amendments, the objection is hereby withdrawn.
In light of the amendments, the 35 U.S.C. 112(b) rejection is hereby withdrawn.
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:
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.
Claims 1-10, 12-16 and 18 are rejected under 35 U.S.C. 103 as being unpatentable over Huang et al. (WO2019/126182A1, published 06/27/2019, 892 dated 03/09/2026) in view of Shimomura et al. (“New Lipophilic Fluorescent Dyes for Labeling Extracellular Vesicles: Characterization and Monitoring of Cellular Uptake”, Bioconjugate Chemistry, 2021, vol. 32, pgs. 680-684, published 03/13/2021, 892 dated 03/09/2026).
With respect to claims 1 and 3, Huang teaches methods related to the detection and molecular profiling of membrane bound vesicles using Raman extracellular vesicle and the method makes use of highly sensitive and specific enhanced Raman scattering technology to label and detection membrane bound vesicles that are captured on a miniaturized device based on the protein expression on the surface of the membrane bound vesicle (see abstract). Huang teaches the method involves the use of highly sensitive and specific surface enhanced Raman scattering (SERS) nanotags (e.g., SERS gold nanorod (AuNR) tags) to detect and quantify surface proteins on membrane bound vesicles that are captured on a substrate (e.g., an array, Au-coated glass microscope slide, bead).
In particular, Fig. 12A-C teach a method for characterizing membrane bound vesicle with a gold-coated substrate comprising an antibody fixed to a surface of the slide, thereby fixing the exosomes to the surface of the slide and contacting the exosomes with a second antibody fixed to a surface of a metal nanoparticle, wherein the second antibody specifically binds to a surface marker of interest (see step 12A, after step 3 or Fig. 27). Huang further teaches in Fig. 27 that white light and Raman laser are used to produce dark field image and SERS image, which produce image analysis to quantify the targeted protein on single exosomes. Huang teaches the target image (Fig. 32B) was added to the outlined mask using the ROI manager (Fig. 32D) to form an overlay image with the target image (Fig. 32E) and the mean pixel intensity of the outlined areas of this overlap image was extracted and the values were subtracted with the background from a blank neighboring area (noise) to give corrected signal intensities (see pg. 43, para. 1; and Figs. 32). Huang further teaches analyzing a number of exosomes, a histogram was generated that shows the distribution of the pixel intensity among all examined exosomes and the population density histogram represents the expression profile of the targeted protein on single EXOs (see pg. 43, para. 1). Huang teaches CD44 antibody and isotype IgG (see pg. 43, bottom of para. 1; and Fig. 32G). Huang teaches SERS nanotags are plasmonic nanoparticles (gold or silver nanoparticles) such as gold nanoparticles coated with Raman reporters such as organic dyes (see pg. 18, lines 14-16, and Figs. 14-15). Fig. 19 teaches measured by nanoparticle tracking analysis (also see pg.13, lines 25-27). Huang teaches the detection and protein profiling of MM231 EXOs (see pg. 11, lines 13-15).
Although Huang teaches the use of organic and inorganic dyes (e.g., pg. 3, lines 20-26) and fluorescent images of EXOs that are captured using CD63 antibodies (e.g., pg. 12, lines 16-20), Huang does not teach a lipophilic dye.
Shimomura teaches lipophilic dyes are for extracellular vesicular-membrane-binding fluorescent probes and performed nanoparticle tracking (see abstract). Shimomura teaches that extracellular vesicle (EV) labeling with small molecules is easy, compatible with biomolecules, and allows versatile functionalization and lipophilic dyes have been widely used (see pg. 680, right col., para. 1). Shimomura further teaches improving the dyes by employing polyethylene glycol and that it is important that EV labeling does not change any EV characteristics (e.g., size, population) (see pg. 681, left col., para. 1). Shimomura teaches that the Mem dyes have stable structures suitable for use in experiments monitoring EV uptake and the Mem dyes do not change the zeta potential of the stained sEVs (see pg. 682, Discussion, right col., para. 2).
It would have been obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to have used the dual immunoassay detection as taught by Huang with a lipophilic dye as taught by Shimomura because Huang teaches image analysis to quantify the targeted proteins on single exosomes and track exosomes through dual imaging with organic dyes and Shimomura teaches lipophilic dyes are easily incorporated into extracellular vesicles for tracking and extracellular vesicles would be clearly identified. Because lipophilic dyes are well understood in the art to be incorporated for tracking extracellular vesicle nanoparticles, it would have been obvious to add the lipophilic dye for fluorescence imaging of the extracellular vesicle nanoparticles of Huang.
The person would have a reasonable expectation of success incorporating a lipophilic dye into the immunoassay detection of Huang because it has been well understood in the art to incorporate lipophilic dyes into the extracellular vesicles.
With respect to claims 2 and 4, Fig. 27 of Huang teaches image analysis to quantify the targeted proteins on single exosomes, which would read on computationally analyzing the images to determine the fraction of vesicles that are positive for the surface of interest and the level of expression of the surface marker of the surface marker of interest on the positive vesicles.
With respect to claim 5, Huang teaches gold nanorod (see pg. 2, line 7 and Fig. 27).
With respect to claim 6, Huang teaches body fluid is blood (see pg. 2, lines 10-11).
With respect to claim 7, Huang teaches the capture molecules are antibody.
With respect to claim 8, Huang teaches EXOs that are captured using CD63 antibodies (see pg. 12, lines 17-18).
With respect to claim 9, as stated above, Huang does not teach the lipophilic dye. Shimomura teaches the lipophilic dyes having an alkyl chain and an affinity for a lipid bilayer of an extracellular vesicle (see abstract and abstract figure). Because lipophilic dyes are well understood in the art to track extracellular vesicle nanoparticles, it would have been obvious to incorporate the lipophilic dye for fluorescence imaging of the extracellular vesicle nanoparticles.
With respect to claim 10, although Huang does not teach the lipophilic dye, it does teach the EVs are immobilized on an array using a lipophilic chemical layer wherein the lipophilic molecules with an alkyl chain have high affinity for the lipid bilayer of molecules through hydrophobic interactions between the lipid membrane of the target and lipophilic molecules on the substate and it is 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-conjugated polyethylene glycol thiol (pg. 20, para. 2). Therefore, it would have been obvious to have used a dye with the claimed lipophilic molecule because Huang has already established and recognized the structural affinity to attach molecules to EV molecules.
With respect to claims 12-13, Huang teaches EXOs were excited with the He laser at 632.8 nm (see pg. 42, lines 25-27).
With respect to claim 14, Fig. 27 of Huang teaches the nanorod is bound to the vesicle or exosome via an antibody linked to the nanoparticle, wherein the antibody specifically binds to a marker present on the vesicle or exosome.
With respect to claim 15, Huang teaches different surface proteins such as EpCAM, CD44, HER2 are detected (see pg. 11, lines 13-16).
With respect to claim 16, Huang teaches detect many types of diseases (see pg. 17, line 4).
With respect to claim 18, Huang teaches antibody-conjugated SERS AuNR tags were prepared by HS-PEG-Ab with HS-PEG-NHS (see pg. 36, lines 3-6).
Claims 11 and 17 are rejected under 35 U.S.C. 103 as being unpatentable over Huang et al. and Shimomura et al., as applied to claim 1 or 9 above, and further in view of Jia et al. (“Efficient cell surface labelling of live zebrafish embryos: wash-free fluorescence imaging for cellular dynamics tracking and nanotoxicity evaluation”, Chemical Science, 2019, vol. 10, pgs. 4062-4068, 892 dated 03/09/2026).
Huang and Shimomura have been discussed above. However, the references do not teach the lipophilic dye comprises cholesterol-PEG-Cy5.
Jia teaches imaging the dynamics and behaviors of plasma membranes and the development of a universal red-fluorescent probe Chol-PEG-Cy5 for wash free plasma membrane labelling both in vitro and in vivo (see abstract and Scheme 1). Jia further teaches that chol-PEG-Cy5 enables rapid, stable and high-quality in vitro cell surface imaging (see abstract). Fig. 1 depicts the structure of chol-PEG-Cy5. Jia also teaches exosome (see pg. 4062, left col., para. 1). Jia teaches fluorescence emission ~670 nm of Cy5, the cho1-PEG-Cy5 probe is suitable for fluorescence imaging (see pg. 4065, left col., last paragraph).
It would have been obvious to have used the modified immunoassay of Huang and Shimomura with a cholesterol-peg-Cy5 of Jia for fluorescence imaging because Jia teaches that cholesterol-peg-Cy5 enables rapid, stable, and high-quality surface imaging and the structure naturally incorporates into the lipid bilayer for detection.
The person would have a reasonable expectation of success in using cholesterol-peg-Cy5 for fluorescence imaging because it has been recognized that cholesterol-peg-Cy5 is for lipid bilayer imaging.
Response to Arguments
Applicant's arguments filed 06/04/2026 have been fully considered but they are not persuasive.
With respect to the obviousness rejection of claims 1-10, 12-16 and 18, Applicant argues on page 8 that the pending claims recite a different dual-imaging arrangement in which fluorescent provides vesicle localization image and dark-field imaging of nanoparticles that provides the marker-presence image. Applicant further argues that Huang’s organic dyes refer to a different use of organic dyes than the claimed lipophilic-dye fluorescence localization. Applicant further argues on page 9 that Huang uses dark-field imaging for localization and SERS/RAMAN imaging for marker detection and quantification. Whereas, in the claims, fluorescence imaging performs localization and darkfield imaging performs marker characterization. Applicant also argues that the rejection does not identify a teaching or suggestion in either Huang or Shimomura that would have led the person to reassign those imaging functions in the specific manner recited in the independent claims.
The arguments are not found persuasive for the following reasons. Huang teaches elements (a) and (b), which have all the components for using dark field imaging to characterize the presence or absence of the surface marker of interest on the vesicles (for example, 12A). In particular, Fig. 27 teaches EXOs will be imaged under dark field with white light illumination to detection and localize EXOs (also see pg. 15, para. 3 under para. for FIG. 27). Further, Huang teaches that dark field is used for optical excitation and detection (see pg. 42, para. 1). Huang’s dark field imaging through white light in the presence of AuNR (gold nanoparticle) would also identify the presence or absence of AuNR optical properties. Thus, dark field imaging is detecting both localization and the presence or absence of AuNR specifically binding to surface marker of interest on the vesicles. Meanwhile, the use of AuNR on the vesicle with two (duo) light sources (while light and Raman laser) would have overlapping detection that may conflict at low sensitivities. Thus, the use of lipophilic dye would distinguish the detection between localizing and the presence of the marker on the vesicle. In particular, Shimomura teaches lipophilic dyes are easily incorporated into extracellular vesicles for tracking and extracellular vesicles would be clearly identified. Meanwhile, Huang also recognizes using fluorescence dyes and measuring fluorescence signals in the presence of vesicles.
Although the claim recites computationally analyzing overlap signals, the extraction is merely the pixels from the nanoparticles of dark field imaging. Huang dark field through white light would have also produced the pixels from the nanoparticles. Also, the claims do not limit to dual imaging as it recites “comprising”. The rejection above does not limit to substitution of Raman for lipophilic dye. Meanwhile, the incorporation of lipophilic dyes would not interfere with using dark field and SERS, as AuNR is specifically binding to protein markers whereas the lipophilic dyes at the lipid sites.
With respect to the obviousness rejection of claims 11 and 17, Applicant argues, page 10, that MPEP 2141.01(a) states that art must be shown to be either from the same field of endeavor or reasonably pertinent to the problem faced by the inventor. Applicant further argues, page 11, that why a person of ordinary skill in the art would have selected the Chol-PEG-Cy5 dye of Jia, rather than the extracellular vesicle-specific Mem dyes of Shimomura.
The arguments are not found persuasive for the following reasons. Jia is within the same field of endeavor as the reference recognizes the cholesterol-polyethylene glycol-Cy5 for lipid incorporation. Although the person of ordinary skill in the art would use Mem dyes of Shimomura, the person would also recognize using a structure that enables a quicker incorporation while retain stability for high-quality images of lipid structures.
Conclusion
No claim is allowed.
THIS ACTION IS MADE FINAL. 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.
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/N.P.N/Examiner, Art Unit 1678
/SHAFIQUL HAQ/Primary Examiner, Art Unit 1678
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