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 .
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and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114.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 04/03/2026 has been entered.
Status of the Claims
Claims 1-3 and 10-11 are pending. Claims 12, 18, and 19 are withdrawn. Claims 1 and 11 are amended. Accordingly, claims 1-3 and 10-11 are examined herein.
Priority
The present application, filed 04/03/2026 is an RCE of U.S. Patent Application 17/788,429 filed, 06/23/2022, which is a 371 of PCT/JP2020/048510, filed 12/24/2020, which claims foreign priority of JP2019-238075 and JP2019-238071, filed 12/27/2019.
Information Disclosure Statement
The Information Disclosure Statement(s) filed 02/02/2026 are acknowledged and have been considered.
Maintained Claim Rejections - 35 USC § 103
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. Considering objective evidence present in the application indicating obviousness or nonobviousness.
This application currently names joint inventors. In considering patentability of the claims the examiner presumes that the subject matter of the various claims was commonly owned as of the effective filing date of the claimed invention(s) absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and effective filing dates of each claim that was not commonly owned as of the effective filing date of the later invention in order for the examiner to consider the applicability of 35 U.S.C. 102(b)(2)(C) for any potential 35 U.S.C. 102(a)(2) prior art against the later invention.
Claims 1-3 and 10-11 are rejected under 35 U.S.C. 103 as being unpatentable Daaboul et al., International Publication No. WO 2020/160402 A1 in view of Golden et al., International Publication No. WO 2021/062057 A1 and Amidzadeh et al. (Amidzadeh, Zahra, et al. "Assessment of different permeabilization methods of minimizing damage to the adherent cells for detection of intracellular RNA by flow cytometry." Avicenna journal of medical biotechnology 6.1 (2014): 38.).
Regarding claims 1 and 10, Daaboul discloses a method of detecting a particular protein contained in an extracellular vesicle (para. [0039]), the method comprising the steps of:
(A) capturing the extracellular vesicle using a carrier capable of binding to an extracellular-vesicle-specific marker present on the surface of the extracellular vesicle (paras. [0039] and [0049]);
(B) carrying out membrane permeabilization treatment for the extracellular vesicle captured by the carrier, using a membrane permeabilization treatment agent (para. [0039]);
(C) introducing a reagent capable of detecting the particular protein contained in the extracellular vesicle, into the membrane-permeabilized extracellular vesicle (paras. [0014], [0039] and [0052]), and
wherein the Step (B) is carried out such that the particular protein does not leak to the outside of the extracellular vesicle, and such that the reagent can be introduced into the extracellular vesicle (paras. [0047] and [0048]).
While Daaboul does not explicitly say that the proteins do not leak out of the vesicle, it is disclosed that damaging and/or rupturing the membrane is avoided and the integrity of the membrane is maintained, thereby suggesting that the proteins are not able to leak outside of the vesicle. Further, the method disclosed by Daaboul is the same as that claimed in the instant application and contains the same parameters: capturing the vesicle using a carrier that can specifically bind to a surface marker (i.e., magnetic beads (para. [0039])), permeabilizing the membrane using a permeabilization reagent comprising an organic solvent (i.e., acetone (para. [0014])) and/or a detergent (i.e., saponin (para. [0014])) at specified concentrations, and introducing a reagent capable of detecting a particular protein contained in the extracellular vesicle (i.e., ALIX (para. [0050)). Because the methods are the same, it can be concluded that the limitation that the proteins do not leak outside of the vesicle while allowing detection reagents to enter is an inherent characteristic of the method disclosed. Daaboul also discloses that the membrane permeabilization treatment agent is a surfactant and/or an organic solvent such as ethanol or acetone (para. [0014]).
Daaboul discloses the method as detailed above, and teaches an organic solvent but is silent on the concentration of the organic solvent used for permeabilization.
However, Golden teaches cargo loading of a vesicle (e.g., an extracellular vesicle (para. [0072]), utilizing a membrane destabilizing agent to permeabilize the vesicle (para. [0005] and [00155])). Golden teaches that the membrane destabilizing agent used to permeabilize the vesicle membrane can be an organic solvent such as acetone (para [00156]) or ethanol (para. [00157]). Golden teaches that the membrane destabilizing organic solvent can be present at a concentration between 0.1% to 50% (v/v), preferably about 0.5% to 30% v/v, more preferably about 5% to 20% v/v, etc. (para. [00159]), which overlaps the concentration ranges of ethanol and acetone in amended claim 1.
Amidzadeh establishes that ranges of reagent concentrations for effective membrane permeabilization are result effective variables, i.e., a variable which achieves a recognized result (see MPEP § 2144.05), that are selected in order to achieve a desirable result for detecting contents enclosed within a membrane (page 39, column 1, paragraph 3). Further, Amidzadeh teaches that one skilled in the art can adjust the reagent concentrations, exposure times, and temperatures in order to optimize permeabilization extent depending on cell or membrane type and intended target (page 39, column 1, paragraph 3).
It would have been prima facie obvious to one of ordinary skill in the art at the time the claimed invention was effectively filed to have modified Daaboul in order to have arrived at the instantly claimed concentration values (i.e., values within the claimed ranges) for the organic solvent (as taught by Daaboul) as an obvious matter of routine optimization of experimental conditions. In particular, Daaboul recognized that organic solvent can be used as a permeabilization agent, Golden teaches concentration ranges that overlap a substantial portion of the claimed organic solvent range for the permeabilization of vesicle membranes, and Amidzadeh supports that permeabilization reagent concentration level is a result effective variable (i.e., a variable which achieves a recognizable result, which is permeabilization of membrane). In the case where the claimed ranges overlap or lie inside ranges disclosed by the prior art, a prima facie case of obviousness exists, see MPEP 2144.05 regarding overlap of ranges, and MPEP 2131.03 regarding anticipation of ranges. In the present case, the claimed range (20-50% v/v or 20%-70% v/v) lies inside the prior art taught range of .1-50% v/v). One having ordinary skill in the art would have found it obvious to have selected from values within the prior art disclosed range in order to determine the optimum/workable conditions for permeabilization because it is obvious to choose from a finite number of identified, predictable solutions with a reasonable expectation of success as the concentration range was recognized in the art to be a result-effective variable that may be adjusted by the investigator, specific to a particular membrane depending on the size and composition of the extracellular vesicle, it would have been obvious to optimize such values in order to achieve a desired level of permeabilization and/or to achieve an optimum value. For this reason, because determining appropriate reagent concentration values is well within the skill level of the ordinary artisan (as supported by Amidzadeh), one having ordinary skill would have had a reasonable expectation of success optimizing the values to arrive at values within the claimed ranges through routine experimentation during efforts to uncover the optimal/desired concentration of organic solvent to achieve the proper amount of permeabilization since the prior art already disclosed ranges substantially similar to, and overlapping the claimed range, intended for the same purpose as Daaboul (the purpose of permeabilization).
Regarding claim 2, Daaboul discloses that extracellular vesicles are washed after being captured by the surface antigen-specific carrier to remove unbound or non-specifically bound particles (para. [0254]), corresponding the Step (A) of the present application. The captured extracellular vesicles were also washed after permeabilization (para. [0257]), corresponding to Step (B) of the present application. Therefore, Daaboul discloses the method of claim 1, further comprising a step of removing an impurity after the Step (A) and/or after the Step (B).
Regarding claim 3, Daaboul discloses a step of detecting the particular protein using the introduced reagent after the Step (C). Specifically, Daaboul discloses contacting the vesicles with one or more fluorescent cargo labels (e.g., antibody) wherein each fluorescent cargo label is specific to a particular biomolecule of interest (e.g., protein) potentially present in at least a portion of the vesicles (para. [0163]). The fluorescent label bound to the target protein is then excited with a light source and one or more detectors then detect the fluorescent light emitted from the fluorescent cargo label, allowing for detection and/or quantification of the biomolecule of interest present within the vesicle (para. [0163]).
Regarding claim 11, Daaboul discloses the method of claim 1, wherein the carrier is a magnetic particle (para. [0039]).
The rejection below under 35 U.S.C. 103 has been updated in view of Applicant’s amendments to claim 1. In particular, claim 1 has been amended to recite that “the carrier is a particle.” In response to this amendment, Fujii et al. has been applied to teach the use of particles, including magnetic particles, as carriers for capturing extracellular vesicles. The modification reflects the inclusion of an additional known element performing the same function, and does not alter the underlying rationale of the rejection.
Claims 1-3 and 10-11 are rejected under 35 U.S.C. 103 as being unpatentable over Unlu et al., WO 2017/053516 A1 in view of Golden et al., WO 2021/062057 A1, Amidzadeh et al., and Fujii et al., US Patent No. 10,139,402 B2.
Regarding claims 1 and 10-11, Unlu discloses a method of detecting a particular protein contained in an extracellular vesicle (para. [0127] and [0204]), the method comprising the steps of:
(A) capturing the extracellular vesicle using a carrier capable of binding to an extracellular-vesicle-specific marker present on the surface of the extracellular vesicle (para. [0120] and [0204], captured on a sensor using an extracellular vesicle-specific probe, e.g., an antibody, peptide, aptamer);
(B) carrying out membrane permeabilization treatment for the extracellular vesicle captured by the carrier, using a membrane permeabilization treatment agent (para. [0128] and [0205], following capture, treated with an organic solvent or detergent to permeabilize);
(C) introducing a reagent capable of detecting the particular protein contained in the extracellular vesicle, into the membrane-permeabilized extracellular vesicle (para. [127] and [0205]-[0206], stained using for example, labeled antibody, aptamer or peptides), and
wherein the Step (B) is carried out such that the particular protein does not leak to the outside of the extracellular vesicle, and such that the reagent can be introduced into the extracellular vesicle (paras. [0059] and [0209] and FIG.6). Although Unlu does not explicitly state that the protein does not leak out of the vesicle, they disclose a fixation step before permeabilizing (para. [0205]) that keeps the membrane intact for intra-vesicular staining.
Unlu teaches an organic solvent that is ethanol or acetone, but is silent on the concentration of organic solvent used for permeabilization.
However, Golden teaches cargo loading of a vesicle (e.g., an extracellular vesicle (para. [0072]), utilizing a membrane destabilizing agent to permeabilize the vesicle (para. [0005] and [00155])). Golden teaches that the membrane destabilizing agent used to permeabilize the vesicle membrane can be an organic solvent such as acetone (para [00156]) or ethanol (para. [00157]). Golden teaches that the membrane destabilizing organic solvent can be present at a concentration between 0.1% to 50% (v/v) (para. [00159]), preferably about 0.5% to 30% v/v, more preferably about 5% to 20% v/v, etc. (para. [00159]), which overlaps the concentration ranges of both ethanol and acetone cited in claim 1.
Amidzadeh establishes that ranges of reagent concentration for effective membrane permeabilization is a result effective variable, i.e., a variable which achieves a recognized result (see MPEP § 2144.05), that are selected in order to achieve a desirable result for detecting contents enclosed within a membrane (page 39, column 1, paragraph 3). Further, Amidzadeh teaches that one skilled in the art can adjust the reagent concentrations, exposure times, and temperatures in order to optimize permeabilization extent depending on cell or membrane type and intended target (page 39, column 1, paragraph 3).
Fujii also teaches a similar method of detecting a particular protein contained in an extracellular vesicle (col. 12, lines 10-15), the method comprising the steps of:
(A) capturing the extracellular vesicle using a carrier capable of binding to an extracellular-vesicle-specific marker present on the surface of the extracellular vesicle (col. 7, lines 43-50);
(B) carrying out membrane permeabilization treatment for the extracellular vesicle captured by the carrier, using a membrane permeabilization treatment agent (col. 22, lines 52-60); and
(C) introducing a reagent capable of detecting the particular protein contained in the extracellular vesicle, into the membrane-permeabilized extracellular vesicle (col. 22, line 64 – col. 23, line 15). Fujii further teaches that the carrier is a magnetic particle (col. 9, lines 9-23 and col. 22, lines 31-34), which is a type of particle.
It would have been prima facie obvious to one of ordinary skill in the art at the time the claimed invention was effectively filed to have modified Unlu to utilize a particle, such as a magnetic particle as taught by Fujii, as the carrier for capturing extracellular vesicles, and further to have arrived at the instantly claimed concentration values (i.e., values within the claimed ranges) for the organic solvent as an obvious matter of routine optimization of experimental conditions. In particular, Unlu recognized that organic solvent can be used as a permeabilization agent, Golden teaches concentration ranges that overlap a substantial portion of the claimed organic solvent range for the permeabilization of vesicle membranes, Amidzadeh supports that permeabilization reagent concentration level is a result effective variable (i.e., a variable which achieves a recognizable result, which is permeabilization of membrane). First, Unlu’s method and Fujii’s method both share the goal of isolating and separating extracellular vesicles for the subsequent determination of proteins contained within, however use slightly different techniques to achieve this. While Unlu uses an immobilized substrate, Fujii uses magnetic particles that can be manipulated by an applied magnetic field. One would be motivated to modify the immobilized substrate taught by Unlu with magnetic particles taught by Fujii because “unreacted substances or impurities in a biological sample can be more efficiently washed and separated, and removed from a surface of magnetic particles” (col. 11, lines 22-31). One would have a reasonable expectation of success by using magnetic particles because both techniques result in the same goal of capturing an extracellular vesicle that is subsequently assayed for protein contents.
Second, in the case where claimed ranges overlap or lie inside ranges disclosed by the prior art, a prima facie case of obviousness exists, see MPEP 2144.05 regarding overlap of ranges, and MPEP 2131.03 regarding anticipation of ranges. In the present case, the claimed range (20-50% v/v or 20%-70% v/v) lies inside the prior art taught range of .1-50% v/v). One having ordinary skill in the art would have found it obvious to have selected from values within the prior art disclosed range in order to determine the optimum/workable conditions for permeabilization because it is obvious to choose from a finite number of identified, predictable solutions with a reasonable expectation of success as the concentration range was recognized in the art to be a result-effective variable that may be adjusted by the investigator, specific to a particular membrane depending on the size and composition of the extracellular vesicle, it would have been obvious to optimize such values in order to achieve a desired level of permeabilization and/or to achieve an optimum value. For this reason, because determining appropriate reagent concentration values is well within the skill level of the ordinary artisan (as supported by Amidzadeh), one having ordinary skill would have had a reasonable expectation of success optimizing the values to arrive at values within the claimed ranges through routine experimentation during efforts to uncover the optimal/desired concentration of organic solvent to achieve the proper amount of permeabilization since the prior art already disclosed ranges substantially similar to, and overlapping the claimed range, intended for the same purpose as Unlu (the purpose of permeabilization).
Regarding claim 2, Unlu teaches the method of claim 1, further comprising a step of removing impurities after Step (A) (para. [0126], contacting captured vesicles with probe and washing unbound probe away, see for example para [0038] of the originally filed specification, this step of washing away as taught by the art is consistent with Applicant’s meaning of the limitation “removing impurity”, see Applicant’s specification indicates “impurity” may mean substances not captured on the carrier).
Regarding claim 3, Unlu teaches the method of claim 1, further comprising a step of detecting the particular protein using the introduced reagent after Step (C) (para. [0209], step (vi), measuring fluorescence of the introduced probe).
For the reasons stated above, all claims are rejected.
Response to Arguments
Applicant’s arguments filed 04/03/2026 have been fully considered but are not persuasive for the reasons set forth below.
Arguments Regarding Daaboul in view of Golden and Amidzadeh
First, Applicant argues that Golden’s disclosed range (0.1–50% v/v) does not represent specific operative concentrations and instead teaches preferred lower ranges (8-12% v/v), and that one of ordinary skill in the art would instead be led to the preferred lower range. This argument is not persuasive. Golden explicitly discloses that an organic solvent may be present at a concentration of about 0.1–50% v/v. It is well established that a prior art reference is relevant for all that it teaches, including nonpreferred embodiments, and is not limited to preferred embodiments (see MPEP § 2123). The mere fact that a reference identifies a preferred range does not negate or disclaim the broader disclosed range. Therefore, one of ordinary skill in the art would have reasonably considered the entire disclosed range, including values within the claimed range (e.g., 20–50%).
Second, Applicant argues that Golden does not disclose particle-based capture and therefore does not provide guidance for solvent concentrations in such systems. This argument is not persuasive. Golden is relied upon for teaching solvent concentration ranges for permeabilization, not for the specific carrier format. The selection of solvent concentration relates to the permeabilization of vesicle membranes, which is independent of the specific physical form of the carrier used to capture the vesicles. The use of different known carrier formats does not alter the fundamental chemical interaction between solvent and membrane. Furthermore, it is well established that the substitution of one known element for another performing the same function is an obvious design choice. As discussed in the rejection, particle-based capture is taught by the art (e.g., Daaboul), and applying known permeabilization conditions to vesicles captured by such carriers would have been within the level of ordinary skill.
Third, Applicant argues that Amidzadeh is limited to HeLa cells and does not disclose extracellular vesicles or combinations with carriers. This argument is not persuasive. Amidzadeh is not relied upon for teaching extracellular vesicles or carrier systems. Rather, Amidzadeh is relied upon for establishing that reagent concentration is a result-effective variable that may be optimized. This teaching is not limited to a specific cell type, but instead reflects a general principle applicable to membrane permeabilization processes. The applicability of such general principles across different biological membrane systems would have been recognized by one of ordinary skill in the art.
Fourth, Applicant argues that the cited references merely suggest general optimization without providing sufficient guidance to arrive at the claimed ranges. This argument is not persuasive. Golden provides a specific numerical range (0.1–50% v/v) for organic solvent concentration. This constitutes a finite set of identified, predictable solutions. Where such a range is disclosed, selecting values within that range to achieve a desired result is considered routine optimization. As stated in MPEP § 2144.05, where claimed ranges overlap or lie inside ranges disclosed by the prior art, a prima facie case of obviousness exists. Additionally, Amidzadeh explicitly teaches that such parameters are routinely adjusted to achieve optimal permeabilization.
Lastly, Applicant argues that the claimed solvent concentrations produce unexpected results, including suppression of aggregation and improved detection efficiency, particularly in particle-based systems. This argument is not persuasive. The alleged advantages relied upon by Applicant (e.g., suppression of aggregation, improved detection efficiency, or reduced reaction inefficiency) are not recited in the claims. The claims are directed to a method defined by process steps and solvent concentration ranges, but do not require any specific performance outcome. Accordingly, these asserted advantages are not commensurate in scope with the claims and cannot establish patentability. Furthermore, where a claimed invention differs from the prior art only in the selection of a value of a result-effective variable, optimization of that variable is considered to produce predictable results. As discussed above, Amidzadeh teaches that solvent concentration affects permeabilization, and therefore any improvement resulting from adjusting this parameter would have been expected.
Arguments Regarding Unlu in view of Golden and Amidzadeh
Applicant presents arguments similar to those above with respect to Unlu. These arguments are not persuasive for the same reasons discussed above. Additionally, although Unlu discloses capture of extracellular vesicles using a sensor substrate, the use of alternative known capture formats, including particle-based carriers, is well known in the art (e.g., Fujii) and represents a substitution of one known element for another performing the same function. As such, the application of known permeabilization conditions to vesicles captured using different carrier formats would have been obvious.
Arguments Regarding Claim 11 (Unlu in view of Golden, Amidzadeh, and Fujii)
Applicant argues that Fujii does not disclose membrane permeabilization using ethanol or acetone and instead addresses aggregation using surfactants. This argument is not persuasive. Fujii is not relied upon for teaching solvent-based permeabilization. Rather, Fujii is relied upon solely for teaching that extracellular vesicles may be captured using magnetic particles, as recited in claim 11. A rejection under 35 U.S.C. 103 may properly rely on different references for different claim limitations. The fact that Fujii addresses a different aspect (e.g., aggregation mitigation using surfactants) does not negate its teaching of particle-based carriers. Furthermore, the combination of Unlu, Golden, Amidzadeh, and Fujii collectively teaches all limitations of the claims, and the substitution of a particle-based carrier as taught by Fujii represents the use of a known technique to improve or facilitate vesicle isolation.
Lastly, Applicant repeats arguments regarding aggregation and improved detection efficiency. These arguments are not persuasive for the reasons discussed above, as such alleged advantages are not recited in the claims and are not commensurate in scope.
Ultimately, for the reasons set forth above, Applicant’s arguments have been fully considered but are not persuasive, and the rejection of claims 1–3 and 10–11 under 35 U.S.C. 103 is therefore maintained.
Conclusion
No claims are allowable.
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/E.O./Examiner, Art Unit 1677
/BAO-THUY L NGUYEN/Supervisory Patent Examiner, Art Unit 1677 April 28, 2026
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