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 .
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and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114.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 04/22/2026 has been entered.
Claims 1, 3-12 and 14-24 are pending in this application and were examined on their merits.
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.
Claims 1, 3, 6, 7, 8, 10 and 23 are rejected under 35 U.S.C. § 103 as being unpatentable over Fortunato et al. (2021), cited in the IDS, in view of Jang et al.
(2013), of record, and Cao et al. (2021).
Fortunato et al. teaches a method comprising:
concentrating cell-derived, secreted extracellular vesicles (sEV) in a biological fluid (HT29 or HEK293 cell conditioned medium/CCM) by centrifugal ultrafiltration (Pg. 15, Paragraph 4.1);
determining a concentration of the sEVs to be 1x10⁷ to 1x10⁸ particles/µl (or 1x10¹⁰ to 1x1011 particles/ml) (Pg. 15, Paragraph 4.1);
incubating the sEVs with various stains including CFSE or anti-CD9, anti-CD63 and anti-CD81 antibodies to generate a labeled sEV population (Pgs. 15-16, Paragraph 4.2.1 and Pg. 16, Paragraph 4.2.2.);
passing the stained sEVs though an ultrafiltration centrifugal device (NANOSEP® 300K, having a molecular weight cutoff of 300 Kda polyethersulfone filter media (see the Specification as published at Pg. 4, Paragraph [0036]);
and analyzing/recovering the labeled sEVs (Pgs. 15-16, Paragraph 4.2.1 and Pg. 4, Fig. 1), and reading on Claims 1, 6, 7, 8 and 10.
The teachings of Fortunato et al. were discussed above.
Fortunato et al. did not teach a method comprising preparing a suspension of nucleated mammalian cells and conducting a serial extrusion of the nucleated cells by sequentially passing them through filters with diminishing micro-size pores to produce a biological fluid comprising CDVs retaining the same membrane topology as that of the nucleated mammalian cells, as required by Claim 1;
wherein the serial extrusion comprises serially passing the nucleated mammalian cells through membrane filters with a pore size of about 10 µm, about 3 µm, and about 0.4 µm, as required by Claim 3;
or wherein the CDVs comprise a therapeutic agent, as now required by Claim 23.
Jang et al. teaches a method wherein human umbilical vein cells (HUVECs) are suspended in chemotherapeutics and then sequentially extruded three times through 10, 5 and 1 µm polycarbonate membrane filters to produce a biological fluid comprising chemotherapeutic loaded nanovesicles (CDVs), which is then ultracentrifuged to obtain the chemotherapeutic loaded nanovesicles (Pg. 7707, Column 1, Lines 1-3 and 18-28 and Pg. 7699, Fig. 1A).
The Jang reference further teaches that:
…most mammalian cells release relatively low quantities of exosomes and purification of exosomes is cumbersome which results in a relatively low yield. Therefore, the generation of exosome-mimetic vesicles with a substantially greater yield is attractive for the development of future nanosized drug delivery systems. Here we developed bioengineered and bioinspired cell-derived nanocarriers, coined as exosome-mimetic nanovesicles (NV), to combine the characteristics of cells and nanocarriers and applied to targeted delivery of anticancer chemotherapeutics. By subjecting cells of different origin to serial extrusion through filters with diminishing pore sizes after the cells had been loaded with chemotherapeutic agents, we generated high quantities of exosome-mimetic NV carrying sheltered drugs (Pg. 7699, Column 2, Lines 11-26).
Cao et al. teaches that cell derived nanovesicles (CDVs) can be prepared by serial extrusion of a homogenate of HEK293 cells through 1 µm, 400 nm and 200 nm filters followed by ultracentrifugation (Pg. 3, Fig. 1 and Pg. 10, Column 1, Lines 1-25).
It would have been obvious to those of ordinary skill in the art before the effective filing date of the claimed invention to modify the method of Fortunato et al. of isolating labeled sEV/exosomes from HEK293 cells with the method of Jang et al. and Cao et al. of producing isolated chemotherapeutic loaded nanovesicles (CDVs) because this would produce isolated, labeled, chemotherapeutically loaded CDVs. Those of ordinary skill in the art would have been motivated to make this modification because Jang et al. teaches the method generates high quantities of exosome-mimetic nanovesicles (CDVs) carrying sheltered drugs.
There would have been a reasonable expectation of success in making this modification because all of the references are reasonably drawn to the same field of endeavor, that is, the isolation of useful cell derived vesicles.
With regard to the limitation of Claim 1, “wherein the CDVs comprise one or more CDV-enriched protein markers selected from: lysosome membrane protein (SCARB2); lysosome-associated membrane glycoprotein 1 (LAMP1); lysosome-associated membrane glycoprotein 2 (LAMP2); nicastrin (NCSTN); Ras-related protein Rab-7a (RAB7A); kinectin (KTN1); sodium/potassium transporting ATPase subunit beta-3 (ATP1B3); basigin (BSG); and integrin beta-1 (ITGB1)”, the Examiner notes that the instant Specification obtains CDVs/exosomes from HEK293 cells at Pgs. 13-14, Paragraph [0044] (which are the same cells used by Fortunato to obtain sEVs/exosomes) and determining CDV/exosome markers thereof (Pg. 14, Paragraph [0049]-[0050]). As Fortunato teaches the same cell type to obtain secreted extracellular vesicles/sEVs/exosomes as the claimed invention, Jang et al. teaches serial extrusion of cells to prepare cell-derived vesicles and Cao et al. teaches that cell-derived nanovesicles can be derived from HEK293 cells, the prior art obtained/enriched sEVs/exosomes/CDVs would be expected to comprise the same CDV-enriched protein markers as claimed.
With regard to the limitation of Claim 1, that the "CDVs retaining the same
membrane topology as that of the nucleated mammalian cells", this is an inherent
feature of the serial extrusion of nucleated mammalian cells through filters with diminishing micro-size pores. As Jang et al. teaches the same process using the same cells as claimed, the same characteristics and properties of the obtained CDVs would be expected.
With regard to Claim 3, the claimed membrane filters with a pore size of about 10
µm, about 3 µm, and about 0.4 µm are made prima facie obvious by the similar sized
10, 5 and 1 µm polycarbonate membrane filters of the prior art such that those of skill in
the art would expect them to have the same properties, particularly as the Specification
as published at Pg. 1, Paragraph [0011] states that the term "about" can encompass up
to an approximately 20% difference. See the MPEP at 2144.05, I.
Claims 1, 3, 4, 5, 6, 7, 8, 10 and 23 are rejected under 35 U.S.C. § 103 as
being unpatentable over Fortunato et al. (2021), cited in the IDS, in view of Jang et al. (2013), of record, and Cao et al. (2021), as applied to Claims 1, 3, 6, 7, 8, 10 and 23 above, and further in view of Gibbings (US 2021/0163550 A1), of record.
The teachings of Fortunato et al., Jang et al. and Cao et al. were discussed above.
Fortunato et al. did not teach a method wherein the concentrating comprises
passing the biological fluid through a tangential flow filter (TFF), as required by Claim 4;
or wherein the TFF has a molecular weight cutoff of about 300-750 kD, as
required by Claim 5.
Gibbings teaches that CDV/exosomes can be enriched (e.g. concentrated) using
size-based filtration using tangential flow filtration or centrifugal filtration (Pg. 20,
Paragraph [0185]).
It would have been obvious to those of ordinary skill in the art to modify the method of Fortunato et al. and Jang et al. comprising concentrating labeled, secreted extracellular vesicles (CDVs) comprising therapeutic agents in a biological fluid by centrifugal ultrafiltration with an ultrafiltration centrifugal device (NANOSEP® 300K, having a molecular weight cutoff of 300 Kda polyethersulfone filter media (see the Specification as published at Pg. 4, Paragraph [0036]) to use TFF with the same molecular weight cutoff as the concentration method because Gibbings teaches that TFF and the centrifugal ultrafiltration method of Fortunato are art-recognized equivalent techniques for concentration exosomes from a sample and Fortunato teaches a particular desired molecular weight cutoff value. See the MPEP at 2144.06, II. Those of ordinary skill in the art would have been motivated to make this modification based on the availability of filters and artisan preference.
There would have been a reasonable expectation of success in making this modification because the art recognizes both filtering techniques as being suitable for the same purpose and Fortunato already teaches a 300 kDa molecular weight cutoff.
Claims 1, 3, 6, 7, 8, 9, 10 and 23 are rejected under 35 U.S.C. § 103 as being unpatentable over Fortunato et al. (2021), cited in the IDS, in view of Jang et al. (2013), of record, and Cao et al. (2021), as applied to Claims 1, 3, 6, 7, 8, 10 and 23 above, and further in view of Doherty et al. (US 2021/092193 A1), of record.
The teachings of Fortunato et al., Jang et al. and Cao et al. were discussed above.
None of the above references taught a method wherein the concentration of CDVs in the biological fluid is determined using a flow cytometer, as required by Claim 9.
Doherty et al. teaches determining the concentration of exosomes by counting the number of complexes in a population by microscopy, flow cytometry or hemacytometry (Pgs. 43-44, Paragraph [0149]).
It would have been obvious to those of ordinary skill in the art at the time of the instant invention to modify the method of Fortunato et al. and Jang et al. whom teach determining the concentration of exosomes by an unspecified method to use the flow cytometry method of Doherty et al. because the reference teaches flow cytometry as a suitable method for determining exosome concentration. Those of ordinary skill in the art would have been motivated to make this modification because Fortunato is silent with regard to the method used to determine exosome concentration and Doherty et al. teaches specific methods for doing so. There would have been a reasonable expectation of success because all of the references are reasonably drawn to the same field of endeavor, that is, exosomes/extracellular vesicles.
Claims 1, 3, 6, 7, 8, 10, 11 and 23 are rejected under 35 U.S.C. § 103 as being unpatentable over Fortunato et al. (2021), cited in the IDS, in view of Jang et al. (2013), of record, and Cao et al. (2021), as applied to Claims 1, 3, 6, 7, 8, 10 and 23 above, and further in view of Larocca et al. (WO 2021/151029), of record.
The teachings of Fortunato et al., Jang et al. and Cao et al. were discussed above.
None of the above references taught a method wherein the labeled CDVs are passed though the centrifugal filter for at least 10 minutes at a centrifugal force of at least 10,000 g, as required by Claim 11.
Larocca et al. teaches a method wherein exosomes are isolated using a centrifugal filter at a centrifugal force of 10,000 g for 15 minutes (Pg. 23, Paragraph [0140]).
It would have been obvious to those of ordinary skill in the art at the time of the instant invention to modify the method of Fortunato et al. and Jang et al. whom teaches isolating labeled, chemotherapeutic loaded CDVs with centrifugal ultrafiltration for an unspecified time and force to use the particular operating parameters for centrifugal filtration taught by Larocca et al. because Larocca teaches a particular force and time as a suitable for exosome isolation/recovery. Those of ordinary skill in the art would have been motivated to make this modification because Fortunato is silent with regard to the time and force used to recover/isolate exosomes by centrifugal filtration and Larocca et al. teaches specific operating parameters for doing so. There would have been a reasonable expectation of success because all of the references are reasonably drawn to the same field of endeavor, that is, exosomes/extracellular vesicles.
Claims 1, 3, 6, 7, 8, 10, 12, 13, 14, 15, 16, 17, 18, 19, 22, 23 and 24 are rejected under 35 U.S.C. § 103 as being unpatentable over Fortunato et al. (2021), cited in the IDS, in view of Jang et al. (2013), of record, and Cao et al. (2021), as applied to Claims 1, 3, 6, 7, 8, 10 and 23 above, and further in view of Gibbings (US 2021/0163550 A1) and Doherty et al. (WO 2021/092192), both of record.
The teachings of Fortunato et al., Jang et al. and Cao et al. were discussed above. The Examiner notes that the rationale for a finding of obviousness for the new limitations of Claim 1, the limitations of Claim 3 and the limitations of Claim 23 are applicable to the new limitations of Claim 12, the limitations of Claim 14 and the limitations of Claim 24.
None of the above references taught a method wherein the CDVs are concentrated with a tangential flow filter and analyzing the recovered, labeled CDV population using a flow cytometer, as required by Claim 12;
or wherein the concentration of CDVs in the sample is determined using a flow cytometer, as required by Claim 18.
Gibbings teaches that CDV/exosomes can be enriched (e.g. concentrated) using size-based filtration using tangential flow filtration or centrifugal filtration (Pg. 20, Paragraph [0185]).
Doherty et al. teaches determining the concentration of exosomes by counting the number of complexes in a population by microscopy, flow cytometry or hemacytometry (Pgs. 43-44, Paragraph [0149]).
It would have been obvious to those of ordinary skill in the art to modify the method of Fortunato et al. and Jang et al. comprising concentrating secreted extracellular vesicles (sEV) comprising chemotherapeutic agents in a biological fluid by centrifugal ultrafiltration with an ultrafiltration centrifugal device (NANOSEP® 300K, having a molecular weight cutoff of 300 Kda polyethersulfone filter media (see the Specification as published at Pg. 4, Paragraph [0036]) to use TFF as the concentration method because Gibbings teaches that TFF and the centrifugal ultrafiltration method of Fortunato and Jang are art-recognized equivalent techniques for concentration exosomes from a sample. See the MPEP at 2144.06, II. Those of ordinary skill in the art would have been motivated to make this modification based on the availability of filters and artisan preference. There would have been a reasonable expectation of success in making this modification because the art recognizes both filtering techniques as being suitable for the same purpose.
It would have been further obvious to those of ordinary skill in the art at the time of the instant invention to modify the method of Fortunato et al., Jang et al. and Gibbings whom teach determining the concentration of exosomes by an unspecified method to use the flow cytometry method of Doherty et al. because the reference teaches flow cytometry as a suitable method for determining exosome concentration. Those of ordinary skill in the art would have been motivated to make this modification because Fortunato and Gibbings are silent with regard to the method used to determine exosome concentration and Doherty et al. teaches specific methods for doing so.
There would have been a reasonable expectation of success because all of the references are reasonably drawn to the same field of endeavor, that is, exosomes/extracellular vesicles.
With regard to Claims 15 and 16, Fortunato teaches incubating the sEVs with various stains including CFSE or anti-CD9, anti-CD63 and anti-CD81 antibodies to generate a labeled sEV population (Pgs. 15-16, Paragraph 4.2.1 and Pg. 16, Paragraph 4.2.2.).
With regard to Claim 17, Fortunato teaches concentrating secreted extracellular vesicles (sEV) in a biological fluid (HT29 cell conditioned medium/CCM) by centrifugal ultrafiltration (Pg. 15, Paragraph 4.1).
With regard to Claim 19, Fortunato teaches determining a concentration of the sEVs to be 1x10⁷ to 1x10⁸ particles/µl (or 1x1010 to 1x1011 particles/ml) (Pg. 15, Paragraph 4.1).
With regard to Claim 22, Fortunato teaches analyzing the recovered, labeled exosomes for labeling percentage (efficiency) (Pg. 4, Fig. 1A).
Claims 1, 3, 6, 7, 8, 10, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 23 and 24 are rejected under 35 U.S.C. § 103 as being unpatentable over Fortunato et al. (2021), cited in the IDS, in view of Jang et al. (2013), of record, Cao et al. (2021), Gibbings (US 2021/0163550 A1) and Doherty et al. (WO 2021/092192), both of record, as applied to Claims 1, 3, 6, 7, 8, 10, 12, 13, 14, 15, 16, 17, 18, 19, 22 and 23 above, and further in view of Anderson et al. (US 2020/0113943 A1), of record.
The teachings of Fortunato et al., Jang et al., Cao et al., Gibbings and Doherty et al. were discussed above.
None of the above references taught a method wherein the CDVs are concentrated using a 750 kDa molecular weight cutoff tangential flow filter, as required by Claim 20.
Anderson et al. teaches the isolation and/or purification of a population of cell- derived vesicles by applying tangential flow filtration (TFF) to a conditioned medium and concentrating the cell-derived vesicle fraction (Pg. 1, Paragraph [0011]) and wherein the tangential flow filter has a molecular weight cutoff between about 50-750 kDa (thereby encompassing the claimed value) (Pg. 6, Paragraph [0039]).
It would have been obvious to those of ordinary skill in the art at the time of the instant invention to modify the method of Fortunato et al., Jang et al., Gibbings and Doherty et al. whom teach isolating labeled, chemotherapeutic loaded extracellular vesicles with tangential flow filtration to use a tangential flow filter with a molecular weight cutoff of 750 kDa as taught by Anderson et al. because Anderson et al. teaches a particular molecular weight cutoff range as a suitable for exosome isolation/recovery. Those of ordinary skill in the art would have been motivated to make this modification because while Fortunato teaches ultrafiltration with a molecular weight cutoff of 300 kDa and Gibbings teaches exosome isolation/recovery using TFF, only Anderson et al. teaches that exosomes can be isolated/purified using TFF with about a 750 kDa molecular weight cutoff. There would have been a reasonable expectation of success because all of the references are reasonably drawn to the same field of endeavor, that is, exosomes/extracellular vesicles.
Claims 1, 3, 6, 7, 8, 10, 12, 13, 14, 15, 16, 17, 18, 19, 21, 22, 23 and 24 are
rejected under 35 U.S.C. § 103 as being unpatentable over Fortunato et al. (2021), cited in the IDS, in view of Jang et al. (2013), of record, Cao et al. (2021), Gibbings (US 2021/0163550 A1) and Doherty et al. (WO 2021/092192), both of record, as applied to Claims 1, 3, 6, 7, 8, 10, 12, 13, 14, 15, 16, 17, 18, 19, 22 and 23 above, and further in view of Larocca et al. (WO 2021/151029 A1), of record.
The teachings of Fortunato et al., Jang et al., Cao et al., Gibbings and Doherty et al. were discussed above.
None of the above references taught a method wherein the labeled CDVs are
passed though the centrifugal filter for at least 10 minutes at a centrifugal force of at
least 10,000 g, as required by Claim 21.
Larocca et al. teaches a method wherein exosomes are isolated using a
centrifugal filter at a centrifugal force of 10,000g for 15 minutes (Pg. 23, Paragraph
[0140]).
It would have been obvious to those of ordinary skill in the art at the time of the
instant invention to modify the method of Fortunato et al., Jang et al., Gibbings and
Doherty et al. whom teach isolating labeled, chemotherapeutic loaded extracellular
vesicles with centrifugal ultrafiltration for an unspecified time and force to use the
particular operating parameters for centrifugal filtration taught by Larocca et al. because
Larocca teaches a particular force and time as a suitable for exosome
isolation/recovery. Those of ordinary skill in the art would have been motivated to make
this modification because Fortunato is silent with regard to the time and force used to
recover/isolate exosomes by centrifugal filtration and Larocca et al. teaches specific
operating parameters for doing so. There would have been a reasonable expectation of
success because all of the references are reasonably drawn to the same field of
endeavor, that is, exosomes/extracellular vesicles.
Response to Arguments
Applicant's arguments filed 04/22/2026 have been fully considered but they are not persuasive.
The Applicant argues that neither Fortunato or Jang disclose the limitations of amended Claim 1. Applicant asserts that Jang describes “nanovesicles” and “exosomes” while Fortunato is drawn to “small extracellular vesicles”. Applicant states that the Examiner has not articulated a rationale for modifying Fortunato in view of Jang to arrive at the claimed invention. Applicant notes that Jang teaches natural exosome yield is low and purification cumbersome which provided impetus for the claimed invention. Applicant further asserts that the ordinary artisan would not look to modify Fortunato in view of Jang with a reasonable expectation of success (Remarks, Pg. 7, Lines 17-29 and Pg. 8, Lines 1-17).
This is not found to be persuasive for the reasoning provided in the above rejections which address the amended limitations to the broad claims. The Examiner has provided explicit rationale as to why the ordinary artisan would choose to modify the method of isolating sEVs from HEK293 cells of Fortunato with the use of serial filter extrusion to produce exosome mimetic nanovesicles (or cell-derived vesicles) of Jang et al. above. That is, exosomes while useful for targeted drug delivery are difficult to obtain as most mammalian cells release relatively low quantities of exosomes and the purification of exosomes is cumbersome, which results in a relatively low yield.
The generation of desired exosome-mimetic vesicles with a substantially greater yield is attractive for the development of future nanosized drug delivery systems. There would have been a reasonable expectation of success in making this modification because both references are reasonably drawn to the same field of endeavor, that is, the isolation of useful cell derived vesicles.
The Applicant argues that the claims are allegedly non-obvious because of the unexpected effect of yielding CDVs with a unique surface profile from exosomes, Applicant cites the Specification at Paragraphs [0049]-[0050] wherein the serial extrusion of HEK293 cells produced CDVs with CDV protein markers and exosomes. Applicant notes the CDVs were enriched with markers as compared to the exosomes (Fig. 1) (Remarks, Pg. 8, Lines 18-30 and Pg. 9, Lines 1-9).
This is not found to be persuasive for the following reasons, initially the Examiner notes that the claims only require the CDVs comprise one or more of the claimed protein markers and not any particular amount thereof. Thus, the exosomes isolated from HEK293 cells of Fortunato and Cao et al. would be expected to have the same protein markers as claimed. This is evidenced by Figure 1 of the disclosure. Secondly, when combined with Jang and Cao, the serial extrusion of the HEK293 cells of Fortunato would therefore be expected to produce both exosomes and CDVs having the same markers, as they are derived from the same cells as exemplified in the disclosure.
The Applicant argues that Gibbings, Doherty, Lawrence, Gibbings/Doherty, Anderson, Larocca do not remedy the alleged deficiencies of Fortunato and Jang
(Remarks, Pg. 9, Lines 23-28 and Pgs. 10-13).
This is not found to be persuasive for the reasoning provided in the above rejections and the Response to Arguments. The Examiner notes that the references were only cited to address limitations in the dependent claims and that the broad claims remain obvious over Fortunato in view of Jang and Cao.
No claims are allowed.
Any inquiry concerning this communication or earlier communications from the Examiner should be directed to PAUL C MARTIN whose telephone number is (571)272-3348. The Examiner can normally be reached Monday-Friday 12pm-8pm EST.
Examiner interviews are available via telephone, in-person, and video conferencing using a USPTO supplied web-based collaboration tool. To schedule an interview, Applicant is encouraged to use the USPTO Automated Interview Request (AIR) at http://www.uspto.gov/interviewpractice.
If attempts to reach the Examiner by telephone are unsuccessful, the Examiner’s supervisor, Sharmila G Landau can be reached at (571) 272-0614. The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300.
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/PAUL C MARTIN/ Examiner, Art Unit 1653 06/24/2026
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