DETAILED ACTION
Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
Continued Examination Under 37 CFR 1.114
A request for continued examination under 37 CFR 1.114, including the fee set forth in 37 CFR 1.17(e), was filed in this application after final rejection. Since this application is eligible for continued examination under 37 CFR 1.114, and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114. Applicant's submission filed on 07/13/2026 has been entered.
Priority
This application is a U.S. National Stage (371) application of PCT/US20/20529 filed on 02/28/2020 which claims priority to U.S. Provisional Application No. 62/962,722 filed on 01/17/2020 and U.S. Provisional Application No. 62/812,878 filed on 03/01/2019.
Information Disclosure Statement
The information disclosure statement (IDS) submitted on 08/20/2025 has been received. The submission is in compliance with the provisions of 37 CFR 1.97. Accordingly, the information disclosure statement is being considered by the examiner, and all references are considered except where they were lined through.
Claim Status
Claims 1-119, 123-124, 128-129 and 132-133 are cancelled. Claims 120-122, 125-127, 130-131 and 134-143 are previously presented.
Thus, claims 120-122, 125-127, 130-131 and 134-143 are under examination.
Withdrawn Rejections
The previous rejections of claims 120-122, 125-127, 130-131 and 134-143 under 35 U.S.C. 103, regarding obviousness, is withdrawn in lights of Applicant’s response filed 07/13/2026.
New Rejections
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 (PHOSITA) 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.
Claims 120-122, 125-127, 130-131 and 134-140 are rejected under 35 U.S.C. 103 as being unpatentable over Routenberg et al. (US 2021/0382043 A1, priority to 10/23/2018) in view of Fredriksson et al. (US 10,781,473 B2).
Claim 120 recites:
“A claim comprising:
capturing an extracellular vesicle (EV) by binding a first biomarker on the EV to a first binder linked to a support;
introducing, to the captured EV, a second binder and a third binder, wherein the second binder and the third binder bind to a second biomarker and a third biomarker, wherein the second and third biomarkers are surface protein biomarkers present on the EV, the second binder being linked to a first oligonucleotide and a third binder linked to a second oligonucleotide, wherein the first oligonucleotide and the second oligonucleotide each comprise a double-stranded portion and a single-stranded overhang, the single-stranded overhangs being complementary to each other;
directly ligating the first oligonucleotide to the second oligonucleotide when the single-stranded overhang portions hybridize due to co-localization of the second and third biomarkers on the captured EV, thereby forming a ligation product; and
detecting the ligation product, wherein detection of the ligation product indicates co-occurrence of the first biomarker, the second biomarker, and the third biomarker on the EV”.
Regarding claim 120, Routenberg teaches a method of capturing an extracellular vesicle (EV) by binding a first biomarker on the EV to a first binder linked to a support (Abstract; Sheet 1 of 72, FIG. 1A, “Capture Antibody”; [0013])
Routenberg teaches introducing to the captured EV a second binder and a third binder ([0269]; [0351], “Multiple binding reagents may also be desired for binding to different surface markers on the same EV”).
Routenberg teaches that the second binder and the third binder bind to a second biomarker and a third biomarker [0351].
Routenberg teaches that the second and third biomarkers are surface protein biomarkers present on the EV ([0269]; [0315] “EV surface proteins are identified and measured, for example, if no specific intracellular proteins are available for a cell type, but they should not be one of the same proteins used in the specific EV isolation”; [0351]; [0437], “produce a large number of EVs expressing a high level of CD81. These EVs also express the surface proteins CD9 and CD63 to a lesser degree”; [0475]).
Routenberg teaches introducing a second binder linked to a first oligonucleotide (Sheet 38 of 72, FIG. 32, “Antibody 1”).
Routenberg teaches introducing a third binder linked to a second oligonucleotide (Sheet 38 of 72, FIG. 32, “Antibody 2”).
Routenberg further teaches that the first and second oligonucleotides are complementary to each other ([0213], “the method further comprises binding the surface marker displaying agent of interest to a second binding reagent, wherein the first and second binding reagents each comprises an oligonucleotide, and the oligonucleotide of the first binding reagent and the oligonucleotide moiety of the anchoring reagent each comprises a sequence that is complementary to the oligonucleotide of the second binding reagent”).
Routenberg teaches ligating the first oligonucleotide to the second oligonucleotide to form a ligation product (Sheet 38 of 72, FIG. 32, “Ligation Site”; [0088]; [380]; [406]).
Routenberg teaches that the ligation of the first oligonucleotide to the second oligonucleotide happens due to the co-localization of the second and third biomarkers on the captured EV (Sheet 38 of 72, FIG. 32, “Ligation Site”; [0088]).
Routenberg teaches detecting the ligation product ([0193], “In embodiments, the amplification technique is proximity ligation amplification (PLA) using RCA”; [0268]; [0282]).
Routenberg teaches that the detection of the ligation product indicates the cooccurrence of the first, second and third biomarkers on the EV by what is referred to as “EV-stapling” [0087-0088].
Regarding claim 121, Routenberg teaches that the first binder is an antibody directed to an EV-associated membrane bound polypeptide ([0137]; [0160-0161]).
Regarding claim 122, Routenberg teaches that the second binder and the third binder are antibodies directed to different surface protein biomarkers ([0269]; [0351], “Multiple binding reagents may also be desired for binding to different surface markers on the same EV”).
Regarding claim 125, Routenberg teaches that the support is a magnetic bead (Sheet 38 of 72, FIG. 32; [0150]; [0171]).
Regarding claim 126, Routenberg teaches that the first, second, and third biomarkers are part of a target biomarker signature for a disease or condition ([0456-0457]; [0580]).
Regarding claim 127, Routenberg teaches that the disease is cancer [0458-0459].
Regarding claim 130, Routenberg teaches further comprising amplifying the ligation product prior to the detecting step ([0193], “In embodiments, the amplification technique is proximity ligation amplification (PLA) using RCA”).
Regarding claim 131, Routenberg teaches that the amplifying step comprises quantitative PCR (qPCR) [0582].
Regarding claim 134, Routenberg teaches that the method further comprises removing binders not bound to the EV after the capturing step ([0043]; [0586]).
Regarding claim 135, Routenberg teaches that the first binder is an aptamer ([0372], “In embodiments, the binding reagent is an aptamer, and a single oligonucleotide is synthesized containing the aptamer and the detection sequence”).
Regarding claim 136, Routenberg teaches that the second and third biomarkers are tumor-associated antigens ([0448]; [0459], “To develop a 10-plex of assays for tumor-associated EVs, 16 antibodies were arrayed against 13 different antigens suspected to be present on tumor-derived EVs in two 8-plexes”).
Regarding claim 137, Routenberg teaches that the ligating step can be performed using a T7 DNA ligase [0406].
Regarding claim 138, Routenberg teaches fixing the captured EV prior to the introducing step ([0039]; Sheet 33 of 72, Fig. 27A).
Regarding claim 139, Routenberg teaches purifying the EV from a sample using size exclusion chromatography prior to the capturing step [0066].
Regarding claim 140, Routenberg teaches that the sample is plasma [0465].
Regarding claim 120, Routenberg does not teach that the first oligonucleotide and the second oligonucleotide each comprise a double-stranded portion and a single-stranded overhang portion. Also, Routenberg does not teach that the single-stranded overhangs are complementary to each other.
Regarding claim 120, Fredriksson teaches that the first oligonucleotide and the second oligonucleotide each comprise a double-stranded portion and a single-stranded overhang portion as in the proximity ligation (Column 10, lines 1-14).
Fredriksson teaches that the single-stranded overhang portions of the first and second oligonucleotides are complementary to each other (Column 12, lines 19-26).
It would have been obvious for a PHOSITA before the effective filing date of the application to use the proximity probes of Fredriksson in the proximity ligation method of Routenberg because Fredriksson manufactured a pair of proximity probes that bind simultaneously to a target analyte and directly interact with each other upon binding to the target analyte (column 3, lines 14-23). A skilled artisan would have had a reasonable expectation of success in adapting the proximity probes of Fredriksson for the method of Routenberg for the detection of extracellular vesicles that are involved in cancer for example because such an adaptation of methods would have resulted in a predictable and advantageous use of prior art elements according to their established functions as described in their methods and discussions. When known elements are simply arranged with each performing the same function it had been known to perform and yields no more than one would expect from such an arrangement, the combination is obvious. A PHOSITA would have had a reasonable expectation of success in combining the methods and discussions of Fredriksson and Routenberg because the methods are directed to detecting target analytes by using proximity ligation assays.
It would have been obvious for a PHOSITA to further improve the proximity probes of proximity ligation assays by directly ligating the two probes. When a work is available in one field of endeavor, design incentives and other market forces can prompt variations of it, either in the same field or a different one. If a person of ordinary skill can implement a predictable variation, § 103 may bar its patentability. When considering obviousness of a combination of known elements, the operative question is thus “whether the improvement is more than the predictable use of prior art elements according to their established functions.” (MPEP 2141. I.) In the instant case, it has been well established that proximity ligation assay can expand the range of DNA amplification applications to include the sensitive, accurate and robust identification of protein markers and Extracellular Vesicles (Fredriksson et al. and Routenberg et al.). A skilled artisan would have been motivated to combine the two methods of Fredriksson and Routenberg to produce a proximity ligation assay that is sensitive and specific for the target analyte.
Claim 141 is rejected under 35 U.S.C. 103 as being unpatentable over Routenberg et al. (US 2021/0382043 A1, priority to 10/23/2018) and Fredriksson et al. (US 10,781,473 B2) as applied to claim 120 above, and further in view of Schallmeiner et al. (Nature Methods, VOL.4, NO.2, February 2007).
Claim 141 recites:
“The method of claim 120, further comprising introducing an inhibitor oligonucleotide complementary to at least one of the single-stranded overhang portions, wherein the inhibitor oligonucleotide lacks a primer binding site”.
Regarding claim 141, Routenberg and Fredriksson teach all of the limitations of claim 141 as previously discussed.
Regarding claim 141, Routenberg does not teach introducing an inhibitor oligonucleotide complementary to at least one of the single-stranded overhang portions.
Routenberg does not teach that the inhibitor oligonucleotide lacks a primer binding site.
Regarding claim 141, Schallmeiner teaches using blocking oligonucleotides that can hybridize to the ligatable 3´ and 5´ ends of the proximity probes (Page 135, right column, “locking oligonucleotides that hybridize to the …”). Schallmeiner teaches that these blocking oligonucleotides ligate to the ends of proximity probes that did not hybridize to the oligonucleotide on a third proximity probe (Page 135, right column, “These blocking oligonucleotides ligate to the ends of proximity probes …”). Schallmeiner teaches that the blocking oligos do not have a primer binding site as indicated by the displacement of blocking oligos by the third proximity probe containing the template sequence (Page 135, Figure 1, (b), “the blocking oligonucleotides are outcompeted by the third proximity probe containing the template sequence”)
It would have been obvious for a PHOSITA before the effective filing date of the application to combine Schallmeiner’s blocking oligonucleotides with the methods of Fredriksson and Routenberg to improve the specificity of proximity ligation assay for detecting EVs because Schallmeiner describes how to use blocking oligos in the proximity ligation assay (Page 135, Figure 1, “(a) Blocking of free proximity probes by competition oligonucleotides”). A PHOSITA would have had a reasonable expectation of success in combining the methods and discussions of Schallmeiner, Fredriksson and Routenberg because the methods are directed to detecting analytes by using proximity ligation assays.
Claims 142-143 are rejected under 35 U.S.C. 103 as being unpatentable over Routenberg et al. (US 2021/0382043 A1, priority to 10/23/2018) and Fredriksson et al. (US 10,781,473 B2) as applied to claim 120 above, and further in view of Lehman et al. (Science, Volume 186, 29 November 1974, 790-797).
Claim 142 recites:
“The method of claim 120, wherein the step of directly ligating creates a newly formed phosphodiester bond that covalently joins the first oligonucleotide to the second oligonucleotide”.
Regarding claim 142-143, the teachings of Fredriksson and Routenberg are previously discussed.
Additionally, regarding claims 142-143, Routenberg teaches that the step of directly ligating creates newly formed phosphodiester bond that covalently joins the first oligonucleotide to the second oligonucleotide and is catalyzed by a ligase enzyme ([380]; [406]).
Regarding claims 142-143, Routenberg does not teach that ligase enzyme forms a covalent bond between the 3’ hydroxyl group of one oligonucleotide and a 5’ phosphate group of the other oligonucleotide to form a phosphodiester bond.
Regarding claims 142-143, Lehman teaches that ligase enzyme forms a covalent bond between the 3’ hydroxyl group of one oligonucleotide and a 5’ phosphate group of the other oligonucleotide to form a phosphodiester bond (Page 791, middle column, third paragraph, “the T4 DNA ligase, but not the E. coli enzyme, can catalyze the joining of oligodeoxynucleotides or oligoribonucleotides in RNA-DNA hybrid duplexes”; page 797, left column, Summary, “Both enzymes catalyze the synthesis of phosphodiester bonds between adjacent 5'-phosphoryl and 3'-hydroxyl groups in nicked duplex DNA”).
It would have been obvious for a PHOSITA before the effective filing date of the application to combine the ligase activity of Lehman with the combined methods of Fredriksson and Routenberg to improve the proximity ligation assay for detecting EVs because Lehman teaches the efficient ligation of oligos to each other by ligases by forming a phosphodiester bond (Page 791, middle column, third paragraph; page 797, left column, Summary) and the ability to construct recombinant DNA molecules in vitro (Page 796, middle column, third paragraph). A PHOSITA would have had a reasonable expectation of success in combining the methods and discussions of Lehman, Fredriksson and Routenberg because the methods are directed to using ligation assays for recombinant DNA.
Double Patenting
The nonstatutory double patenting rejection is based on a judicially created doctrine grounded in public policy (a policy reflected in the statute) so as to prevent the unjustified or improper timewise extension of the “right to exclude” granted by a patent and to prevent possible harassment by multiple assignees. A nonstatutory double patenting rejection is appropriate where the conflicting claims are not identical, but at least one examined application claim is not patentably distinct from the reference claim(s) because the examined application claim is either anticipated by, or would have been obvious over, the reference claim(s). See, e.g., In re Berg, 140 F.3d 1428, 46 USPQ2d 1226 (Fed. Cir. 1998); In re Goodman, 11 F.3d 1046, 29 USPQ2d 2010 (Fed. Cir. 1993); In re Longi, 759 F.2d 887, 225 USPQ 645 (Fed. Cir. 1985); In re Van Ornum, 686 F.2d 937, 214 USPQ 761 (CCPA 1982); In re Vogel, 422 F.2d 438, 164 USPQ 619 (CCPA 1970); In re Thorington, 418 F.2d 528, 163 USPQ 644 (CCPA 1969).
A timely filed terminal disclaimer in compliance with 37 CFR 1.321(c) or 1.321(d) may be used to overcome an actual or provisional rejection based on nonstatutory double patenting provided the reference application or patent either is shown to be commonly owned with the examined application, or claims an invention made as a result of activities undertaken within the scope of a joint research agreement. See MPEP § 717.02 for applications subject to examination under the first inventor to file provisions of the AIA as explained in MPEP § 2159. See MPEP § 2146 et seq. for applications not subject to examination under the first inventor to file provisions of the AIA . A terminal disclaimer must be signed in compliance with 37 CFR 1.321(b).
The filing of a terminal disclaimer by itself is not a complete reply to a nonstatutory double patenting (NSDP) rejection. A complete reply requires that the terminal disclaimer be accompanied by a reply requesting reconsideration of the prior Office action. Even where the NSDP rejection is provisional the reply must be complete. See MPEP § 804, subsection I.B.1. For a reply to a non-final Office action, see 37 CFR 1.111(a). For a reply to final Office action, see 37 CFR 1.113(c). A request for reconsideration while not provided for in 37 CFR 1.113(c) may be filed after final for consideration. See MPEP §§ 706.07(e) and 714.13.
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 120-122, 125-127, 130-131 and 134-143 are rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1-3, 8-9, 11, 13, 14-15, 17, 19 and 20-23 of U.S. Patent No. US 11,085,089 B2 in view of Routenberg et al. (US 2021/0382043 A1, priority to 10/23/2018) and of Lehman et al. (Science, Volume 186, 29 November 1974, 790-797).
Regarding claim 120 pertaining to a method comprising capturing an extracellular vesicle (EV) by binding a first biomarker on the EV to a first binder linked to a support, introducing to the captured EV, a second binder and a third binder, wherein the second binder and the third binder bind to a second biomarker and a third biomarker, wherein the second and third biomarkers are surface protein biomarkers present on the EV, the second binder being linked to a first oligonucleotide and a third binder linked to a second oligonucleotide, wherein the first oligonucleotide and the second oligonucleotide each comprise a double-stranded portion and a single-stranded overhang, the single-stranded overhangs being complementary to each other, directly ligating the first oligonucleotide to the second oligonucleotide when the single stranded overhang portions hybridize due to co-localization of the second and third biomarkers on the captured EV, thereby forming a ligation product, and detecting the ligation product, wherein detection of the ligation product indicates co-occurrence of the first biomarker, the second biomarker, and the third biomarker on the EV, ‘089 teaches a method of capturing an extracellular vesicle by using three targets with three detection probes wherein each detection probe comprises an oligonucleotide domain that comprise a double stranded portion and a single stranded overhang that is complementary to each other. ‘089 further teaches that the first single stranded overhang and the second single stranded overhang are complementary to each other and are hybridized to each other to ligate the two oligos for detecting the ligation product to indicate the presence of the three targets (See claims 1, 13, 21 and 22 of ‘089).
Regarding claim 121 pertaining to that the first binder is an antibody directed to an EV-associated membrane bound polypeptide, ‘089 teaches that the first binder is an antibody directed to an EV-associated membrane bound polypeptide (See claims 1, 8, 19, 21 and 22 of ‘089).
Regarding claim 122 pertaining to that the second binder and the third binder are antibodies, ‘089 teaches that the second binder and the third binder are antibodies (See claims 8-9 of ‘089).
Regarding claim 126 pertaining to that the first, second, and third biomarkers are part of a target biomarker signature for a disease or condition, ‘089 teaches that one of the captured extracellular vesicles expresses a target biomarker signature for a disease, disorder, or condition (See claims 14-15 of ‘089).
Regarding claim 127 pertaining to that the disease is cancer, ‘089 teaches that the disease, disorder, or condition is cancer (See claim 15 of ‘089).
Regarding claim 130 pertaining to amplifying the ligation product prior to the detecting step, ‘089 teaches that detecting comprises performing amplification of the ligated template and detecting the presence of the amplification product (See claim 2 of ‘089).
Regarding claim 131 pertaining to that the amplifying step comprises quantitative PCR (qPCR), ‘089 teaches that the amplification is or comprises quantitative polymerase chain reaction (See claim 3 of ‘089).
Regarding claim 139 pertaining to purifying the EV from a sample using size exclusion chromatography prior to the capturing step, ‘089 teaches that the sample has been subjected to size exclusion chromatography to isolate nanoparticles having a size range of interest that includes extracellular vesicles (See claim 20 of ‘89).
Regarding claim 140 pertaining to that the sample is plasma, ‘089 teaches that the sample is plasma or blood derived (See claims 17 and 23 of ’89).
Regarding claim 141 pertaining to introducing an inhibitor oligonucleotide complementary to at least one of the single-stranded overhang portions, wherein the inhibitor oligonucleotide lacks a primer binding site, ‘089 teaches a set of detection probes that further comprises a control probe that is characterized in that binding of the control probe to the entity of interest inhibits generation of a ligated
template and/or inhibits amplification of a ligated template from a non-target biological entity (See claim 11 of ‘089).
Regarding claim 125 pertaining to that the support is a magnetic bead, ‘089 does not teach that the bead is magnetic.
Regarding claim 134 pertaining to removing binders not bound to the EV after the capturing step, 089 does not teach removing binders not bound to the EV after the capturing step.
Regarding claim 135 pertaining to that the first binder is an aptamer, ‘089 does not teach that the first binder is an aptamer.
Regarding claim 136 pertaining to that the second and third biomarkers are tumor-associated antigens, ‘089 does not teach that the second and third biomarkers are tumor-associated antigens.
Regarding claim 137 pertaining to that the ligating step is performed using a T7 DNA ligase or a T4 DNA ligase, 089 does not teach that the ligating step is performed using a T7 DNA ligase or a T4 DNA ligase.
Regarding claim 138 pertaining to fixing the captured EV prior to the introducing step, ‘089 does not teach fixing the captured EV prior to the introducing step.
Regarding claims 142 pertaining to that the step of directly ligating creates a newly formed phosphodiester bond that covalently joins the first oligonucleotide to the second oligonucleotide, ‘089 does not teach the step of directly ligating creates a newly formed phosphodiester bond that covalently joins the first oligonucleotide to the second oligonucleotide.
Regarding claim 143 pertaining to that the directly ligating step is catalyzed by a ligase enzyme that forms a covalent bond between a 3' hydroxyl group of one oligonucleotide and a 5' phosphate group of the other oligonucleotide, ‘089 does not teach that the directly ligating step is catalyzed by a ligase enzyme that forms a covalent bond between a 3' hydroxyl group of one oligonucleotide and a 5' phosphate group of the other oligonucleotide.
Regarding claim 125, Routenberg teaches that wherein the support is a magnetic bead (Sheet 38 of 72, FIG. 32; [0150]; [0171]).
Regarding claim 134, Routenberg teaches that the method further comprises removing binders not bound to the EV after the capturing step ([0043]; [0586]).
Regarding claim 135, Routenberg teaches that the first binder is an aptamer ([0372], “In embodiments, the binding reagent is an aptamer, and a single oligonucleotide is synthesized containing the aptamer and the detection sequence”).
Regarding claim 136, Routenberg teaches that the second and third biomarkers are tumor-associated antigens ([0448]; [0459], “To develop a 10-plex of assays for tumor-associated
EVs, 16 antibodies were arrayed against 13 different antigens suspected to be present on tumor-derived EVs in two 8-plexes”).
Regarding claim 137, Routenberg teaches that the ligating step can be performed using a T7 DNA ligase [0406].
Regarding claim 138, Routenberg teaches fixing the captured EV prior to the introducing step ([0039]; Sheet 33 of 72, Fig. 27A).
Regarding claims 142-143, Routenberg teaches that the step of directly ligating creates newly formed phosphodiester bond that covalently joins the first oligonucleotide to the second oligonucleotide and is catalyzed by a ligase enzyme ([380]; [406]).
Regarding claims 142-143, Lehman teaches that ligase enzyme forms a covalent bond between the 3’ hydroxyl group of one oligonucleotide and a 5’ phosphate group of the other oligonucleotide to form a phosphodiester bond (Page 791, middle column, third paragraph, “the T4 DNA ligase, but not the E. coli enzyme, can catalyze the joining of oligodeoxynucleotides or oligoribonucleotides in RNA-DNA hybrid duplexes”; page 797, left column, Summary, “Both enzymes catalyze the synthesis of phosphodiester bonds between adjacent 5'-phosphoryl and 3'-hydroxyl groups in nicked duplex DNA”).
It would have been obvious to one of ordinary skill in the art at the time the application was filed to add or combine the magnetic beads of Routenberg with the biomarker testing of EVs of ‘089 because Routenberg noted that magnetic beads are commonly used for purification purposes ([0150] and [0171]). The ordinary artisan would have been motivated to do so because Routenberg noted the specificity of their method in isolating EVs of interest from samples [0073]. Furthermore, a skilled artisan would have been further motivated to combine the ligase activity of Lehman with the combined methods of Routenberg and ’89 because Lehman teaches the efficient ligation of oligos to each other by ligases by forming a phosphodiester bond (Page 791, middle column, third paragraph; page 797, left column, Summary) and the ability to construct recombinant DNA molecules in vitro (Page 796, middle column, third paragraph).Therefore, such a combination would be considered an advantageous additive to ‘089 which also recognizes a need for improving measurements.
Claims 120-122, 125-127, 130-131 and 134-143 are rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1-10 of U.S. Patent No. US 12,674,190 B2 in view of Routenberg et al. (US 2021/0382043 A1, priority to 10/23/2018), Fredriksson et al. (US 10,781,473 B2), Schallmeiner et al. (Nature Methods, VOL.4, NO.2, February 2007) and Lehman et al. (Science, Volume 186, 29 November 1974, 790-797).
Although the claims at issue are not identical, they are not patentably distinct from each other because:
Regarding claim 120 pertaining to a method comprising capturing an extracellular vesicle (EV) by binding a first biomarker on the EV to a first binder linked to a support, introducing to the captured EV, a second binder and a third binder, wherein the second binder and the third binder bind to a second biomarker and a third biomarker, wherein the second and third biomarkers are surface protein biomarkers present on the EV, the second binder being linked to a first oligonucleotide and a third binder linked to a second oligonucleotide, wherein the first oligonucleotide and the second oligonucleotide each comprise a double-stranded portion and a single-stranded overhang, the single-stranded overhangs being complementary to each other, directly ligating the first oligonucleotide to the second oligonucleotide when the single stranded overhang portions hybridize due to co-localization of the second and third biomarkers on the captured EV, thereby forming a ligation product, and detecting the ligation product, wherein detection of the ligation product indicates co-occurrence of the first biomarker, the second biomarker, and the third biomarker on the EV, ‘190 teaches a method of capturing an extracellular vesicle (EV) by using a capturing agent that binds to a first protein on the surface of EV and two detection probes wherein each detection probe comprises an oligonucleotide domain that comprise a double stranded portion and a single stranded overhang that is complementary to each other. ‘089 further teaches that the first single stranded overhang and the second single stranded overhang are complementary to each other and are hybridized to each other so that the two oligos are ligated for detecting a ligation product to indicate the presence of the three proteins (See claims 1, 2 and 3 of ‘190).
Regarding claim 130 pertaining to amplifying the ligation product prior to the detecting step, ‘190 teaches that detecting comprises performing amplification of the ligated template and detecting the presence of the amplification product (See claim 6 of ‘190).
Regarding claim 131 pertaining to that the amplifying step comprises quantitative PCR (qPCR), ‘190 teaches that the amplification is or comprises quantitative polymerase chain reaction (See claim 7 of ‘190).
Regarding claim 121 pertaining to that the first binder is an antibody directed to an EV-associated membrane bound polypeptide, 190 does not teach that the first binder is an antibody directed to an EV-associated membrane bound polypeptide.
Regarding claim 122 pertaining to that the second binder and the third binder are antibodies, 190 does not teach that the second binder and the third binder are antibodies.
Regarding claim 125 pertaining to that the support is a magnetic bead, 190 does not teach that the bead is magnetic.
Regarding claim 126 pertaining to that the first, second, and third biomarkers are part of a target biomarker signature for a disease or condition, 190 does not teach that one of the captured extracellular vesicles expresses a target biomarker signature for a disease, disorder, or condition.
Regarding claim 127 pertaining to that the disease is cancer, 190 does not teach that the disease, disorder, or condition is cancer.
Regarding claim 134 pertaining to removing binders not bound to the EV after the capturing step, 190 does not teach removing binders not bound to the EV after the capturing step.
Regarding claim 135 pertaining to that the first binder is an aptamer, 190 does not teach that the first binder is an aptamer.
Regarding claim 136 pertaining to that the second and third biomarkers are tumor-associated antigens, ‘190 does not teach that the second and third biomarkers are tumor-associated antigens.
Regarding claim 138 pertaining to fixing the captured EV prior to the introducing step, ‘190 does not teach fixing the captured EV prior to the introducing step.
Regarding claim 139 pertaining to purifying the EV from a sample using size exclusion chromatography prior to the capturing step, 190 does not teach that the sample has been subjected to size exclusion chromatography to isolate nanoparticles having a size range of interest that includes extracellular vesicles.
Regarding claim 140 pertaining to that the sample is plasma, 190 does not teach that the sample is plasma or blood derived.
Regarding claim 141 pertaining to introducing an inhibitor oligonucleotide complementary to at least one of the single-stranded overhang portions, wherein the inhibitor oligonucleotide lacks a primer binding site, 190 does not teach a set of detection probes that further comprises a control probe that is characterized in that binding of the control probe to the entity of interest inhibits generation of a ligated
template and/or inhibits amplification of a ligated template from a non-target biological entity.
Regarding claims 142-143 pertaining
Regarding claims 142 pertaining to that the step of directly ligating creates a newly formed phosphodiester bond that covalently joins the first oligonucleotide to the second oligonucleotide, 190 does not teach the step of directly ligating creates a newly formed phosphodiester bond that covalently joins the first oligonucleotide to the second oligonucleotide.
Regarding claim 143 pertaining to that the directly ligating step is catalyzed by a ligase enzyme that forms a covalent bond between a 3' hydroxyl group of one oligonucleotide and a 5' phosphate group of the other oligonucleotide, ‘190 does not teach that the directly ligating step is catalyzed by a ligase enzyme that forms a covalent bond between a 3' hydroxyl group of one oligonucleotide and a 5' phosphate group of the other oligonucleotide.
Regarding claim 121, Routenberg teaches that wherein the first binder is an antibody directed to an EV-associated membrane bound polypeptide ([0137]; [0160-0161]).
Regarding claim 122, Routenberg teaches that wherein the second binder and the third binder are antibodies directed to different surface protein biomarkers ([0269]; [0351], “Multiple binding reagents may also be desired for binding to different surface markers on the same EV”).
Regarding claim 125, Routenberg teaches that wherein the support is a magnetic bead (Sheet 38 of 72, FIG. 32; [0150]; [0171]).
Regarding claim 126, Routenberg teaches that wherein the first, second, and third biomarkers are part of a target biomarker signature for a disease or condition ([0456-0457]; [0580]).
Regarding claim 127, Routenberg teaches that wherein the disease is cancer [0458-0459].
Regarding claim 134, Routenberg teaches that the method further comprise removing binders not bound to the EV after the capturing step ([0043]; [0586]).
Regarding claim 135, Routenberg teaches that the first binder is an aptamer ([0372], “In embodiments, the binding reagent is an aptamer, and a single oligonucleotide is synthesized containing the aptamer and the detection sequence”).
Regarding claim 136, Routenberg teaches that the second and third biomarkers are tumor-associated antigens ([0448]; [0459], “To develop a 10-plex of assays for tumor-associated
EVs, 16 antibodies were arrayed against 13 different antigens suspected to be present on tumor-derived EVs in two 8-plexes.”).
Regarding claim 138, Routenberg teaches fixing the captured EV prior to the introducing step ([0039]; Sheet 33 of 72, Fig. 27A).
Regarding claim 139, Routenberg teaches purifying the EV from a sample using size exclusion chromatography prior to the capturing step [0066].
Regarding claim 140, Routenberg teaches that the sample is plasma [0465].
Regarding claims 142-143, Routenberg teaches that the step of directly ligating creates newly formed phosphodiester bond that covalently joins the first oligonucleotide to the second oligonucleotide and is catalyzed by a ligase enzyme ([380]; [406]).
Regarding claim 141, Schallmeiner teaches using blocking oligonucleotides that can hybridize to the ligatable 3´ and 5´ ends of the proximity probes (Page 135, right column, “locking oligonucleotides that hybridize to the …”). Schallmeiner teaches that these blocking oligonucleotides ligate to the ends of proximity probes that did not hybridize to the oligonucleotide on a third proximity probe (Page 135, right column, “These blocking oligonucleotides ligate to the ends of proximity probes …”). Schallmeiner teaches that the blocking oligos do not have a primer binding site as indicated by the displacement of blocking oligos by the third proximity probe containing the template sequence (Page 135, Figure 1, (b), “the blocking oligonucleotides are outcompeted by the third proximity probe containing the template sequence”).
Regarding claims 142-143, Lehman teaches that ligase enzyme forms a covalent bond between the 3’ hydroxyl group of one oligonucleotide and a 5’ phosphate group of the other oligonucleotide to form a phosphodiester bond (Page 791, middle column, third paragraph, “the T4 DNA ligase, but not the E. coli enzyme, can catalyze the joining of oligodeoxynucleotides or oligoribonucleotides in RNA-DNA hybrid duplexes”; page 797, left column, Summary, “Both enzymes catalyze the synthesis of phosphodiester bonds between adjacent 5'-phosphoryl and 3'-hydroxyl groups in nicked duplex DNA”).
It would have been obvious to one of ordinary skill in the art at the time the application was filed to add or combine the magnetic beads of Routenberg with the biomarker testing of EVs of ‘190 because Routenberg noted that magnetic beads are commonly used for purification purposes ([0150] and [0171]). The ordinary artisan would have been motivated to do so because Routenberg noted the specificity of their method in isolating EVs of interest from samples [0073]. The ordinary artisan would be further motivated to combine the inhibitory oligos of Schallmeiner with the method of ‘190 because it will prevent non-specific binding and false detection of target entities. Also, a skilled artisan would have been further motivated to combine the ligase activity of Lehman with the combined methods of Schallmeiner, Routenberg and ‘190 because Lehman teaches the efficient ligation of oligos to each other by ligases by forming a phosphodiester bond (Page 791, middle column, third paragraph; page 797, left column, Summary) and the ability to construct recombinant DNA molecules in vitro (Page 796, middle column, third paragraph). Therefore, such a combination would be considered an advantageous additive to ‘190 which also recognizes a need for improving measurements.
Response to Arguments
Applicant’s arguments, see pages 7-8 of Applicant’s Remarks, filed 07/13/2026, with respect to the rejections of claims 120-122, 125-127, 130-131 and 134-143 under 35 U.S.C. 103, regarding obviousness, have been fully considered and are persuasive.
Specifically, the Applicant noted that Greenwood did not teach in a proximity ligation assay (PLA) that the first oligonucleotide and the second oligonucleotide each comprise a double-stranded portion and a single-stranded overhang portion. Also, the Applicant further noted that Greenwood did not teach in a PLA directly ligating the two oligos nor did it teach or suggest that they are complementary to each other. The Applicant concluded that if Greenwood taught these limitations, they are part of a proximity extension assay (PEA) and not PLA. And none of the secondary references teach or suggest these limitations. Therefore, the previous rejections of claims 120-122, 125-127, 130-131 and 134-143 under 35 U.S.C. 103 have been withdrawn.
However, upon further consideration, new grounds of rejection are made in view of Fredriksson et al. (US 10,781,473 B2). Specifically, Fredriksson teaches in a PLA that the first oligonucleotide and the second oligonucleotide each comprise a double-stranded portion and a single-stranded overhang portion as in the proximity ligation (Column 10, lines 1-14). Fredriksson also teaches that the single-stranded overhang portions of the first and second oligonucleotides are complementary to each other and are directly ligated (Column 3, lines 14-23; column 12, lines 19-26).
Conclusion
No claims are allowed.
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/OMAR RAMADAN/Examiner, Art Unit 1678
/GREGORY S EMCH/Supervisory Patent Examiner, Art Unit 1678
