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 .
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.
Continued Examination Under 37 CFR 1.114
A request for continued examination under 37 CFR 1.114, including the fee set forth in 37 CFR 1.17(e), was filed in this application after final rejection. Since this application is eligible for continued examination under 37 CFR 1.114, and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114. Applicant's submission filed on 05/26/2026 has been entered.
Priority
The present application claims benefit under 35 U.S.C. 119(e) to provisional application No. 63/249,705, filed 09/29/2021.
Status of the Claims
Claims 1-27 are pending; claims 1, 2, 13 and 24-27 are amended; no claims are withdrawn. Claims 1-27 are examined below.
Withdrawn Objections/Rejections
The previous rejections of claims under 35 U.S.C. 112(b) are withdrawn in response to Applicant’s amendments to the claims.
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.
Claim(s) 1-3, 5, 7-13, 15, 17, 18 and 20-23 are rejected under 35 U.S.C. 103 as being unpatentable over Oscar et al., WO2019/081474A1 in view of Busatto et al., Tangential Flow Filtration for Highly Efficient Concentration of Extracellular Vesicles from large Volumes of Fluid, Cells, 7(273), (2018), p. 1-11, Morales-Kastresana et al., Flow Cytometry Analysis of Extracellular vesicles, Methods Mol. Biol., 1545, (2017), p. 215-225, Artinger et al., WO2016/154286A1, Carney et al., Targeting Tumor-Associated Exosomes with Integrin-Binding Peptides, Adv. Biosyst., 1(5), (2017), 28 pages and Pall Life Sciences. “Microsep™ Advance Centrifugal Devices”. (2014), https://www.pall.com/content/dam/pall/japan/laboratory/literature-library/non-gated/Microsep_PII.pdf. 16 pages (Accessed: 09/08/2025).
Although Oscar’s invention is directed to affinity chromatography isolation and purification of EVs, Oscar et al. also teach (by example) methods which encompass processing extracellular vesicles consistent with the claimed method. In particular, see Oscar demonstrated in their examples a comparative example comprising method steps comprising a step of concentrating extracellular vesicles in a conditioned media using tangential flow filtration with a 300 kd hollow fiber columns (see Example 1 at page 33, para 3), passed through 10kD spin filters (also page 33, para 3, Oscars describes these steps as concentrating EVs). At this example, Oscar followed the concentrating step by by electron microscopy and flow cytometry to assess Fc-binding EVs (see page 33, para 4).
Regarding the flow cytometry at the referenced Example of Oscar, see end of page 33 to page 34, Oscar et al. teach EVs were incubated with antibody coated capture beads and AlexaFluor647 conjugated anti-human IgG Fc fragments (contacted with a fluorescent labeled antibody for a surface marker, incubating to generate a labeled EV population).
Oscar teaches performing flow cytometry, and although Oscar is silent as to stating a quantitative measure/determining concentration, prior to the contacting step, see for example Figure 3 referenced by Oscar’s example 1 at page 33, Oscar is determining the concentration of the concentrated EVs (the amount of EVs comprising Fc binding polypeptides). Oscar is teaching EVs from complex biological fluids, such as cell culture medium (see abstract).
Additionally, as noted at Example Oscar demonstrate a combination of tangential flow filtration and ultrafiltration using spin filters (i.e., a type of centrifugation filtration) for concentration. The instant claims recite “wherein… not purified via size exclusion chromatography column or other filtration media other than tangential flow filter”; the originally filed specification throughout clearly excludes a prior step of “size exclusion chromatography”, however, see also para [0038], regarding context for the claimed language, the specification at para [0038] recites “As described herein, it has surprisingly been found that it is not necessary to purify the extracellular vesicles, prior to contacting them with the fluorescent dye or antibodies, to label the EVs. Traditionally, EVs had to be purified from the cellular growth media solution, prior to labeling. However, it has been determined, as described herein, that simply concentrating the EVs (e.g., via centrifugation or tangential flow filtering, or a combination thereof), without purification, leads to an EV sample that can be labeled, then later purified, recovered and analyzed, resulting in a high concentration of EVs for analysis and providing reproducible analytic results of the EV characteristics.”. Oscar is using a combination of ultra filtration and tangential flow filtering to “concentrate” consistent with Applicant’s invention. As such, Oscar’s example 1 is using a form of filtration centrifugation, and as such differs from the claim in that it fails to exclude “other filtration media other than tangential flow”.
Oscar et al. fails to teach, a step of determining concentration prior to contacting step c, and fails to teach, following the contacting step, passing the contacted (labeled) EVs through a centrifugal filter comprising a 200-750kD molecular weight cut-off, polyethersulfone filter media, to separate the labeled extracellular vesicle population from excess antibody, recovering the labeled population.
See further Busatto et al. teach, regarding concentration of EVs from biological fluids, that tangential flow filtration achieves highly efficient isolation of EVs from large volumes of sample, that it is even more efficient, scalable and more gentle than ultracentrifugation, which according to Busatto, is most widely used method for EV concentration (see abstract). See at page 2, paragraph 2, Busatto teach common isolation techniques such as UC, density gradients and SEC can be limited by input volume, time consumption, and EV yield, that UC result in low recovery rates, and have time consuming centrifugation steps, and frequently damage EV structure. Busatto teach advantages of TFF are that fluid flow tangentially across the surface, avoiding filter cake formation, and are not subject to clogs of the membrane pores (page 2, paragraph 3).
Morales-Kastresana et al. teach it is important to determine an approximate EV concentration (a first step of isolation and quantification) before capture and binding to beads for flow cytometry (see end of page 2 and page 3, first paragraph).
Regarding flow cytometry, see Artinger et al. teaching the presence of background signals from unbound fluorophore stains is a significant concern, that there are some techniques that may be used in order to ensure that a detected fluorescent signal is associated with the cell or microsphere of interest, rather than combing from stained debris or from free, unbound fluorescent stain molecules that remain in solution. See Artinger et al. teach, after staining, separating particles from liquid that contains residual stain molecules in solution, such as by filtration, the separate cells or microsphere particles then resuspended (i.e., recovered for cytometry/detection), see page 5, lines 14-30. See also page 7, lines 10-13 (and page 17, lines 26-32, page 22, lines 18-31), the principles of Artinger apply also to virus sized particles, such as exosomes and other nanoparticles.
As an example of filtration, Artinger teach for example spin chromatography in a centrifuge (page 20, lines 10-11).
See however, as another filtration means for removing unlabeled components from labeled exosomes, Carney teach the technique of centrifugal filtration (see page 14, last paragraph, Carney describe remove of free components using centrifugal filtration).
See also PALL Life Science teach examples of commercially available polyethersulfone centrifugal filter devices come in various sizes depending on one’s need (see page 3, Introduction, see also page 5, guide for selection).
It would have been prima facie obvious to one having ordinary skill in the art before the effective filing date of the claimed invention, to have modified Oscar’s example 1 to have performed merely tangential flow filtration alone (omitting the ultrafiltration/spin filtration) in order to concentration EVs because tangential flow filtration alone is considered an effective technique for concentration of EVs, thereby simplifying the concentration step as in performed in Oscar’s example 1, one motivated to perform TFF only because it is considered an efficient, scalable technique that is considered more gentle, and without the need for a filter which can be subject to clogs (at the filter membrane), see Busatto. Additionally, one would be motivated to omit the ultrafiltration step, as this is considered a form of centrifugation (see as requires spin filters), and Busatto teach centrifugation steps can be time consuming. One having ordinary skill in the art would have a reasonable expectation of success because based on Busatto, tangential flow filtration alone would be considered a sufficient technique for concentration of EVs on its own.
It would have been prima facie obvious to one having ordinary skill before the effective filing date of the claimed invention, to have modified Oscar et al., in order to perform a step to quantitate/determine EV approximate concentration prior to contacting with label/bead for flow cytometry, as taught by Morales-Kastresana et al., because this was recognized as in the art as an important first step when performing flow cytometry (see as cited above), and further to add a step after the contacting step c. that is a step of filtration (filtering out unbound component) to separate the labeled extracellular vesicles population from excess fluorescent staining/antibody in order to reduce the presence of background signal, thereby ensuring detected signal is specific to the captured target and not residual unbound label, as in Artinger et al.
One having ordinary skill in the art would have had a reasonable expectation of success modifying Oscar with Morales-Kastresana considering this reference is specific to details for performing flow cytometry of EVs (Oscar is teaching flow cytometry of concentrated EVs), and with Artinger because Artinger teach the presence of background signal from unbound component is a significant concern. As a result, one would expect that removing the unbound components (label/antibody) would similarly improve signal detection in Oscar, as it does in Artinger.
Further, regarding the limitation “centrifugal filter”, it would have been prima facie obvious to one having ordinary skill in the art before the effective filing date of the claimed invention, to have modified Oscar and the cited art, to use a polyethersulfone centrifugal filter (as in Pall Life Sciences) having a molecular weight cutoff of 300Kd, in place of the spin chromatography as taught by Artinger as a simple substitution of one art recognized suitable filtration means for another. Both techniques were recognized for the same purpose, as suitable or applicable ways to filter out unbound binding reagent (Artinger and Carney). One having ordinary skill would predictably, based on Carney, expect the centrifugal filter to work to remove unbound component, thereby improving non-specific signal as in Artinger, because Carney specifically disclose this technique as capable for this purpose.
Further it would have been prima facie obvious to have relied on that structure as taught by Pall Life sciences, as an obvious matter of a known material for its art recognized intended purpose, specifically teaching having a cutoff of 300 kD, because Oscar already disclose 300 as a suitable size for filtering/retaining targeted extracellular vesicles in a sample. One having ordinary skill would have a predictable expectation of success first using the filter system of Pall since it is a commercially available centrifugal filter (Carney does not specify a required type, and as such is considered to leave open the ability to us any suitable commercially available system), and further one would expect success selecting a Pall centrifugal filter having a cutoff of 300 kD because Pall teach one is able to select a filter size (see as cited above).
Regarding independent claim 13, Oscar et al. teach EVs typically have a nano-sized hydrodynamic radius (see page 7, mid page; also page 1, background paragraph 1). Also, Oscar does not teach concentration by size exclusion chromatography (method without purifying by size exclusion). As a result, the methods of Oscar et al. in view of the cited art are performing analysis on a targeted structure that is considered to be a nanoparticle. Therefore, when given broadest reasonable interpretation, the method of Oscar and the cited art is determining concentration using a flow cytometer for “nanoparticle analysis” (since the target being analyzed is considered to be on the nanometer size range). Nonetheless, in the interest of compact prosecution, see below, claim 13 is further addressed with additionally cited prior art.
Regarding claim 2, see as cited above, the concentrating as taught by the cited prior art is by passing fluid through a tangential flow filter.
Regarding claim 3, see further the combination of the cited art teaches a tangential flow filter with a molecular weight cut-off of 300 kD (a value that reads on the claimed range).
Regarding claims 5 and 15, see Oscar at page 15, first paragraph, Oscar teach exosomal polypeptides such as CD9, CD63 and CD81; however, further see page 4, regarding Figure 3 (using IgG antibody).
Regarding claims 7 and 17, as discussed above, Oscar teaches for flow cytometry, the antibody labeled reagent incubated for an hour at room temperature (thereby addressing at least 30 minutes), however fails to teach EVs labeled antibody conjugate incubated at a temperature of about 30-40°C (claim 7 and 17).
Regarding the claimed incubation duration and temperature for forming preformed complex (for combining antibody and fragment), and the claimed antibody concentration, generally, differences in concentration or temperature will not support the patentability of subject matter encompassed by the prior art unless there is evidence indicating such concentration or temperature is critical. "[W]here the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimum or workable ranges by routine experimentation." In re Aller, 220 F.2d 454, 456, 105 USPQ 233, 235 (CCPA 1955). MPEP 2144.05.
In the present case, the general conditions were known in the prior art, differing in terms of the incubation temperature (the prior art teaching a similar temperature that is just outside the claimed range, namely room temperature). Based on MPEP 2144.05 and Oscar et al., it would have been prima facie obvious to one having ordinary skill in the art before the effective filing date of the claimed invention to have arrived at a temperature within 30-40°C by routine optimization of experimental conditions, to uncover the optimum workable conditions for binding and detection, one having ordinary skill would have a reasonable expectation of success because the prior art disclosed temperature (referring to Oscar, who also teaches methods comprising flow cytometry) is similar (room temperature is just below the claimed range, the prior art and the claimed invention teaching temperatures on the same order of magnitude).
Regarding claims 8, 11, 18 and 21 see at page 33, Example 1, Oscar et al. teach HEK293T derived extracellular vesicles.
Regarding claims 9 and 19, Oscar et al. teach EVs typically have a nano-sized hydrodynamic radius (see page 7, mid page; also page 1, background paragraph 1). As a result, the methods of Oscar et al. in view of the cited art are performing analysis on a targeted structure that is considered to be a nanoparticle. Therefore, when given broadest reasonable interpretation, the method of Oscar and the cited art is determining concentration using a flow cytometer for “nanoparticle analysis” (since the target being analyzed is considered to be on the nanometer size range). Nonetheless, in the interest of compact prosecution, see below, claim 9 is further addressed with additionally cited prior art.
Regarding claims 10 and 20, see Oscar at page 7 (end of page) teaching EVs can be present in concentrations such as 1010.
Regarding claims 12 and 22, Oscar et al. fails to teach passing through the centrifugal filter for at least 10 minutes at a force of at least 10,000 x g. However, see further Pall Life Sciences teach at page 7, step 3, to spin the device, typically 30-90 minutes at 3000-7500 x g for the required length of time. Pall Life Sciences teach, for optimal performance, it is recommended that spin time and g-force be determined for each application, referring to pages 10 and 11 for protocol. Based on Pall Life Sciences, duration and g-force are variables considered to be result effective variables, namely variables that achieve a recognized result (i.e., filtration of a given composition), Pall teaching these are variables that should be optimized. It would have been prima facie obvious to one having ordinary skill in the art before the effective filing date to have optimized the duration and g-force, thereby arriving at the claimed duration and force, at the direction of Pall Life Sciences, one motivated to experiment with the conditions to uncover the optimum workable variables to achieve the desired filtration. See MPEP 2144.05.
Regarding claim 23, see the combination of the cited art is teaching amount of EVs (concentration, see Oscar et al. cited in detail above).
Claim(s) 4 and 14 are rejected under 35 U.S.C. 103 as being unpatentable over Oscar et al. in view of Busatto et al., Morales-Kastresana et al., Artinger et al., Carney et al. and Pall Life Sciences, as applied to claims 1 and 13 above, and further in view of Barilani et al., WO2019/111197 A1.
Oscar et al. and the cited art teach a method substantially as claimed (see as cited in detail previously above).
However, Oscar et al. is performing flow cytometry using labeled antibody conjugate and as such fails to teach EVs labeled by contact with CFSE (claims 4 and 14)
See Barilani et al., teaching in order to investigate EV integrity, staining with a stain such as CFDA-SE, that is converted into a fluorescent molecule, CFSE, by cytoplasmic esterases (see page 2, description of Figure 3, showing EVs are intact cytoplasm containing enclosed particles; page 53, lines 20-30).
It would have been further prima facie obvious to one having ordinary skill in the art to have modified Oscar et al. and the cited art to stain EVs with CFSE in order to assess EV integrity, namely in order to identify those that are intact EVs, as in Barilani et al. One having ordinary skill int eh art would have a reasonable expectation of success because CSFE was already known as a fluorescent indicator for EV integrity.
Claim(s) 6 and 16 are rejected under 35 U.S.C. 103 as being unpatentable over Oscar et al. in view of Busatto et al., Morales-Kastresana et al., Artinger et al., Carney et al. and Pall Life Sciences, as applied to claims 1 and 13 above, and further in view of Barilani et al., WO2019/111197 A1 and Skalnikova et al., Isolation and Characterization of Small Extracellular Vesicles from Porcine Blood Plasma, Cerebrospinal Fluid, and Seminal Plasma, Proteomes, 7(17), (2019), (14 pages).
As discussed above, Oscar fails to teach EVs labeled by contact with CFSE (claims 4 and 14), and further fails to teach fluorescent staining dye for at least 1 hour at a temperature of about 30-40°C (claim 6 and 16).
As discussed above, see Barilani et al., teaching in order to investigate EV integrity, staining with a stain such as CFDA-SE, that is converted into a fluorescent molecule, CFSE, by cytoplasmic esterases (see page 2, description of Figure 3, showing EVs are intact cytoplasm containing enclosed particles; page 53, lines 20-30).
It would have been further prima facie obvious to one having ordinary skill in the art to have modified Oscar et al. and the cited art to stain EVs with CFSE for the reason indicated in detail above (see as indicated above, as the same reasoning applies presently).
Regarding claim 6, Barilani et al. cited above (page 53) teach staining EVs at room temperature for 30 minutes. However, see MPEP 2144.04, generally, differences in concentration or temperature will not support the patentability of subject matter encompassed by the prior art unless there is evidence indicating such concentration or temperature is critical. "[W]here the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimum or workable ranges by routine experimentation." In re Aller, 220 F.2d 454, 456, 105 USPQ 233, 235 (CCPA 1955).
See further Skalnikova et al., who also teach labeling EVs with CFSE for flow cytometry, see at page 4, last paragraph, teaching incubation time of 60 min at 37°C.
It would have been prima facie obvious to one having ordinary skill in the art before the effective filing date of the claimed invention to have modified methods labeling EVs with CFSE as taught by the combination of the cited art to have arrived at an incubation of 60 min for 37°C out of routine optimization of experimental conditions, namely trying from art recognized incubation conditions (temperatures and times) to uncover the optimum workable conditions. One would have a reasonable expectation of success, considering such conditions were known in the art at the time particularly for EV staining using CFSE (Skalnikova et al.).
Claim(s) 9 and 19 are rejected under 35 U.S.C. 103 as being unpatentable over Oscar et al. in view of Busatto et a., Morales-Kastresana et al., Artinger et al., Carney et al. and Pall Life Sciences, as applied to claim 1 above, and further in view of Inokuma et al., US PG Pub No. 2018/0224460A1.
Oscar and the cited art teach a method substantially as claimed (see as cited above).
However, Oscar and the cited art fail to teach, for the flow cytometry, beads that are nanometer sized beads (claim 9, interpreting “nanoparticle analysis” to be in reference to the size of the attached bead).
However, see Inokuma et al. at para [0037] teaching particles, such as beads, for methods of flow cytometry, Inokuma teaching structures having a diameter in the nanometer to micrometer range as suitable for use in flow cytometry.
It would have been further prima facie obvious to one having ordinary skill in the art at the time the claimed invention was effectively filed, to have modified Oscar et al. and the cited art, to have used nm diameter sized beads as an obvious matter of applying a known bead for its intended purpose in flow cytometry. One having ordinary skill in the art would have had a reasonable expectation of success because based on Inokuma et al. it appears that either nanometer or micrometer sized beads are suitable for this type of assay.
Claim(s) 13, 15, 18, 21-23 are rejected under 35 U.S.C. 103 as being unpatentable over Oscar et al. in view of Busatto et al., Morales-Kastresana et al., Artinger et al., Carney et al., Pall Life Sciences and Inokuma et al.
Oscar et al. is as cited teaching a method substantially as claimed (see above).
As indicated above, Oscar fails to teach only tangential flow filtration (excluding SEC and other filtration), fails to teach determining a concentration before the contacting step, and fails to teach following the contacting step, passing the contacted (labeled) EVs through a centrifugal filter comprising a 200-750kD molecular weight cut-off, polyethersulfone filter media, to separate the labeled extracellular vesicle population from excess antibody, recovering the labeled population. Further, for the flow cytometry, Oscar et al. fails to teach beads that are nanometer sized beads (claim 13, interpreting “nanoparticle analysis” to be in reference to the size of the attached bead).
Busatto et al., Morales-Kastresana et al., Artinger et al., Carney et al. and Pall Life Sciences are cited as set forth in detail above, it would have been obvious to have modified Oscar in view of these references for the reasons as indicated in detail above.
See also Inokuma et al. at para [0037] teaching particles, such as beads, for methods of flow cytometry, Inokuma teaching structures having a diameter in the nanometer to micrometer range as suitable for use in flow cytometry.
It would have been further prima facie obvious to one having ordinary skill in the art at the time the claimed invention was effectively filed, to have modified Oscar et al. and the cited art, to have used nm diameter sized beads as an obvious matter of applying a known bead for its intended purpose in flow cytometry. One having ordinary skill in the art would have had a reasonable expectation of success because based on Inokuma et al. it appears that either nanometer or micrometer sized beads are suitable for this type of assay.
Regarding claim 15, see Oscar at page 15, first paragraph, Oscar teach exosomal polypeptides such as CD9, CD63 and CD81; however, further see page 4, regarding Figure 3 (using IgG antibody).
Regarding claims 18 and 21 see at page 33, Example 1, Oscar et al. teach HEK293T derived extracellular vesicles.
Regarding claim 22, Oscar et al. fails to teach passing through the centrifugal filter for at least 10 minutes at a force of at least 10,000 x g. However, see further Pall Life Sciences teach at page 7, step 3, to spin the device, typically 30-90 minutes at 3000-7500 x g for the required length of time. Pall Life Sciences teach, for optimal performance, it is recommended that spin time and g-force be determined for each application, referring to pages 10 and 11 for protocol. Based on Pall Life Sciences, duration and g-force are variables considered to be result effective variables, namely variables that achieve a recognized result (i.e., filtration of a given composition), Pall teaching these are variables that should be optimized. It would have been prima facie obvious to one having ordinary skill in the art before the effective filing date to have optimized the duration and g-force, thereby arriving at the claimed duration and force, at the direction of Pall Life Sciences, one motivated to experiment with the conditions to uncover the optimum workable variables to achieve the desired filtration. See MPEP 2144.05.
Regarding claim 23, see the combination of the cited art is teaching amount of EVs (concentration, see Oscar et al. cited in detail above).
Claim(s) 24 and 25 are rejected under 35 U.S.C. 103 as being unpatentable over Oscar et al. in view of Busatto et al., Morales-Kastresana et al., Flow Cytometry Analysis of Extracellular vesicles, Methods Mol. Biol., 1545, (2017), p. 215-225 (presently cited as Morales-Kastresana et al. (1) below) and Morales-Kastresana et al., Labeling Extracellular vesicles for Nanoscale Flow Cytometry, Nature Scientific Reports, 7(1878), (2017), (10 pages) (presently cited as Morales-Kastresana et al. (2)).
Oscar et al. is as cited in detail previously above, Oscar et al. is teaching a method substantially as claimed (see above), including concentrating EVs, contacting with labeled antibody, incubating to generate a labeled population. Oscar does recover the labeled EVs and subject them to flow cytometry analysis. Claim 24 differs from the independent claims detailed above in that it does not require a step of passing EVs through a centrifugal filter, rather claim 24 merely requires, following the contacting and labeling steps, a step of diluting (noted above) and further recovering the labeled EV population. It would have been obvious to have modified Oscar in view of the cited art as indicated in detail previously above (as the same reasoning applies presently, for example regarding Busatto et al., etc.).
Oscar fails to teach determining and a concentration before the contacting step (determining vesicles to be at least 5 x 1010 EVs/ml). Further Oscar fails to teach “diluting the population by at least a factor 1:300” after the step of incubating
Morales-Kastresana et al. (1) teach it is important to determine an approximate EV concentration (a first step of isolation and quantification) before capture and binding to beads for flow cytometry (see end of page 2 and page 3, first paragraph).
Morales-Kastresana et al. (2) further discuss the issue of swarm detection when detecting EVs, see at page 2, para 1 (first full paragraph), teaching to confirm individual EV detection without swarming or coincident event detection, performing serial dilution analysis to determine an operational range where the event rate increase is proportional to the EV concentration (See also Figure 2 caption, E).
It would have been prima facie obvious to one having ordinary skill in the art before the effective filing date of the claimed invention to have modified Oscar et al., to quantitate/determine EV approximate concentration prior to contacting with label/bead for flow cytometry, as taught by Morales-Kastresana et al. (1), because this was recognized as in the art as an important first step when performing flow cytometry. Further, it would have been obvious to have modified to arrive at a dilution as claimed (such as 1:300), out of routine optimization of experimental conditions, one motivated to perform serial dilutions to uncover the optimum workable range, as in Morales-Kastresana et al. (2), in order to achieve detection without swarm detection or coincidental detection, to uncover the optimum dilution to achieve single event detection for accurate determination of concentration and EV assessment. One having ordinary skill in the art would have had a reasonable expectation of success performing these modifications because based on both Morales-Kastresana references, these are techniques encouraged for performing flow cytometry on EV targets.
See as cited in detail previously and above (referring to the citations of Oscar et al. previously at claim 1, and also immediately above regarding claim 24), the combination of the cited art addresses the limitations of claim 25.
Regarding claim 24 and 25, Oscar et al. appears to be consistent with the meaning of “not purified prior to the contacting at step c”, as claimed, as Oscar is performing a step of concentration (by tangential flow filter, and not by other techniques considered to be “chromatography column or other filtration media” as claimed (however, see the rejection above, under 35 U.S.C. 112(b), as tangential flow filtration is a technique considered to involve “other filtration media”). Based on the originally filed specification and present claims, it appears Oscar in view of the cited art is performing methods without purification consistent with the claimed meaning.
Claim(s) 26 and 27 are rejected under 35 U.S.C. 103 as being unpatentable over Oscar et al. in view of Busatto et al., Morales-Kastresana et al., Flow Cytometry Analysis of Extracellular vesicles, Methods Mol. Biol., 1545, (2017), p. 215-225 (presently cited as Morales-Kastresana et al. (1) below), Morales-Kastresana et al., Labeling Extracellular vesicles for Nanoscale Flow Cytometry, Nature Scientific Reports, 7(1878), (2017), (10 pages) (presently cited as Morales-Kastresana et al. (2)), Mirmira et al., US PG Pub No. 2019/0226006A1 and ThermoFisher Scientific. “Nucleic Acid Stains – Section 8.1”., 2018, Internet Archive: https://web.archive.org/web/20180822113410/https://www.thermofisher.com/us/en/home/references/molecular-probes-the-handbook/nucleic-acid-detection-and-genomics-technology/nucleic-acid-stains.html (Accessed 9/9/2025).
Regarding claims 26 and 27, the claims are substantially similar to claim 25 above, however differs in that the claim further recites the extracellular vesicles are stained with an RNA-specific dye (step c. claim 26), further Green fluorescent or red fluorescent RNA stain (step c. claim 27).
The combination of Oscar et al. in view of Busatto et al., Morales-Kastresana et al. (1) and (2) addresses the method substantially as claimed (see as cited in detail above, it would have been obvious to have modified Oscar et al. for the reasons as indicated in detail above).
Mirmira et al. teach analyzing EVs for miRNA content for diagnostic purposes (predict risk for developing hyperglycemia or type 1 diabetes, see abstract, paras [0004], [0007]).
ThermoFisher teach examples of commercially available fluorescent dyes known for nucleic acid staining (see for example, page 3, referring to cyanine dyes as among the highest affinity fluorescent probes for staining, see further page 2, these probes available in red and green).
It would have been further prima facie obvious to have modified Oscar et al. and the cited prior art in order to target and detect EV related miRNA in order to achieve predictive diagnostics, such as predicting risk for developing hyperglycemia or type 1 diabetes as in Mirmira et al., specifically to have used commercially available, known fluorescent RNA stains, such as the fluorescent green probe or fluorescent red probe as in ThermoFisher, as an obvious matter of applying a known stain for its art intended purpose (i.e., to stain and detect RNA target as in Mirmira).
One having ordinary skill would have had a reasonable expectation of success because Oscar et al. is teaching detection of EVs (as such, one would expect success modifying to detect EVs for another specific purpose, such as predicting risk related to hyperglycemia and diabetes), and further one would expect success modifying the stain for those of ThermoFisher because these stains were specifically known to have high affinity for RNA (ThermoFisher).
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.
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Claims 1-5, 9-10, 12-15, 18-20, 22 and 23 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1, 3-12 and 14-24 of copending Application No. 17/936,027 (reference application). Although the claims at issue are not identical, they are not patentably distinct from each other because:
‘027 recites methods for processing extracellular vesicles (CVDs), comprising a. concentrating in a biological fluid, b. determining a concentration, c. contacting with fluorescent staining dye or antibody, d. incubating to generate a labeled population, e, passing through a centrifugal filter comprising 200-500 kD molecular weight cutoff, polyethersulfone filter media to separate as claimed, and recovering the labeled population (see ‘027 claim 1). See further copending claims 4 and 5, concentrating by tangential flow filter, the method of the copending does not include a step of purifying by chromatography column or other filtration media (other than tangential flow filtration as claimed) prior to step c.
Regarding independent claim 13, see ‘027 at claim 1, further claims 1-22 performed without size exclusion chromatography.
Regarding claim 2, see ‘027 claims 4 and 5.
Regarding claim 3, see ‘027 at claim 5 recites tangential flow filter with a molecular weight cutoff of about 300-750 kD, which is a range overlapping with the claimed range. It would have been prima facie obvious to ne having ordinary skill int eh art before the effective filing date of the claimed invention to have arrived at cutoff molecular weights within the claimed range by selecting and trying those within the disclosed range by ‘027. See MPEP 2144.04, In the case where the claimed ranges "overlap or lie inside ranges disclosed by the prior art" a prima facie case of obviousness exists. In re Wertheim, 541 F.2d 257, 191 USPQ 90 (CCPA 1976); In re Woodruff, 919 F.2d 1575, 16 USPQ2d 1934 (Fed. Cir. 1990).
Regarding claims 4 and 14, see ‘027 at claim 6.
Regarding claims 5 and 15, see ‘027 at claim 7.
Regarding claims 8 and 18 , see ‘027 at claim 8.
Regarding claims 9 and 19, see ‘027 at claim 9.
Regarding claims 10 and 20, see ‘027 at claim 10.
Regarding claim 12 and 22, see ‘027 at claim 11.
Regarding claim 23, see ‘027 at claim 9, determining concentration.
Claims 6 and 16 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1, 3-12 and 14-24 of copending Application No. 17/936,027 (reference application) in view of Skalnikova et al.
‘027 recites a method substantially as claimed (see above); however, ‘027 fails to recite fluorescent staining dye incubated for at least 1 hour at a temperature of about 30-40°C (claims 6 and 16).
Regarding claims 6 and 16, see MPEP 2144.04, Generally, differences in concentration or temperature will not support the patentability of subject matter encompassed by the prior art unless there is evidence indicating such concentration or temperature is critical. "[W]here the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimum or workable ranges by routine experimentation." In re Aller, 220 F.2d 454, 456, 105 USPQ 233, 235 (CCPA 1955).
See further Skalnikova et al., who also teach labeling EVs with CFSE for flow cytometry, see at page 4, last paragraph, teaching incubation time of 60 min at 37°C.
It would have been prima facie obvious to one having ordinary skill in the art before the effective filing date of the claimed invention to have modified methods labeling EVs with CFSE as taught by the combination of the cited art to have arrived at an incubation of 60 min for 37°C out of routine optimization of experimental conditions, namely trying from art recognized incubation conditions (temperatures and times) to uncover the optimum workable conditions. One would have a reasonable expectation of success, considering such conditions were known in the art at the time particularly for EV staining using CFSE (Skalnikova et al.).
Claims 7, 11, 17 and 21 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1, 3-12, and 14-24of copending Application No. 17/936,027 (reference application) in view of Oscar et al.
‘027 recites a method substantially as claimed (see above); however, ‘027 fails to recite antibody incubated for at least 1 hour at a temperature of about 30-40°C (claims 7 and 17).
Regarding claims 7 and 17, as discussed above, see further Oscar who teaches method similar to ‘027, Oscar et al. teach for flow cytometry, the antibody labeled reagent incubated for an hour at room temperature (thereby addressing at least 30 minutes), however fails to teach EVs labeled antibody conjugate incubated at a temperature of about 30-40°C (claim 7 and 17).
Regarding the claimed incubation duration and temperature for forming preformed complex (for combining antibody and fragment), and the claimed antibody concentration, generally, differences in concentration or temperature will not support the patentability of subject matter encompassed by the prior art unless there is evidence indicating such concentration or temperature is critical. "[W]here the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimum or workable ranges by routine experimentation." In re Aller, 220 F.2d 454, 456, 105 USPQ 233, 235 (CCPA 1955). MPEP 2144.05.
In the present case, the general conditions were known in the prior art, differing in terms of the incubation temperature (the prior art teaching a similar temperature that is just outside the claimed range, namely room temperature). Based on MPEP 2144.05 and Oscar et al., it would have been prima facie obvious to one having ordinary skill in the art before the effective filing date of the claimed invention to have arrived at a temperature within 30-40°C by routine optimization of experimental conditions, to uncover the optimum workable conditions for binding and detection, one having ordinary skill would have a reasonable expectation of success because the prior art disclosed temperature (referring to Oscar, who also teaches methods comprising flow cytometry) is similar (room temperature is just below the claimed range, the prior art and the claimed invention teaching temperatures on the same order of magnitude) .
Regarding claims 11 and 21, ‘027 recites a method substantially as claimed (see above); however, ‘027 fails to recite biologic fluid that is conditioned cell growth medium (claims 11 and 21). However, see further Oscar et al. teach detection for EVs from cells consistent with those as claimed (e.g., HEK, etc., see as cited above), Oscar further teaching samples may include cultured growth medium (cited above). It would have been further obvious to have detected in samples such as cultured growth medium as in Oscar as an obvious matter of a known sample fluid in which EVs are targeted for detection. One having ordinary skill would have a reasonable expectation of success because Oscar teach similar methods for detection of EVs in this biological sample type.
Claims 24 and 25 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1, 3-12 and 14-24of copending Application No. 17/936,027 (reference application) in view Morales-Kastresana et al., Labeling Extracellular vesicles for Nanoscale Flow Cytometry, Nature Scientific Reports, 7(1878), (2017), (10 pages).
‘027 recites a method substantially as claimed (see above, substantially addresses claims 24 and 25); however, ‘027 fails to recite dilution of labeled EVs as 1:300 (claims 24 and 25).
Morales-Kastresana et al. further discuss the issue of swarm detection when detecting EVs, see at page 2, para 1 (first full paragraph), teaching to confirm individual EV detection without swarming or coincident event detection, performing serial dilution analysis to determine an operational range where the event rate increase is proportional to the EV concentration (See also Figure 2 caption, E).
It would have been prima facie obvious to one having ordinary skill in the art before the effective filing date of the claimed invention to have modified ‘027 in order to have arrived at a dilution as claimed (such as 1:300), out of routine optimization of experimental conditions, one motivated to perform serial dilutions to uncover the optimum workable range, as in Morales-Kastresana et al. (2), in order to achieve detection without swarm detection or coincidental detection, to uncover the optimum dilution to achieve single event detection for accurate determination of concentration and EV assessment. One having ordinary skill in the art would have had a reasonable expectation of success performing these modifications because based on both Morales-Kastresana references, these are techniques encouraged for performing flow cytometry on EV targets.
Claims 26 and 27 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1, 3-12 and 14-24 of copending Application No. 17/936,027 (reference application) in view Mirmira et al. and ThermoFisher.
‘027 recites a method substantially as claimed (see above); however, ‘027 fails to recite RNA specific dye (claims 26); namely green or red fluorescent RNA stain (claim 27).
Mirmira et al. teach analyzing EVs for miRNA content for diagnostic purposes (predict risk for developing hyperglycemia or type 1 diabetes, see abstract, paras [0004], [0007]).
ThermoFisher teach examples of commercially available fluorescent dyes known for nucleic acid staining (see for example, page 3, referring to cyanine dyes as among the highest affinity fluorescent probes for staining, see further page 2, these probes available in red and green).
It would have been further obvious to have modified the method for detection extracellular vesicles of ‘027 in order to target and detect EV related miRNA in order to achieve predictive diagnostics, such as predicting risk for developing hyperglycemia or type 1 diabetes as in Mirmira et al., specifically to have used commercially available, known fluorescent RNA stains, such as the fluorescent green probe or fluorescent red probe as in ThermoFisher, as an obvious matter of applying a known stain for its art intended purpose (i.e., to stain and detect RNA target as in Mirmira).
One having ordinary skill would have a reasonable expectation of success because ‘027 et al. is teaching detection of EVs (as such, one would expect success modifying to detect EVs for another specific purpose, such as predicting risk related to hyperglycemia and diabetes), and further one would expect success modifying the stain for those of ThermoFisher because these stains were specifically known to have high affinity for RNA (ThermoFisher).
This is a provisional nonstatutory double patenting rejection because the patentably indistinct claims have not in fact been patented.
Response to Arguments
Applicant's arguments filed 05/26/2026 have been fully considered but they are not persuasive for the following reasons:
The previous rejection of claims under 35 U.S.C. 112(b) is withdrawn in response to Applicant’s amendments to the claims.
Additionally, Applicant’s remarks at page 9 are acknowledged, it does appear that para [0038] of the originally filed specification supports Applicant’s position that the specification supports excluding size exclusion chromatography column or other filtration media. As such, no new matter rejection is being made.
Regarding the rejection of claims under 35 U.S.C. 103 (remarks pages 9-11), applicant argues Oscar’s example 1 requires both tangential flow filtration followed by ultrafiltration, the latter constituting an additional filtration step, as is excluded by the claimed invention. However, in response, Applicant’s arguments (through remarks page 11) are not persuasive in light of the new grounds of rejection set forth in detail above (referring further to Bursatto who support that tangential flow filtration alone is considered a sufficient technique for EV concentration in samples).
The remaining arguments (through page 16), refer back to the arguments specific to the exclusion of additional filtration as addressed above. The argument is not persuasive for the reasons as indicated in detail above.
Further no new arguments specific to the rejection on the ground of non-statutory double patenting are presented (referring to remarks pages 16-17, Applicant refers to previous arguments and amendments to the claims). The rejection is maintained as indicated above.
For all of these reasons, Applicant’s arguments are not persuasive.
Correspondence
Any inquiry concerning this communication or earlier communications from the examiner should be directed to ELLEN J MARCSISIN whose telephone number is (571)272-6001. The examiner can normally be reached M-F 8:00am-4:30pm.
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/ELLEN J MARCSISIN/Primary Examiner, Art Unit 1677
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