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.
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Claims 1-27 are pending; claims 1, 13, 24-27 are amended; no claims are withdrawn. Claims 1-27 are examined below.
Claim Rejections - 35 USC § 112
The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
Claims 1-27 are rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
Claim 1 recites at step f. “wherein the extracellular vesicles are not purified via a chromatography column or other filtration media that separates unwanted components from the extracellular vesicles, resulting in purified extracellular vesicles prior to the contacting in c”, however prior to step c., at step a., the claim recites “concentrating extracellular vesicles in a biological fluid, wherein the concentrating comprises centrifugation, tangential flow filtering, or a combination thereof”. Although step a. is a step of concentrating and step f. is a step of recovering, it is the case that at least “tangential flow filtering” is a technique that involves a type of “filtration media that separates” and as such, would be expected (to at least some extent) separate unwanted components from the targeted extracellular vesicles. As a result, the claims are indefinite because the claims both include a step that involves filtration media that separates before step c. and simultaneously exclude steps that involve filtration media that separates before step c.
This rejection similarly applies to claim 13, as step a. recites “a tangential flow filter” and step g. recites “not purified via a chromatography column or other filtration media that separates unwanted components from the extracellular vesicles…prior to the contacting in c; as well as claims 24-27.
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 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).
Oscar et al. teach methods of processing extracellular vesicles, the methods of Oscar demonstrated in examples as 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), followed by electron microscopy and flow cytometry to assess Fc-binding EVs (see page 33, para 4). Although Example 1 cited above teaches tangential flow filtration followed by passing through a 10kD spin filter, see also Oscar et al. at page 25, teaching their methods may comprise EV purification steps prior to affinity capture using methods selected from those presented at page 25, the list including spin filtration and tangential flow filtration, “or any combination thereof” (referring to combinations of techniques as the list at page 25). Therefore, although the reference teaches advantages regarding purity when using techniques in combination, the reference also encompasses using any one technique from those listed at page 25 (the finite list at page 25, which includes, for example tangential flow filtration). Regarding the flow cytometry, 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, 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).
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.
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 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 step a., although Oscar et al. teaches at their examples (for example, Example 1 cited above), relying on combinations of purifying techniques, inclusive of spin filtration and tangential flow filtration (teaching advantages regarding purity when used in combination) prior to affinity capture, Oscar’s invention also encompasses the method performed using any of those purifying techniques recited from the finite list of techniques at page 25, as a result, the method of Oscar encompasses, for example, performing a step of concentrating by performing tangential flow filtration, one having ordinary skill in the art motivated to arrive at tangential flow filtration as an obvious matter to try, namely by selecting from the finite list of suitable alternatives listed by Oscar (referring to page 25, Oscar teaching any of those techniques or combinations thereof).
One having ordinary skill in the art would have had a reasonable expectation of success arriving at tangential flow filtration given that Oscar specifically lists this as a suitable technique and because Oscar’s invention encompasses any one of the techniques in alternative to the combination. See the rejection of claims under 35 U.S.C. 112(b) as set forth in detail above (because tangential flow filtration is considered “other filtration media”); however, in the interest of compact prosecution, assuming it is not Applicant’s intent to include “tangential flow filtration” as a type of “other filtration media”, it is noted that the combination of Oscar et al. and the cited art addresses the claim because the combination of the cited art results in a method comprising, at step a., tangential flow filtration, i.e., not purification by chromatography column or other filtration medium that separates unwanted components, as appears to be claimed.
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., 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., 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., 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., 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 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).
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 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.
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 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 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 12/09/2025 have been fully considered but they are not persuasive for the following reasons.
Regarding the rejection of claims under 35 U.S.C. 112(b) (remarks page 8), Applicant refers to amendments to the claims to recite concentrating comprises centrifugation, tangential flow filtering, or a combination thereof; and purifying comprises use of a chromatography column or other filtration media that separates unwanted components from extracellular vesicles, resulting in purified extracellular vesicles. Applicant asserts that the two processes are distinct. However, while the intention of “tangential flow filtering” is recited as being for concentrating, not purifying, it is the case that it is still a technique that achieves at least some degree of purification (in that it filters/collects), and it would be encompassed by language such as “other filtration media”. As a result, the claim is indefinite because the limitations are contradictory (encompass both a step prior to c comprising “other filtration media”).
Regarding the rejection of claims under 35 U.S.C. 103 (remarks pages 8-9), Applicant argues the combination of the cited art fails to teach all the elements of claims 1 and 13, that the claimed method recites a multi-step method for processing extracellular vesicles, comprising contacting the extracellular vesicles with a fluorescent staining dye or an antibody, wherein the extracellular vesicles are not purified via a chromatography column or other filtration media that separates unwanted components from extracellular vesicles, resulting in purified extracellular vesicles prior to the contacting. Applicant specifically refers to Oscar at Example 1, that discloses an exemplary embodiment comprising EVs isolated from medium using tangential flow filtration, followed by ultrafiltration for concentration, the latter constituting a purification step according to the present claims.
However, this argument is not persuasive in light of the amended grounds of rejection detail above, which also cites page 25 of Oscar, Oscar’s methods encompass using any of those techniques cited at page 25, either alone or in combination. While the exemplary embodiment of Oscar encompasses the combination of tangential flow filtering and spin filtration, the invention of Oscar is not as narrowly limited, and rather encompasses any of those techniques at page 25, either alone or in combination, and as such it would have been obvious to arrive at only tangential flow as an obvious matter to try, namely by selecting from the finite list of suitable alternatives. While the combination at Oscar’s example 1 is an advantageous embodiment, Oscar also encompasses using tangential flow filtration alone, and therefore the combination of the cited art detailed above addresses the claim.
At remarks page 10 Applicant argues that it was surprisingly demonstrated by the inventors that it is not necessary to purify the extracellular vesicles prior to contacting them with the fluorescent dye or antibodies to label the extracellular vesicles. This argument is not persuasive in view of the cited art, particularly Oscar et al. Additionally, although Applicant refers to the tangential flow filtration as contributing to the step of concentrating and not purifying, applying the technique to concentrate EVs (i.e., remove other agents, gather the targeted EVs) is still an act that, to at least some degree, achieves purification.
Applicant assert that traditionally EVs had to be purified from cellular growth media solution, prior to labeling, further asserting that Applicant has determined hat simply concentrating the EVs without purification leads to a sample that can be labeled, then later purified, recovered and analyzed, resulting in high concentration, providing reproducible results (Applicant referencing para [0083], para [0083] description of Fig. 1A-1H, reported as both size exclusion chromatography and NanoSep filtration provided comparable staining, that filtration based methods reduced the time of separation and provided increase in concentration).
Although Applicant’s arguments assert surprising or unexpected results, this argument is also not persuasive. Whether evidence shows unexpected results is a question of fact and the party asserting unexpected results has the burden of proving that the results are unexpected. In re Geisler, 116 F.3d 1465, 1469-70, 43 USPQ2d 1362, 1364-5 (Fed. Cir. 1997). The evidence must be (1) commensurate in scope with the claimed subject matter, In re Clemens, 622 F.2d 1019, 1035, 206 USPQ 289, 296 (CCPA 1980), (2) show what was expected, to "properly evaluate whether a … property was unexpected", and (3) compare to the closest prior art. Pfizer v. Apotex, 480 F.3d 1348, 1370-71, 82 USPQ2d 1321, 1338 (Fed. Cir. 2007).
The burden of demonstrating unexpected results rests on the party asserting them, and “it is not enough to show that results are obtained which differ from those obtained in the prior art: that difference must be shown to be an unexpected difference.” In re Klosak, 455 F.2d 1077, 1080 (CCPA 1972). Moreover, it has been long held that “even though applicant’s modification results in great improvement and utility over the prior art, it may still not be patentable if the modification was within the capabilities of one skilled in the art, unless the claimed ranges ‘produce a new and unexpected result which is different in kind and not merely in degree from the results of the prior art.” In re Huang, 100 F.3d 135, 139 (Fed. Cir. 1996) (quoting In re Aller, 220 F.2d 454, 456 (1955), and citing In re Woodruff, 919 F.2d 1575, 1578 (Fed. Cir. 1990)).
In the present case, Applicant has not provided any evidence to support that the observed difference is an unexpected difference (there is no evidence as to why the results may be characterized as unexpected or surprising). For example, regarding Oscar, it is not necessarily surprising that performing one technique for concentrating EVs as compared to performing a combination of different techniques would result in less time (two or more techniques would be expected to take longer, time to perform each). Even further, the combination of the cited art establishes obviousness, the modification was well within the capabilities of one skilled in the art.
At remarks pages 10-15, specific to further dependent and additional other independent claim limitations, refer to the arguments above specific to the independent claims (claims 1 and 13). The arguments are as addressed in detail above.
Regarding the rejection of claims on the ground of non-statutory double patenting, Applicant refers to amendments to the claims in order to overcome the rejection. However, see the rejection as set forth in detail above, the rejection is maintained.
Conclusion
Applicant's amendment necessitated the new ground(s) of rejection presented in this Office action. Accordingly, THIS ACTION IS MADE FINAL. See MPEP § 706.07(a). Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action. In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any nonprovisional extension fee (37 CFR 1.17(a)) pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action. In no event, however, will the statutory period for reply expire later than SIX MONTHS from the mailing date of this final action.
Correspondence
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/ELLEN J MARCSISIN/Primary Examiner, Art Unit 1677