DETAILED ACTION
Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
Priority
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.
Claim Status
Claims 1-119, 123-124, 128-129 and 132-133 are cancelled. Claims 120-122, 125-127, 130-131 and 134-141 are previously presented. Claims 142-143 are new and the Applicant notes that no new matter is added.
Thus, claims 120-122, 125-127, 130-131 and 134-143 are pending and are under examination.
Maintained and 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 Greenwood et al. (Biomolecular Detection and Quantification 4 (2015) 10–16).
Regarding claim 120, the claim 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 singlestranded 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 cooccurrence 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”; Page 2, [0013])
Routenberg teaches introducing to the captured EV a second binder and a third binder (Page 25, [0269]; page 33-34, [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 (page 33-34, [0351]).
Routenberg teaches that the second and third biomarkers are surface protein biomarkers present on the EV (Page 25, [0269]; page 31, [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.”; page 33-34, [0351; page 43, [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”; page 45, [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”).
Regarding claim 120, Routenberg further teaches that the first and second oligonucleotides are complementary to each other (Page 19, [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”; page 7, [0088]; page 36, [380]; page 40, [406]).
Routenberg teaches that the ligation of the first oligonucleotide to the second oligonucleotide happens when the single-stranded overhang portions hybridize due to the co-localization of the second and third biomarkers on the captured EV (Sheet 38 of 72, FIG. 32, “Ligation Site”, Page 7, [0088]).
Routenberg teaches detecting the ligation product (Page 17, [0193], “In embodiments, the amplification technique is proximity ligation amplification (PLA) using RCA”; page 25, [0268]; page 27, [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” (Page 7, [0087-0088]).
Regarding claim 121, Routenberg teaches that wherein the first binder is an antibody directed to an EV-associated membrane bound polypeptide (Page 12, [0137]; page 14, [0160-0161]).
Regarding claim 122, Routenberg teaches that wherein the second binder and the third binder are antibodies directed to different surface protein biomarkers(Page 25, [0269]; page 33-34, [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; page 13, [0150]; page 15, [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 (Page 44, [0456-0457]; page 52, [0580]).
Regarding claim 127, Routenberg teaches that wherein the disease is cancer (Page 44, [0458-0459]).
Regarding claim 130, Routenberg teaches further comprising amplifying the ligation product prior to the detecting step (Page 17, [0193], “In embodiments, the amplification technique is proximity ligation amplification (PLA) using RCA”).
Regarding claim 131, Routenberg teaches that wherein the amplifying step comprises quantitative PCR(qPCR) (Page 53, [0582]).
Regarding claim 134, Routenberg teaches that the method further comprise removing binders not bound to the EV after the capturing step (Page 4, [0043]; page 53, [0586]).
Regarding claim 135, Routenberg teaches that the first binder is an aptamer (Page 36, [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 (Page 44, [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 (Page 40, [0406]).
Regarding claim 138, Routenberg teaches fixing the captured EV prior to the introducing step (Page 3, [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 (Page 5, [0066]).
Regarding claim 140, Routenberg teaches that the sample is plasma (Page 45, [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.
Routenberg does not teach that the single-stranded overhangs are complementary to each other.
Regarding claim 120, Greenwood teaches that wherein the first oligonucleotide and the second oligonucleotide each comprise a double-stranded portion and a single-stranded overhang portion as in the proximity ligation (Page 13, left column, first paragraph, “Configuring the assay through the binding of three independent affinity reagents to the same target molecule can further enhance specificity of signal generation”, “In this triple antibody specific proximity ligation assay, the third proximity ligation probe replaces the connector oligonucleotide as a ligation template.”). Greenwood further teaches the ability to use an overhang as part of one of the two oligos that can hybridize to a second oligo to form a product that can be detected (Page 14, Fig 4., “The 3’-oligonucleotide is double stranded with a 3’-overhang. In the presence of target antigen, the probe oligonucleotides can hybridize to each other”). Greenwood teaches that different combinations of 5’- and 3’-oligonucleotides results in unique sequences that serves as primer sites for target-specific amplification and quantification by qPCR (page 13, left column, third paragraph, “Multiplex PLAs have been developed, where the ligation of numerous PLA probes linked to different combinations of 5’- and 3’-oligonucleotides results in unique sequences that serve as primer sites for target-specific amplification and quantification by qPCR”).
Greenwood teaches that wherein the single-stranded overhang portions of the first and second oligonucleotides are complementary to each other (Page 14, Fig 4., (B), “The 3’-oligonucleotide is double stranded with a 3’-overhang. In the presence of target antigen, the probe oligonucleotides can hybridize to each other”). As stated above, Greenwood showed that different combinations are made for detecting a target molecule with proximity probes.
It would have been obvious for a PHOSITA before the effective filing date of the application to modify the method of Routenberg with Greenwood teachings to improve the proximity ligation assay for detecting EVs because Greenwood noted that proximity probes joined by a DNA ligase suffer from recovery loss in complex biological fluids (Page 13, left column, last paragraph) and further noted that proximity extension assay has the advantages of very low sample consumption, high sensitivity and specificity detection in a homogeneous reaction (Page 13, right column, first paragraph). A skilled artisan would have been motivated to use proximity ligation/ extension in Routenberg’s method because Routenberg suggested using proximity ligation/extension for its ability to identify the target surface molecules on extracellular vesicles (Page 53, [0581]). A skilled artisan would have had a reasonable expectation of success in adapting the methods of Greenwood and Routenberg for the detection of EVs that are involved in cancer for example because such an adaptation of methods would have resulted in a predictable 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 Greenwood and Routenberg because the methods are directed to detecting markers of extracellular vesicles by using proximity ligation/ extension assays.
It would have been obvious for a PHOSITA to further improve the proximity probes of proximity ligation/ extension assays to improve the probe recovery in complex biological fluids. 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 EVs (Greenwood et al. and Routenberg et al.). A skilled artisan would have been motivated to combine the two methods of Greenwood 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 Greenwood et al. (Biomolecular Detection and Quantification 4 (2015) 10–16) as applied to claim 120 above, and further in view of Schallmeiner et al. (Nature Methods, VOL.4, NO.2, February 2007).
Regarding claim 141, the claim 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 Greenwood teaches all of the limitations of claim 141 as described above.
Additionally, regarding claim 141, Greenwood teaches the use of short blocking oligonucleotides to prevent ligation in the absence of the target molecule to minimize background noise (Page 13, left column, second paragraph, “Short blocking oligonucleotides prevent ligation in the absence of the target molecule and considerably minimise background noise”).
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.
Moreover, 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 modify the method of Routenberg with Greenwood and Schallmeiner teachings to improve the specificity of proximity ligation assay for detecting EVs because Greenwood teaches using short blocking oligonucleotides to prevent ligation in the absence of the target molecule to minimize background noise (Page 13, left column, second paragraph) and Schallmeiner further 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 skilled artisan would have had a reasonable expectation of success in adapting the methods of Greenwood, Routenberg and Schallmeiner for the detection of EVs that are involved in cancer because such an adaptation of methods would have resulted in a predictable 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 Greenwood and Routenberg because the methods are directed to detecting markers of extracellular vesicles by using proximity ligation/ extension assays.
It would have been obvious for a PHOSITA to further improve the proximity probes of proximity ligation/ extension assays to improve the probe recovery in complex biological fluids and to minimize nonspecific ligations. 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 EVs (Greenwood et al. and Routenberg et al.). Furthermore, the use of blocking oligo to minimize nonspecific ligations is well known (Greenwood et al. and Schallmeiner et al.). A skilled artisan would have been motivated to combine the two methods of Greenwood and Routenberg to produce a proximity ligation assay that is sensitive and specific for the target analyte.
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 Greenwood et al. (Biomolecular Detection and Quantification 4 (2015) 10–16) as applied to claim 120 above, and further in view of Lehman et al. (Science, Volume 186, 29 November 1974, 790-797).
Regarding claim 142, the claim 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, Routenberg and Greenwood teach or suggest all of the limitations as discussed above.
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 (Page 36, [380]; Page 40, [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 Routenberg and Greenwood 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 skilled artisan would have been motivated to combine the above methods of Lehman, Greenwood and Routenberg to the detection of EVs involved in cancer for example because such an adaptation of methods would have resulted in a predictable 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 Greenwood and Routenberg because the methods are directed to using ligation assays for recombinant DNA.
It would have been obvious for a PHOSITA to further improve the proximity probes of proximity ligation to improve the probe recovery in complex biological fluids. 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 EVs (Greenwood et al. and Routenberg et al.). Also, Lehman teaches that DNA ligases has proved to be an invaluable reagent in the construction of recombinant DNA molecules in vitro (Page 796, middle column, third paragraph). A skilled artisan would have been motivated to combine the methods of Lehman, Greenwood and Routenberg to produce a proximity ligation assay that is sensitive and specific for the target analyte.
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; page 13, [0150]; page 15, [0171]).
Regarding claim 134, Routenberg teaches that the method further comprises removing binders not bound to the EV after the capturing step (Page 4, [0043]; page 53, [0586]).
Regarding claim 135, Routenberg teaches that the first binder is an aptamer (Page 36, [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 (Page 44, [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 (Page 40, [0406]).
Regarding claim 138, Routenberg teaches fixing the captured EV prior to the introducing step (Page 3, [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 (Page 36, [380]; Page 40, [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 provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claims 120, 128, 131-133, 135-138 and 233 of copending Application No. 17/204,773 in view of Routenberg et al. (US 2021/0382043 A1, priority to 10/23/2018), Greenwood et al. (Biomolecular Detection and Quantification 4 (2015) 10–16), Schallmeiner et al. (Nature Methods, VOL.4, NO.2, February 2007) and Lehman et al. (Science, Volume 186, 29 November 1974, 790-797).
This is a provisional nonstatutory double patenting rejection because the patentably indistinct claims have not in fact been patented.
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, ‘773 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 120, 128 and 131 of ‘773).
Regarding claim 130 pertaining to amplifying the ligation product prior to the detecting step, ‘773 teaches that detecting comprises performing amplification of the ligated template and detecting the presence of the amplification product (See claim 135 of ‘773).
Regarding claim 131 pertaining to that the amplifying step comprises quantitative PCR (qPCR), ‘773 teaches that the amplification is or comprises quantitative polymerase chain reaction (See claim 136 of ‘773).
Regarding claim 121 pertaining to that the first binder is an antibody directed to an EV-associated membrane bound polypeptide, ‘773 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, ‘773 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, ‘773 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, ‘773 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, ‘773 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, ‘773 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, ‘773 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, ‘773 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, ‘773 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, ‘773 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, ‘773 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, ‘773 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, ‘773 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, ‘773 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 (Page 12, [0137]; page 14, [0160-0161]).
Regarding claim 122, Routenberg teaches that wherein the second binder and the third binder are antibodies directed to different surface protein biomarkers(Page 25, [0269]; page 33-34, [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; page 13, [0150]; page 15, [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 (Page 44, [0456-0457]; page 52, [0580]).
Regarding claim 127, Routenberg teaches that wherein the disease is cancer (Page 44, [0458-0459]).
Regarding claim 134, Routenberg teaches that the method further comprise removing binders not bound to the EV after the capturing step (Page 4, [0043]; page 53, [0586]).
Regarding claim 135, Routenberg teaches that the first binder is an aptamer (Page 36, [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 (Page 44, [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 (Page 3, [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 (Page 5, [0066]).
Regarding claim 140, Routenberg teaches that the sample is plasma (Page 45, [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 (Page 36, [380]; Page 40, [406]).
Regarding claim 141, Greenwood teaches the use of short blocking oligonucleotides to prevent ligation in the absence of the target molecule to minimize background noise (Page 13, left column, second paragraph, “Short blocking oligonucleotides prevent ligation in the absence of the target molecule and considerably minimise background noise”).
Regarding claim 141, Greenwood teaches the use of short blocking oligonucleotides to prevent ligation in the absence of the target molecule to minimize background noise (Page 13, left column, second paragraph, “Short blocking oligonucleotides prevent ligation in the absence of the target molecule and considerably minimise background noise”).
Moreover, 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 ‘773 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 Greenwood and Schallmeiner with the method of ‘773 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, Greenwood, Routenberg and ‘773 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 ‘773 which also recognizes a need for improving measurements.
Response to Arguments
Applicant's arguments filed 12/11/2025 have been fully considered but they are not persuasive.
The Applicant argued that Reference Routenberg fails to disclose or suggest direct ligation and thus an element of the claims of the instant application is not found in Routenberg.
This argument is not persuasive because Routenberg clearly teaches directly ligating the first oligonucleotide to the second oligonucleotide to form one product (Page 36, [380]; page 40, [406]). The formation of one product without a mediator is a confirmation of a direct ligation and the enzyme used is DNA ligase (Page 40, [406], “the first ligation site of the proximal oligonucleotide and second ligation site of the distal oligonucleotide are capable of being ligated together… Methods of ligating oligonucleotides are known in the art and described herein and include, e.g., using a DNA ligase such as the T4 ligase”).
The Applicant still argued that Greenwood teaches away from using proximity ligation assay (PLA) in complex biological fluids and argues that Greenwood reports that proximity extension assay (PEA) is an alternative to PLA.
This argument is not persuasive because a reference that teaches a way for detecting an analyte among other methods in the reference does not necessarily teach away from those other methods of detecting the same analyte. In this case, Greenwood teaches two methods of proximity assays which are proximity ligation assays (PLA) and proximity extension assays (PEA) (Page 11, right column, proximity ligation assays; page 13, left column, proximity extension assays). Although Greenwood gave a reason for why PEA could be used as an alternative to PLA (Page 13, left column, last paragraph), Greenwood still concluded that PLA has multiple applications and advantages such as being used as a useful method for the validation of potential biomarkers for clinical diagnostic needs (Page 13, right column, second and third paragraph). Thus, Greenwood does not teach away from PLA. Furthermore, per MPEP 2123, the prior art’s mere disclosure of more than one alternative does not constitute a teaching away from alternatives because such disclosure does not criticize, discredit, or otherwise discourage the solution claimed. In re Fulton, 391 F.3d 1195, 1201 (Fed. Cir. 2004). In the instant case, Greenwood is providing a reason for using PEA as compared to PLA without discouraging the use of PLA. Actually, Greenwood acknowledges using PLA for detecting proteins directly from biofluids such as blood (Page 13, first paragraph, “PLA can also be configured … an approach that may be more suitable for detecting protein directly from biofluids such as blood”).
The Applicant argued that the Office relies on hindsight to arrive at the direct ligation of the instant application.
This argument is not persuasive because in response to applicant's argument that the examiner's conclusion of obviousness is based upon improper hindsight reasoning, it must be recognized that any judgment on obviousness is in a sense necessarily a reconstruction based upon hindsight reasoning. But so long as it takes into account only knowledge which was within the level of ordinary skill at the time the claimed invention was made, and does not include knowledge gleaned only from the applicant's disclosure, such a reconstruction is proper. See In re McLaughlin, 443 F.2d 1392, 170 USPQ 209 (CCPA 1971). In the instant case, a skilled artisan would have had a reasonable expectation of success in adapting the methods of Greenwood, Routenberg and Schallmeiner for the detection of extracellular vesicles (EVs) that are involved in cancer because such an adaptation of methods would have resulted in a predictable 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.
The Applicant argued that Schallmeiner neither supplies the missing "direct ligation of complementary overhangs" element nor provides a motivation to alter the cited prior art methods into Applicants' claimed overhang-dependent chemistry.
This argument is not persuasive because Schallmeiner is a secondary reference that is brought to teach the missing limitation of inhibitory oligonucleotides. Furthermore, the Applicant is reminded to look at the references in combination. In response to applicant's arguments against the references individually, one cannot show nonobviousness by attacking references individually where the rejections are based on combinations of references. See In re Keller, 642 F.2d 413, 208 USPQ 871 (CCPA 1981); In re Merck & Co., 800 F.2d 1091, 231 USPQ 375 (Fed. Cir. 1986). In the instant case, Schallmeiner is not the primary reference used in the 103 rejection of claims of the instant application.
The Applicant argued that there is not teaching, suggestion, or motivation to combine the references of Schallmeiner, Greenwood, and Routenberg.
This argument is not persuasive because in response to applicant’s argument that there is no teaching, suggestion, or motivation to combine the references, the examiner recognizes that obviousness may be established by combining or modifying the teachings of the prior art to produce the claimed invention where there is some teaching, suggestion, or motivation to do so found either in the references themselves or in the knowledge generally available to one of ordinary skill in the art. See In re Fine, 837 F.2d 1071, 5 USPQ2d 1596 (Fed. Cir. 1988), In re Jones, 958 F.2d 347, 21 USPQ2d 1941 (Fed. Cir. 1992), and KSR International Co. v. Teleflex, Inc., 550 U.S. 398, 82 USPQ2d 1385 (2007). 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 EVs (Greenwood et al. and Routenberg et al.). Furthermore, the use of blocking oligonucleotides to minimize nonspecific ligations is well known (Greenwood et al. and Schallmeiner et al.). A skilled artisan would have been motivated to combine the methods of Schallmeiner, Greenwood, and Routenberg to produce a proximity ligation assay that is sensitive and specific for the target analyte.
Thus, the previous rejection of claims 120-122, 125-127, 130-131 and 134-140 under 35 U.S.C. 103 is maintained and is made final.
Conclusion
No claims are allowed.
Applicant's amendment necessitated the new ground(s) of rejection presented in this Office action. Accordingly, THIS ACTION IS MADE FINAL. See MPEP § 706.07(a). Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action. In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any nonprovisional extension fee (37 CFR 1.17(a)) pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action. In no event, however, will the statutory period for reply expire later than SIX MONTHS from the mailing date of this final action.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to OMAR RAMADAN whose telephone number is (571)270-0754. The examiner can normally be reached Monday-Friday 8:30 am - 5:00 pm.
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/OMAR RAMADAN/Examiner, Art Unit 1678
/GREGORY S EMCH/Supervisory Patent Examiner, Art Unit 1678
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