DETAILED ACTION
Notice of Pre-AIA or AIA Status
1. The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Election/Restrictions
2. Applicant's election with traverse of Group I in the reply filed on 29 December 2025 is acknowledged. However, Applicant did not in fact provide a traversal over the restriction. Rather, the response merely states:
PNG
media_image1.png
74
638
media_image1.png
Greyscale
Because applicant did not distinctly and specifically point out the supposed errors in the restriction requirement, the election has been treated as an election without traverse (MPEP § 818.01(a)).
Claim Status
3. Claims 1-20 are pending.
Claims 15-20 are withdrawn from further consideration pursuant to 37 CFR 1.142(b), as being drawn to a nonelected invention, there being no allowable generic or linking claim.
Claims 1-14 read on the elected invention and have been examined herein.
Claim Objections
4. Claims 1-14 are objected to because of the following informalities: Claims 1-14 recite both “bioorthogonal” (e.g., claims 1, 7 and 14) and “biorthogonal” (e.g., claims 2, 4, 8 and 10). The claims should consistently recite the same terminology. Appropriate correction is required.
Claim Rejections - 35 USC § 112(b) - Indefiniteness
5. 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 1-14 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.
Claims 1-14 are indefinite over the recitation that the “first molecule from said first bioorthogonal functional group and said capture agent are present in a molar ratio of between 2:1 to 10:1.” It is unclear as to whether the first molecule and capture agent are present at this ratio in the initial sample, or are mixed together in the “functionalizing” step at this ratio, or are present in the activated solution that is deposited onto the capture surface at this ratio.
Claim 6 is indefinite over the recitation of “said first molecule is also able to bond to said molecule comprising said second molecule from said second bioorthogonal functional group” because the phrase “said molecule” lacks proper antecedent basis. While the claim previously refers to a first molecule and a second molecule, the claim does not previously refer generally to a molecule and it is unclear as to what constitutes “said molecule comprising said second molecule.”
Claims 7-13 are indefinite and confusing over the recitation of “said molecule with said first molecule” (see claim 7) because “said molecule” lacks proper antecedent basis and it is unclear as to what constitutes “said molecule.” Similarly, it is unclear as to what constitutes “said molecule comprising said second molecule” in claim 12.
Claim 14 is indefinite over the recitation of “said EV by binding of said molecule with said first molecule” because “said molecule” lacks proper antecedent basis. While the claim previously refers to a first molecule and a second molecule, the claim does not previously refer generally to a molecule and it is unclear as to what constitutes “said molecule comprising said second molecule.”
Double Patenting
6. 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.
The USPTO Internet website contains terminal disclaimer forms which may be used. Please visit www.uspto.gov/patent/patents-forms. The actual filing date of the application in which the form is filed determines what form (e.g., PTO/SB/25, PTO/SB/26, PTO/AIA /25, or PTO/AIA /26) should be used. A web-based eTerminal Disclaimer may be filled out completely online using web-screens. An eTerminal Disclaimer that meets all requirements is auto-processed and approved immediately upon submission. For more information about eTerminal Disclaimers, refer to www.uspto.gov/patents/apply/applying-online/eterminal-disclaimer.
Claims 1-14 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1-15 of copending Application No. 18/854,316 (reference application). Although the claims at issue are not identical, they are not patentably distinct from each other because the present claims and the claims of ‘316 are both inclusive of methods of selectively capturing an extracellular vesicle (EV) from a sample, comprising: functionalizing a capture agent for said EV with a first molecule from a first bioorthogonal functional group such that said capture agent remains attachable to said EV and said first molecule is also able to bond to a second molecule from a second bioorthogonal functional group, said second molecule being complementary to said first molecule; mixing said functionalized capture agent with said sample such that said functionalized capture agent binds to said EV and such that an activated sample is formed; functionalizing a capture surface with said second molecule; and depositing at least a portion of said activated sample on at least a portion of said functionalized capture surface to thereby selectively capture said EV by binding of said second molecule with said first molecule, particularly wherein the first bioorthogonal functional group is trans-cyclooctene (TCO), Alkyne, and a cyclooctyne derivative and the second bioorthogonal functional group is tetrazine (Tz)or azide (claim 13 of ‘316). Note that the present claims recite the open claim language of comprising and thereby are inclusive of additional steps and elements, including the barcodes recited in the claims of ‘316. The claims of ‘316 are also inclusive of methods wherein the disease is cancer since the claims of ‘316 (e.g., claims 6, 7 and 9) determine a cell surface antigen expression signature of HCC (i.e., hepatocellular cancer cells) and determine the stage of a disease. The claims of ‘361 do not recite that the ratio of the first biorthogonal functional group to the capture agent is between 2:1 to 10:1. However, when read in light of the specification of ‘316 (e.g., para [0066]), it is clear that the ratio of the first biorthogonal functional group to the capture agent is intended to be between 2:1 to 10:1. Further, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have optimized the ratio of the first biorthogonal functional group to the capture agent, including to have used a ratio of between 2:1 and 10:1 in order to have ensured the efficiency of capturing the extracellular vesicle with only capture agents that are attached to the first bioorthogonal group while maintaining cost-effectiveness of the assay.
This is a provisional nonstatutory double patenting rejection because the patentably indistinct claims have not in fact been patented.
Claim Rejections - 35 USC § 103
7. 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.
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.
Claim(s) 1-13 is/are rejected under 35 U.S.C. 103 as being unpatentable over Dong et al (herein after “Dong1”; ACS Applied Mater. Interfaces. 20 March 2019. 11: 13973-12983 and Supporting Information, 40 pages total) in view of Dong et al (herein after “Dong2”; Science Adv. “Covalent chemistry on nanostructured substrates enables noninvasive quantification of gene rearrangements in circulating tumor cells” 31 July 2019. 5:eaav9186, p. 1-12 and Supplementary Materials, 23 pages total).
Dong1 teaches a method for capturing an extracellular vesicle (EV) from a sample, comprising: reacting a sample with a capture reagent which comprises anti-EpCAM antibody (which binds to a cell surface antigen on the EV) grafted to a silicon (Si) nanowire array via a (see Figure 1, reproduced in part below; and p. 13976 “Fabrication of NanoVilli Chips).
PNG
media_image2.png
146
264
media_image2.png
Greyscale
As shown in Figure S2, anti-EpCAM is linked to biotin, which binds to streptavidin (SA).
PNG
media_image3.png
308
840
media_image3.png
Greyscale
Thus, in the method of Dong1 - referred to therein as a NanoVilli assay / chip (see abstract and Figure 1) - the capture agent of anti-EpCAM is bound to the first functional group of biotin and the resulting biotinylated anti-EpCAM is contacted with the capture surface (SiNWS) that carries the second functional group of streptavidin. The EV in the sample is then captured onto this anti-EpCAM-grafted SiNWS.
Dong1 does not teach a method for capturing EVs in which the EVs are first contacted with a capture agent (anti-EpCAM) which has been functionalized by contacting anti-EpCAM with a first functional group to form an activated sample, and the activated sample is then deposited on a capture surface that is functionalized with a second functional group.
However, Dong2 teaches a method for capturing a circulating tumor cell (CTC) from a sample, comprising: functionalizing a capture agent (i.e., anti-EpCAM antibody) for a CTC with a first molecule from a first bioorthogonal functional group (i.e., TCO) such that said capture agent remains attachable to the CTC and is also able to bind to a second molecule from a second bioorthogonal functional group (i.e., tetrazine (TZ)), the second molecule (TZ) being complementary to the first molecule (TCO); mixing the functionalized capture agent with the sample so that the functionalized capture agent binds to the CTC and an activated sample is formed; functionalizing a capture surface with the second molecule; and depositing at least a portion of the activated sample on the functionalized capture surface to selectively capture the CTC by binding of said second molecule (TZ) with the first molecule (i.e., the biotin moiety attached to anti-EpCAM; see e.g., Figure 1, reproduced in part below; and p. 3 “CTC capture with Click Chips”).
PNG
media_image4.png
296
350
media_image4.png
Greyscale
Dong2 teaches that this method for capturing / immobilizing and then releasing the cells provides several advantages over prior art methods for capturing cells (p. 2, col. 2). In particular, Dong2 (p. 2, col. 2) states:
“a pair of highly reactive click chemistry motifs, i.e., Tz and TCO, were grafted onto cell capture substrates (via surface modification) and CTCs (via TCO–anti-EpCAM
conjugation), respectively. The ligation between Tz-grafted SiNWS and TCO-grafted CTCs is facile, specific, irreversible, and insensitive to biomolecules, water, and oxygen (22), leading to specific, rapid, and irreversible immobilization of the CTCs with improved capture efficiency and reduced nonspecific immobilization of WBCs compared
with that observed in previously reported anti-EpCAM–mediated approaches (19, 25). After CTC capture, exposure to a disulfide cleavage agent [i.e., 1,4-dithiothreitol (DTT)] (26) results in the prompt release of the CTCs from the SiNWS by cleaving the disulfide bond linking the Tz to the SiNWS. As WBCs are captured nonspecifically, they are not released by freeing Tz from the SiNWS, reducing WBC contamination in the released samples. In addition, mRNA stability is improved as a result of the (i) short CTC purification time (24 min) and (ii) mild processing conditions, enabling downstream mRNA analysis by RT-ddPCR (16).”
Dong2 compares the Click Chip method with prior art methods that rely on capture of CTCs using biotinylated antibodies that are linked to a solid support via streptavidin binding to biotin (i.e., NanoVelcro Assays; see Figure 3 and p. 7). It is stated that “a much smaller quantity of TCO–anti-EpCAM (as low as 0.1 ng)” is required to perform the disclosed method as compared to capture methods that use biotinylated anti-EpCAM bound to streptavidin - i.e., “NanoVelcro assays” (p. 7, and Fig. 3).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of Dong1 so as to have used the nanostructured silicon substrate (“Click Chip”) of Dong2 to capture the EVs present in the sample. Dong2 is considered to be analogous art to the claimed method because the capturing step uses an antibody that binds to a surface protein - i.e., an anti-EpCAM antibody is used in both the method of Dong1 and Dong2 to bind to an EpCAM surface antigen; and the captured entity (cell) is released so that it can be further analyzed. Also Dong2 compares their capture method to that of “NanoVelcro assays” which use the capture assay of Dong1 in which a biotinylated antibody (anti-EpCAM) binds to streptavidin immobilized on a solid support (see Figure 3 and p. 7). The ordinary artisan would have been motivated to have modified the method of Dong1 in this manner because such a modification would have provided the same advantages set forth by Dong2 for cells and particularly would have provided a rapid, specific and highly efficient means for capturing EVs, and an improved means for releasing the captured EVs via disulfide cleavage, which would release only the immobilized EVs and not other materials that may have non-specifically bound to the solid support (SiNWS).
Regarding the recitation in the claims that “said first bioorthogonal functional group and said capture agent are present in a molar ratio of between 2:1 to 10:1,” the combined prior art does not teach this limitation. However, Dong1 does teach “we next attempted to reduce the consumption of our EV capture agent (anti-EpCAM) without compromising the EV capture performance at the optimal flow rate and nanowire
configurations identified earlier” (p. 13977, col. 2).
Further, Dong2 does teach (p. 10, col. 1) the methodology for preparing TCO-anti-EpCAM:
“TCO–anti-EpCAM conjugate was prepared by incubating TCO PEG4-NHS ester (0.5 mM; Click Chemistry Tools Bioconjugate Technology Company) with human EpCAM/TROP-1 antibody [goat immunoglobulin G (IgG), 0.5 ug ul−1; R&D Systems Inc.] in PBS at room temperature for 30 min.”
In view of the guidance provided by Dong2 and the teachings of Dong1 of the need to minimize the quantity of anti-EpCAM used in the capture assay, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have further modified the method of Dong1 so as to have performed the step of generating the TCO-anti-EpCAM conjugate using between a 2:1 to 10:1 molar ratio of TCO (first biorthogonal functional group) to anti-EpCAM (capture agent). The ordinary artisan would have been motivated to have optimized the ratio of the first biorthogonal functional group to the capture agent, including to have used a ratio of between 2:1 and 10:1 in order to have ensured the efficiency of capturing the EVs with the capture agents that are attached to the first biorthogonal group while maintaining cost-effectiveness of the assay.
Regarding claims 2-4 and 8-10, as discussed above, Dong2 teaches that the first biorthogonal group is TCO and the second biorthogonal group is tetrazine (TZ: see, e.g., Figure 1 and p. 2, col. 2). Note that claims 3 and 9 do not limit the first biorthogonal group to DBCO or BARAC but only defines a cyclooctyne derivative.
Regarding claims 5 and 11, as discussed above, Dong2 teaches that the capture surface is a nanostructured surface (e.g., Figure 1 and p. 3, col. 1).
Regarding claims 6 and 12, the combined references do not teach that the method further comprises functionalizing a second capture agent for the EV with the first molecule such that the second capture agent remains attachable to said EV and the first molecule is also able to bind to a molecule comprising the second molecule biorthogonal functional group, and wherein the second capture agent is distinct from said capture agent.
However, Dong1 teaches that EVs derived from different tumor types have different surface antigens and can be captured with different capture agents (p. 13975, col. 1). It is stated that “More recent research efforts have explored the application
of immunoaffinity-based capture techniques for enriching tumor-derived EVs in different solid tumors. For example, pancreatic cancer-derived exosomes can be captured selectively using anti-GPC1-coated beads and isolated via flow cytometry, and the enrichment of glioblastoma-derived exosomes has been demonstrated in herringbone microfluidic devices (i.e., EVHB-Chip) with EGFRvIII antibodies used as the capture
Agent” (p. 13975, col. 1). As discussed above Dong1 exemplifies using anti-EpCAM as a capture agent for NSCLC-derived EVs / EVs from cancers derived from epithelial cells (p. 13976, col. 1).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have further modified the method of Dong1 so as to also functionalized a second, different capture agent that would bind to a different type of EV with the first molecule such that the second capture agent remains attachable to said EV and the first molecule is also able to bind to the second functional group. One would have been motivated to have done so in order to have provided the advantage that additional capture agents could be used to capture and assay the nucleic acids of EVs derived from other types of cancers, such as pancreatic and glioblastoma, as taught by Dong1.
Regarding claims 7-13, Dong2 teaches that following capture of the EVs, the EVs are released from the capture surface and nucleic acid from within the EVS are assayed to determine if the subject has cancer (e.g., p. 13977 “EV-Derived RNA Assay Using NanoVilli Chips”).
Further regarding claim 13, Dong2 teaches that the captured CTC is released from the support using a cleaving agent - i.e., 1,4-dithiothreitol (DTT) to cleave the disulfide bond linking the captured CTC to the Si nanowire (see Figure 1 and p. 2, col. 2). Thus, modification of the method of Dong1 as set forth above would have resulted in a method in which the captured EV was released from the support using a cleaving agent.
8. Claim 14 is rejected under 35 U.S.C. 103 as being unpatentable over Dong et al (herein after “Dong1”; ACS Applied Mater. Interfaces. 20 March 2019. 11: 13973-12983 and Supporting Information, 40 pages total) in view of Dong et al (herein after “Dong2”; Science Adv. “Covalent chemistry on nanostructured substrates enables noninvasive quantification of gene rearrangements in circulating tumor cells” 31 July 2019. 5:eaav9186, p. 1-12 and Supplementary Materials, 23 pages total), and further in view of Reategui et al (Nature Communications. 2018. 9: 175, p. 1-11).
The teachings of Dong1 and Dong2 are presented above.
The combined references do not specifically teach that the method further comprises comparing an expression profile from a plurality of EVs from the sample from the subject with the expression profile of a control; and determining from the comparing whether cancer is present in the subject
However, Dong1 (p. 13975, col. 1) does teach:
“To characterize and/or to quantify the trace amount of mRNA extracted from the enriched tumor-derived EVs, highly sensitive mRNA profiling technologies, for example, next generation sequencing and Droplet Digital PCR (ddPCR), were adopted for downstream detection purposes.”
It is also stated that:
“RNA in the captured EVs can be recovered for downstream molecular analyses by reverse transcription Droplet Digital PCR. We demonstrate that this assay can be applied to monitor the dynamic changes of ROS1 rearrangements and epidermal growth factor receptor T790M mutations that predict treatment response and disease progression in non-small cell lung cancer patients” (see abstract).
Further Reategui teaches methods for capturing EVs from a sample from a patient, determining an RNA expression profile from the captured EVs, comparing the RNA expression profile to that from control samples, and detecting differences in the RNA expression profile of the EVs from the subject as compared to control samples as diagnostic of cancer, and particularly glioblastoma and subtypes of glioblastoma (p. 7, col. 1). Reategui concludes “These results demonstrate that the EVHB-Chip captured tumor EVs containing GBM enriched mRNA signatures and potentially reveal transcriptional heterogeneity in GBM tumors” (p. 7, col. 1).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of Dong1 so as to have also compared the RNA expression profile from the plurality of captured EVs to normal, control samples. One would have been motivated to have done so for the advantage set forth by Reategui of using the EV RNA expression profile to diagnose cancer in a subject and/or to diagnose the type or subtype of cancer.9. Claims 1, 6, 7 and 12 are rejected under 35 U.S.C. 103 as being unpatentable over Theodoraki et al (Clinical Exper Immunol. 2018. 192: 271-283) in view of Sigma-Aldrich (Product Information for ImmunoProbe™ Biotinylation Kit. Technical Bulletin. 2012, available at SigmaAldrich.com).
Theodoraki teaches a method for capturing exosomes (which are a type of EV) wherein the method comprises: contacting anti-CD3 antibodies (i.e. an EV capture agent) with the functional group of biotin to produce biotin-labeled anti-CD3; contacting a sample comprising exosomes with the biotin-labeled anti-CD3 antibodies to form an activated sample; contacting the activated sample with a support having the second functional group of streptavidin bound thereto, such that biotin (the first functional group) binds to streptavidin (the second functional group) and the exosomes (bound to the biotin-labeled anti-CED3 antibodies) are captured (see Figure 1 and p. 274, col. 1).
Thus, Theodoraki teaches a method for capturing exosomes (EVs) from a sample, comprising: functionalizing a capture agent (i.e., anti-CD3 antibody) for an exosome with a first molecule from a first bioorthogonal functional group (i.e., biotin) such that said capture agent remains attachable to the exosome and is also able to bind to a second molecule from a second bioorthogonal functional group (i.e., streptavidin), the second molecule (streptavidin) being complementary to the first molecule (biotin); mixing the functionalized capture agent with the sample so that the functionalized capture agent binds to the exosome and an activated sample is formed; functionalizing a capture surface (beads) with the second molecule (streptavidin); and depositing at least a portion of the activated sample on the functionalized capture surface to selectively capture the exosomes by binding of said second molecule (streptavidin) with the first molecule (i.e., the biotin moiety attached to anti-CD3 antibody).
Theodoraki does not teach that the biotin (first molecule from said first bioorthogonal functional group) and anti-CD3 antibody (capture agent) are contacted with one another at molar ratio of between 2:1 to 10:1.
However, Sigma-Aldrich teaches methods for generating biotin labeled antibodies wherein the molar ratio in the reaction mixture is 5:1 or 10:1 of biotin reagent to antibody (p. 1 first para and p. 3, first para).
In view of the teachings of Sigma-Aldrich, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of Theodoraki so as to have specifically generated the biotinylated anti-CD3 antibody reagent by mixing biotin and anti-CD3 antibody at a molar ratio of 5:1 or 10: 1 since Sigma-Aldrich teaches that this is an effective molar ratio for producing biotin labeled antibodies.
Regarding claims 6 and 12, Theodoraki (p. 274, col. 2) also teaches generating biotinylated anti-CD63 antibody. Thus, the method of Theodoraki is one that further comprises functionalizing a second capture agent (anti-CD63 antibody) for the EV with the first molecule (biotin) such that the second capture agent (anti-CD63 antibody) remains attachable to said EV and the first molecule (biotin) is also able to bind to a second molecule biorthogonal functional group (streptavidin), and wherein the second capture agent (anti-CD63 Ab) is distinct from said capture agent (anti-CD3 Ab).10. Claims 5, 11, 13 and 14 are rejected under 35 U.S.C. 103 as being unpatentable over Theodoraki et al (Clinical Exper Immunol. 2018. 192: 271-283) in view of Sigma-Aldrich (Product Information for ImmunoProbe™ Biotinylation Kit. Technical Bulletin. 2012, available at SigmaAldrich.com) and further in view of Reategui et al (Nature Communications. 2018. 9: 175, p. 1-11).
The teachings of Theodoraki and Sigma-Aldrich are presented above.
Regarding claims 5 and 11, Theodoraki teaches capturing the exosomes on ExoCap™ streptavidin magnetic beads (p. 274, col. 1) but does not teach that the capture surface is a nanostructure.
However, Reategui teaches methods for capturing EVs from a sample from a patient using a nano-structured substrate tumor-specific capture agents / antibodies (e.g., p. 9, col. 2)
Reategui (p. 2, col. 1) states “The nanostructured substrate consists of an ultra-thin (∼150 nm) gelatin membrane functionalized with streptavidin-coated nanoparticles
that when combined with the chaotic mixing resulting from the herringbone grooves, maximizes EV interactions with the tumor specific antibody-coated surfaces.” It is further stated (p. 2 col. 1, final par to col. 2, first para):
“we show that our EVHB-Chip can efficiently capture tumor-derived EVs from plasma and serum. Functionalization of the EVHB-Chip with a cocktail of antibodies allows for specific and rapid isolation of tumor-derived EVs that can subsequently be released allowing for the analysis of their biological.”
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of Theodoraki so as to have immobilized the biotinylated-antibody-exosome complex onto the nanostructured surface comprising a gelatin membrane functionalized with streptavidin-coated nanoparticles, as taught by Reategui. One would have been motivated to have done so for the advantages set forth by Reategui that the nanostructured substrate provides an effective surface for immobilizing and separating the EVs from other materials in the sample and provides for the rapid and simple release of the captured EVs.
Regarding claim 13, Theodoraki does not teach that the exosomes / EVs are released from the capture surface using a cleaving agent.
However, Reategui teaches that the EVs are released from the gelatin membrane using a proteinase K solution, which is a cleaving agent (p. 5, col. 1 final para to col. 2, first para). In particular, Reategui states (p. 5, col. 1): “EVs can be eluted from the surface of the EVHB-Chip by flushing a proteinase K solution (0.05 %) that shaves EVs from the device.”
Accordingly, modification of the method of Theodoraki as set forth above would have resulted in a method wherein the exosomes / EVs are released from the gelatin membrane capture surface using proteinase K (a cleaving agent). One would have been motivated to have released the exosomes / EVs using this method of Reategui since Reategui teaches that this provides an effective means for releasing the captured EVs so that they can be further assayed, including by performing functional studies (p. 5, col. 1 final para) and RNA profiling studies (p. 7, col. 1).
Regarding claim 14, the combined references do not teach that the method further comprises comparing an expression profile from a plurality of EVs captured from the sample from the subject with the expression profile of a control; and determining from the comparing whether cancer is present in the subject
However, Theodoraki does teach assaying the captured exosomes to determine protein profiles (e.g., p. 273, col. 2; p. 274, col.. 2 and Figure 4). It is also stated that “Association of exosome molecular profiles with disease progression supports the exosome potential as future cancer biomarkers” (abstract).
Further, Reategui teaches methods for capturing EVs from a sample from a patient; releasing the captured tumor EVs determining an RNA expression profile from the captured EVs, comparing the RNA expression profile to that from control samples, and detecting differences in the RNA expression profile of the EVs from the subject as compared to control samples as diagnostic of cancer, and particularly glioblastoma and subtypes of glioblastoma (p. 7, col. 1). It is stated at p. 2, col. 1 “Functionalization of the EVHB-Chip with a cocktail of antibodies allows for specific and rapid isolation of tumor-derived EVs that can subsequently be released allowing for the analysis of their biological cargo.” Reategui concludes “These results demonstrate that the EVHB-Chip captured tumor EVs containing GBM enriched mRNA signatures and potentially reveal transcriptional heterogeneity in GBM tumors” (p. 7, col. 1).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of Theodoraki so as to have included the steps of releasing the plurality of exosomes from the capture surface; assaying a plurality of nucleic acid sequences from said plurality of exosomes; creating an expression profile of the nucleic acid sequences, the expression profile comprising a quantification of each of said plurality of nucleic acid sequences; comparing the expression profile with a control; and determining from said comparing of said expression profile with said control whether said cancer is present in said subject. One would have been motivated to have done so for the advantage set forth by Reategui of using the EV RNA expression profile to diagnose cancer in a subject and/or to diagnose the type or subtype of cancer.11. Claims 2-5 and 8-11 are rejected under 35 U.S.C. 103 as being unpatentable over Theodoraki et al (Clinical Exper Immunol. 2018. 192: 271-283) in view of Haun et al (Nature Nanotechnology. 2010. 5:660-665 and Supplementary Information, 21 pages total).
Theodoraki teaches a method for capturing exosomes (which are a type of EV) wherein the method comprises: contacting anti-CD3 antibodies (i.e. an EV capture agent) with the functional group of biotin to produce biotin-labeled anti-CD3; contacting a sample comprising exosomes with the biotin-labeled anti-CD3 antibodies to form an activated sample; contacting the activated sample with a support having the second functional group of streptavidin bound thereto, such that biotin (the first functional group) binds to streptavidin (the second functional group) and the exosomes (bound to the biotin-labeled anti-CED3 antibodies) are captured (see Figure 1 and p. 274, col. 1).
Thus, Theodoraki teaches a method for capturing exosomes (EVs) from a sample, comprising: functionalizing a capture agent (i.e., anti-CD3 antibody) for an exosome with a first molecule from a first bioorthogonal functional group (i.e., biotin) such that said capture agent remains attachable to the exosome and is also able to bind to a second molecule from a second bioorthogonal functional group (i.e., streptavidin), the second molecule (streptavidin) being complementary to the first molecule (biotin); mixing the functionalized capture agent with the sample so that the functionalized capture agent binds to the exosome and an activated sample is formed; functionalizing a capture surface (beads) with the second molecule (streptavidin); and depositing at least a portion of the activated sample on the functionalized capture surface to selectively capture the exosomes by binding of said second molecule (streptavidin) with the first molecule (i.e., the biotin moiety attached to anti-CD3 antibody).
Regarding claims 2-4 and 8-10, Theodoraki teaches that the first functional group is biotin and the second molecule from a second functional group is streptavidin. Theodoraki does not teach that the first functional group is TCO (claims 2, 3, 8 and 9) or the second molecule from a second functional group is tetrazine (TZ; claims 4 and 10).
However, Haun teaches methods for capturing cells wherein the methods comprise: reacting a first functional group (i.e., TCO) with a first capture agent (i.e., an antibody specific to a cell surface antigen) to generate a TCO-Ab; contacting a sample comprising target cells with the TCO-Ab to form an activated sample in which a target cell binds to the TCO-Ab; contacting the activated sample with the capture surface of a magnetic fluorescent nanoparticle (MFNPs) which have been functionalized by binding to a molecule comprising a second functional group that is tetrazine / TZ (see Figure 1; p. 660, col. 1 to p. 661, col. 1; and p. 664 “Methods”). Haun refers to this method as “biorthogonal nanoparticle detection (BOND)” and particularly “BOND-2 (see Figure 1 and p. 660, col. 1). See p. 661, col. 1 “we used a two-step strategy (BOND-2) in which TCO-modified antibodies were used for primary target binding followed by covalent reaction with Tz–MFNP (Fig. 1c).”
Haun compares the BOND-2 method with prior art methods that rely on capture of cells using biotinylated antibodies that are linked to a solid support via streptavidin binding to biotin (e.g., p. 663, col. 1 and p. 664 “Methods”). It is stated that “the covalent Tz/TCO reaction permits more nanoparticles to bind per antibody scaffold. The increased detection sensitivity resulting from amplification and the modular nature of the BOND-2 technique make it ideally suited for clinically oriented molecular profiling applications. We have demonstrated such an application here with magnetic detection of tumour cells using a miniaturized magnetic resonance detector that was designed for point-of-care clinical use.” (p. 664, col. 1).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of Theodoraki so as to have used the BOND method of Haun to capture the EVs present in the sample. Haun is considered to be analogous art to the claimed method because it also relies on capturing an entity (cell) using an antibody that binds to a surface protein, as in the method of Theodoraki; and subsequently releases the entity (cell) so that the isolated entity (cell) can be further analyzed. Also Haun compares their BOND-2 capture method to capture assays in which a biotinylated binds to streptavidin immobilized on a solid support, as encompassed by the method of Theodoraki. The ordinary artisan would have been motivated to have modified the method of Theodoraki in this manner because such a modification would have provided the advantages set forth by Haun of increased sensitivity of capture and detection.
Regarding the recitation in the claims that “said first bioorthogonal functional group and said capture agent are present in a molar ratio of between 2:1 to 10:1,” the combined prior art does not teach this limitation. However, Haun (p. 4 of Supplementary Information) does teach that “(f)or BOND-1, antibodies were modified with 10 equivalents of TCO-NHS as described in the main text.” Haun also teaches varying the quantity of TCO to antibody and found that the degree of TCO modification varied for different antibodies despite identical reaction conditions, and the same effect was observed for biotinylation (p. 2 final para to p. 3 first para of Supplementary Information).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have further modified the method of Theodoraki so as to have used a 2:1 to 10:1 molar ratio of TCO (first biorthogonal functional group) to antibody (capture agent). The ordinary artisan would have been motivated to have optimized the ratio of the first biorthogonal functional group to the capture agent, including to have used a ratio of between 2:1 and 10:1 in order to have ensured the efficiency of capturing the EVs with the capture agents that are attached to the first biorthogonal group while maintaining cost-effectiveness of the assay.
Regarding claims 5 and 11, modification of the method of Theodoraki as set forth above would have resulted in a method in which the exosomes / EVs are captured on nanostructured surface - i.e., the magnetic fluorescent nanoparticle (MFNPs) of Haun.12. Claims 1-13 is/are rejected under 35 U.S.C. 103 as being obvious over Tseng (U.S. 20220163519, priority to 20 March 2019) in view of Haun et al (Nature Nanotechnology. 2010. 5:660-665 and Supplementary Information, 21 pages total) OR over Tseng et al (WO2020191206, priority to 20 March 2019) in view of Haun et al.
The applied Tseng reference has a common inventor with the instant application. Based upon the earlier effectively filed date of the reference, it constitutes prior art under 35 U.S.C. 102(a)(2).
This rejection under 35 U.S.C. 103 might be overcome by: (1) a showing under 37 CFR 1.130(a) that the subject matter disclosed in the Tseng reference was obtained directly or indirectly from the inventor or a joint inventor of this application and is thus not prior art in accordance with 35 U.S.C.102(b)(2)(A); (2) a showing under 37 CFR 1.130(b) of a prior public disclosure under 35 U.S.C. 102(b)(2)(B); or (3) a statement pursuant to 35 U.S.C. 102(b)(2)(C) establishing that, not later than the effective filing date of the claimed invention, the subject matter disclosed and the claimed invention were either owned by the same person or subject to an obligation of assignment to the same person or subject to a joint research agreement. See generally MPEP § 717.02.
Note that U.S. 20220163519 is the publication of U.S. Application 17/440,653 which is the national stage of PCT/US2020/23656, which published as WO20200191206. Citations below are with respect to U.S. 20220163519.
Tseng teaches a method for capturing extracellular vesicles wherein the method comprises: contacting an EV capture agent (i.e., an antibody that binds to EVs and particularly anti-LINGO1) anti-CD3 antibodies (i.e. an EV capture agent) with the first functional group of trans-cyclooctene (TCO) to produce TCO- modified Ab; contacting a sample comprising extracellular vesicles with the TCO-modified Ab to form an activated sample; contacting the activated sample with a capture support (silicon (Si) nanowire-embedded microfluidic chips having a second functional group of tetrazine (TZ) bound thereto, such that TCO (the first functional group) binds to TX (the second functional group) and the extracellular (bound to the TCO-modified Ab) are captured onto the support (see, e.g., Figures 12-17 and para [0024], [0063-0064], [0144-0145]).
Teng is silent as to the ratio of TCO to antibody and thereby does not specifically teach that “said first bioorthogonal functional group and said capture agent are present in a molar ratio of between 2:1 to 10:1.”
However, Haun teaches methods for capturing cells wherein the methods comprise: reacting a first functional group (i.e., TCO) with a first capture agent (i.e., an antibody specific to a cell surface antigen) to generate a TCO-Ab; contacting a sample comprising target cells with the TCO-Ab to form an activated sample in which a target cell binds to the TCO-Ab; contacting the activated sample with the capture surface of a magnetic fluorescent nanoparticle (MFNPs) which have been functionalized by binding to a molecule comprising a second functional group that is tetrazine / TZ (see Figure 1; p. 660, col. 1 to p. 661, col. 1; and p. 664 “Methods”). Haun refers to this method as “biorthogonal nanoparticle detection (BOND)” and particularly “BOND-2 (see Figure 1 and p. 660, col. 1). See p. 661, col. 1 “we used a two-step strategy (BOND-2) in which TCO-modified antibodies were used for primary target binding followed by covalent reaction with Tz–MFNP (Fig. 1c).”
Haun (p. 4 of Supplementary Information) does teach that “(f)or BOND-1, antibodies were modified with 10 equivalents of TCO-NHS as described in the main text.” Haun also teaches varying the quantity of TCO to antibody and found that the degree of TCO modification varied for different antibodies despite identical reaction conditions, and the same effect was observed for biotinylation (p. 2 final para to p. 3 first para of Supplementary Information).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of Tseng so as to have used a 2:1 to 10:1 molar ratio of TCO (first biorthogonal functional group) to antibody (capture agent). The ordinary artisan would have been motivated to have optimized the ratio of the first biorthogonal functional group to the capture agent, including to have used a ratio of between 2:1 and 10:1 in order to have ensured the efficiency of capturing the EVs with the capture agents that are attached to the first biorthogonal group while maintaining the cost-effectiveness of the assay.
Regarding claims 2-4 and 8-10, as discussed above, Tseng teaches that the first biorthogonal group is TCO and the second biorthogonal group is tetrazine (TZ: see, e.g., Figures 12-17 and para [0024], and [0144-0145])). Note that claims 3 and 9 do not limit the first biorthogonal group to DBCO or BARAC but only defines a cyclooctyne derivative.
Regarding claims 5 and 11, as discussed above, Tseng teaches that the capture surface is a nanostructured surface (i.e., a Si Nanowire; see, e.g., Figure 1).
Regarding claims 6 and 12, Tseng teaches that the method further comprises functionalizing a second capture agent (e.g., anti-PSMA) for the EV with the first molecule such that the second capture agent remains attachable to said EV and the first molecule is also able to bind to a molecule comprising the second molecule biorthogonal functional group, and wherein the second capture agent (e.g., anti-PSMA) is distinct from said capture agent (e.g., anti-EpCAM; see, Figure 15).
Regarding claims 7-13, Tseng teaches that following capture of the EVs, the EVs are released from the capture surface and nucleic acid from within the EVS are assayed to determine if the subject has cancer (e.g., para [0064], [0083], [0086] and Figure 15).
Further regarding claim 13, Tseng (para [0148] teaches “he captured EVs can be released specifically via a disulfide cleavage-driven by 1,4 dithiothreitol (DTT).” Thus, the method of Tseng is one in which the captured EV is released from the support using a cleaving agent.13. Claim 14 is rejected under 35 U.S.C. 103 as being obvious over Tseng (U.S. 20220163519, priority to 20 March 2019) in view of Haun et al (Nature Nanotechnology. 2010. 5:660-665 and Supplementary Information, 21 pages total), and further in view of Reategui et al (Nature Communications. 2018. 9: 175, p. 1-11) OR over Tseng et al (WO2020191206, priority to 20 March 2019) in view of Haun et al, and further in view of Reategui et al.
The applied Tseng reference has a common inventor with the instant application. Based upon the earlier effectively filed date of the reference, it constitutes prior art under 35 U.S.C. 102(a)(2).
This rejection under 35 U.S.C. 103 might be overcome by: (1) a showing under 37 CFR 1.130(a) that the subject matter disclosed in the Tseng reference was obtained directly or indirectly from the inventor or a joint inventor of this application and is thus not prior art in accordance with 35 U.S.C.102(b)(2)(A); (2) a showing under 37 CFR 1.130(b) of a prior public disclosure under 35 U.S.C. 102(b)(2)(B); or (3) a statement pursuant to 35 U.S.C. 102(b)(2)(C) establishing that, not later than the effective filing date of the claimed invention, the subject matter disclosed and the claimed invention were either owned by the same person or subject to an obligation of assignment to the same person or subject to a joint research agreement. See generally MPEP § 717.02.
Note that U.S. 20220163519 is the publication of U.S. Application 17/440,653 which is the national stage of PCT/US2020/23656, which published as WO20200191206. Citations below are with respect to U.S. 20220163519.
The teachings of Tseng and Haun are presented above.
The combined references do not specifically teach that the method further comprises comparing an expression profile from a plurality of EVs from the sample from the subject with the expression profile of a control; and determining from the comparing whether cancer is present in the subject
However, Tseng does teach that the captured EVs are released from the capture surface, lysed and EV-derived RNA assayed to detect cancer-specific RNA markers, including prostate cancer specific and HCC-specific RNA markers, for disease profiling (see, e.g., para [0148], [0151] and Figures 15 and 17).
Further Reategui teaches methods for capturing EVs from a sample from a patient, determining an RNA expression profile from the captured EVs, comparing the RNA expression profile to that from control samples, and detecting differences in the RNA expression profile of the EVs from the subject as compared to control samples as diagnostic of cancer, and particularly glioblastoma and subtypes of glioblastoma (p. 7, col. 1). Reategui concludes “These results demonstrate that the EVHB-Chip captured tumor EVs containing GBM enriched mRNA signatures and potentially reveal transcriptional heterogeneity in GBM tumors” (p. 7, col. 1).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of Tseng so as to have also compared the RNA expression profile from the plurality of captured EVs to normal, control samples. One would have been motivated to have done so for the advantage set forth by Reategui of using the EV RNA expression profile to diagnose cancer in a subject and/or to diagnose the type or subtype of cancer.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to CARLA J MYERS whose telephone number is (571)272-0747. The examiner can normally be reached M-Th 6:30-5:00 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, Wu-Cheng Winston Shen can be reached on 571-272-3157. The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300.
Information regarding the status of published or unpublished applications may be obtained from Patent Center. Unpublished application information in Patent Center is available to registered users. To file and manage patent submissions in Patent Center, visit: https://patentcenter.uspto.gov. Visit https://www.uspto.gov/patents/apply/patent-center for more information about Patent Center and https://www.uspto.gov/patents/docx for information about filing in DOCX format. For additional questions, contact the Electronic Business Center (EBC) at 866-217-9197 (toll-free). If you would like assistance from a USPTO Customer Service Representative, call 800-786-9199 (IN USA OR CANADA) or 571-272-1000.
/CARLA J MYERS/Primary Examiner, Art Unit 1682