DETAILED ACTION
Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Continued Examination Under 37 CFR 1.114
A request for continued examination under 37 CFR 1.114, including the fee set forth in 37 CFR 1.17(e), was filed in this application after final rejection. Since this application is eligible for continued examination under 37 CFR 1.114, and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114. Applicant's submission filed on March 19, 2026 has been entered.
Claim Status/Action Summary
This action is in response to the papers filed March 19, 2026.
Claims 98-99 and 101 were canceled in the response. Currently, claims 12-18, 96-97, and 100 are pending.
Claims 12-18, 96-97, and 100 are under examination.
Any objections and rejections not reiterated below are hereby withdrawn.
Priority
This application is a 371 of PCT/US2019/053426, filed September 27, 2019, and claims the benefit of U.S. Provisional Application No. 62/738,537, filed September 28, 2018.
Election/Restrictions
Applicant’s election without traverse of Group II invention, claims 12-20, in the reply filed on July 3, 2024 is acknowledged.
Applicant’s election of species without traverse of “fluorocarbon oil” and “circulating cells” in the reply filed on July 3, 2024 is acknowledged.
Claim Rejections - 35 USC § 103
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
This application currently names joint inventors. In considering patentability of the claims the examiner presumes that the subject matter of the various claims was commonly owned as of the effective filing date of the claimed invention(s) absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and effective filing dates of each claim that was not commonly owned as of the effective filing date of the later invention in order for the examiner to consider the applicability of 35 U.S.C. 102(b)(2)(C) for any potential 35 U.S.C. 102(a)(2) prior art against the later invention.
Claims 12-18, 96, 97 and 100 are/remain rejected under 35 U.S.C. 103 as being unpatentable over Nicol et al. (US 2018/0112212 A1, published April 26, 2018) in view of Griffiths et al. (US 2006/0078888 A1, (published April 13, 2006), Gao et al., US 20140100131 A1 (published April 10, 2014), and Menon V et al. (J Vis Exp. 2014 Dec 18;(94):52241).
Regarding claim 12, Nicol et al. teaches a method of target capture and barcoding in monodisperse droplets. (Nicol, figures 3 and 4). Nicol teaches capture template particles: hydrogel beads comprising “nucleic acid identifiers, (for example a nucleic acid barcode), (i.e. a target-specific capture element genetic identifier comprising a “template-type element” comprising a unique barcode sequence corresponding to the target-specific capture element… attached to a moiety linked to the template particle)” and “target molecule specific binding agents… [such as] an antibody (i.e. a target-specific capture element)” (Nicol, figure 3 and paragraph 0130) as well as “sequences that enable gene specific capture/amplification” (Nicol, figure 3).
Nicol et al. explicitly teach the target-specific capture element genetic identifier comprises a “template-type element” (i.e. that “identifies target particle from which the nucleic acid was derived (instant spec, para 0023)” comprising a barcode sequence corresponding to the target-specific capture element (see Nicol et al., Figure 3, particularly indices Ai-Di)
Nicol et al further explicitly teach that alternative binding agents that are well-known in the art include “aptamer[s]” (Nicol, paragraph 0062) (i.e. the target-specific capture element may comprise an aptamer).
Furthermore, Nicol explicitly teaches “Each bead carries a clonal population of a[n]… index sequence plus sequences that enable gene specific capture/amplification and Illumina library construction and a second population of antibody or protein capture sequences (Nicol, figure 3 caption, see below).
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Nicol et al. teach the target-specific capture element genetic identifier (i.e. the barcode sequences Ai-Di) is attached to a capture element genetic identifier moiety linked to the template particle (see Nicol et al., Figure 3).
Nicol et al. teach the target-specific capture element comprises an antibody (see “X” in figure 3); an aptamer (Nicol et al., paragraph 0062), or “a nucleic acid capture sequence that specifically binds to target nucleic acids”; furthermore, in an example, “the single-stranded part of the hydrogel bead-bound DNA consists of a UMI sequence (i.e. a barcode; a target-specific capture element genetic identifier), followed by a sequence that is antisense to the 3’ end of the target mRNAs. It is well known in the art that mature mRNAs are polyadenylated (i.e. comprise an “(AAA)x” sequence at their 3’ terminus).
Nicol further teaches incubating a mixed population of B cells from patient samples (i.e. a first fluid comprising target particles; circulating cells) with a mixture of DNA-tag labeled (i.e. barcoded) antigens (i.e. a target-specific capture moiety) and hydrogel beads. (Nicol, paragraph 0368). A subset of the B cells display IgM on their surface with affinity to an antigen (HIV gp120). Said hydrogel beads comprise barcoded-primers complementary to the gp120-linked barcodes “the antisense sequence of the TSP [Tag Specific Primer]” and barcoded-primers complementary to specific mRNA target sequences (Nicol, paragraph 0377) (i.e. capture template particles) (Nicol, paragraph 0368 and 0375). Nicol teaches washing the cells “three times in a large volume of PBS” after incubation with the barcoded antigens (Nicol, paragraph 0375) and before encapsulating the cells into monodisperse droplets comprising a reaction buffer and a “hydrogel bead carrying a mix of barcoded-primers complementary to the DNA tags and to [target] mRNAs” (i.e. a/the “plurality of capture template particle” recited in claim 12) using a microfluidic device. (Nicol, paragraph 0368) Nicol teaches that the barcoded antigens (i.e. the capture element) can alternatively be linked to the capture particle (Nicol et al., figure 3) and the capture template particles can be used to determine the expression of cell surface markers on a cell type (or types) of interest. By utilizing an array, or set, of antibodies labeled with indexable nucleic acid identifiers, a population of cells can be analyzed in multiplex to determine the cell surface markers expressed on individual cells, for example individual cells segregated into individual compartments (Nicol et al., paragraph 0111). Nicol further teaches: “Emulsion [Encapsulation] may be achieved by a variety of methods known in the art. (see, for example, [Griffiths et al.], of which paragraphs [0139]-[0143] are incorporated by reference herein)” and “the emulsion may comprise a fluorinated oil” (i.e. a fluorocarbon oil and water mixture – immiscible fluids). (Nicol, paragraph 0157)
Nicol does not teach (i) “generating a supernatant and pellet from the mixture”; “removing the supernatant to separate the plurality of capture template particles and associated target particles from the solution of the first mixture; washing and resuspending the pellet in a reaction buffer…”; “generating a pellet and a supernatant comprises centrifuging” (lines 8 and 11-12 of amended claim 12, and new claim 96); and does not teach that (ii) emulsions can be formed by shearing a mixture of immiscible fluids (lines 16-19 of claim 12).
However, (i) Menon et al. teaches a protocol for labeling cell surface antigens on live or fixed cells (i.e. target particles) with antibodies conjugated to a detectable label (i.e. a target-specific element) prior to fluorescence activated cell sorting. Menon teaches steps for binding the detectable antibody to cell surface antigens (Menon, page 5, protocol 3; step 3-4), centrifuging the binding mixture (Menon, page 6, protocol 3; step 5.1 (i.e. generating a supernatant and pellet), removing the supernatant (Menon, page 6, protocol 3; step 5.2), and washing and resuspending the pellet in a new buffer (Menon, page 6, protocol 3, step 6-7).
Additionally, (i) Gao et al. teaches methods comprising capture and isolation of individual target cells expressing specific cell-surface markers using capture beads comprising target-specific affinity reagents (i.e. target-specific capture elements) such as DNA-indexed antibodies (Gao et al., figure 1). Gao et al. further teach that the target-specific binding molecule (i.e. the capture element) may comprise an aptamer (i.e. Gao et al. teach the target-specific capture element comprises an aptamer) (Gao et al., paragraph 0086).
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Gao et al. further teaches that any suitable method for isolation of captured targets can be used such as magnetic capture or centrifugation prior to downstream analyses (Gao et al., paragraph 0126) and that the methods can comprise one or more washing steps after a contacting step to wash away any leftover reagents from the contacting step and that “such washing steps are well known in the art” (Gao et al., paragraph 0128).
Further, (ii) Griffiths teaches: “Emulsions may be produced from any suitable combination of immiscible liquids… preferably…an aqueous liquid… and an oil.” (Griffiths, paragraph 0140) (i.e. a second mixture and second fluid wherein the second fluid is immiscible with the second mixture) and “Creation of an emulsion generally requires the application of mechanical energy to force the phases together.” (i.e. shearing). (Griffiths, paragraph 0143) The claimed invention describes “Any suitable method or technique may be utilized to apply a sufficient shear force to the second mixture. For example, the second mixture may be sheared by flowing the second mixture through a pipette tip. Other methods include, but are not limited to, shaking the second mixture with a homogenizer (e.g., vortexer), or shaking the second mixture with a bead beater.” (paragraph 00105)
Therefore, it would have been prima facie obvious prior to the effective filing date of the claimed invention for one of ordinary skill in the art to use the routine and explicit centrifugation, washing, and resuspension steps taught by Menon as the wash step taught by Nicol. Alternatively, it would similarly have been obvious to one of ordinary skill in the art to use the routine and explicit centrifugation, washing, and resuspension steps taught by Gao et al. specifically in the context of washing away unbound capture particles after contacting said capture particles with a sample comprising target particles as the wash step taught by Nicol.
The ordinary artisan would have been motivated use the wash protocol taught by Menon (or Gao et al.) because centrifugation is a convenient and widely used method known to those in the art for exchanging the buffer(s) in which particles (i.e. cells, viruses, target particles, etc.) are suspended. Furthermore, Gao et al. specifically teaches centrifuging and washing capture particles bound to target particles are useful for removing non-specifically bound molecules from capture particles prior to further analysis, are well known in the art, and are readily optimized by one of skill in the art. Therefore, the ordinary artisan would have been reasonably confident that steps directed to washing cells (target particles) bound by detectable antibody:antigen or aptamer:ligand complexes (i.e. target-specific elements), or even more particularly, to affinity capture beads such as those taught by Gao et al. for the express purpose of removing non-specifically bound molecules from the capture element, would have been conveniently and successfully practiced by the centrifugation and resuspension method taught by Menon or the method taught by Gao et al.
Further, it would have been prima facie obvious prior to the effective filing date of the claimed invention for one of ordinary skill in the art to encapsulate the target particles and capture template particles in the presence of an immiscible fluid (i.e. a fluorocarbon oil) by the application of mechanical energy (i.e. shearing). The ordinary artisan would have been motivated to encapsulate the particles taught by Nicol in an emulsion comprising a fluorinated oil by applying a shear force because Griffiths teaches that non-fluorinated compounds are essentially insoluble in fluorocarbons (fluorocarbon oils) and that this property is advantageous in the context of emulsion formation. (Griffiths, paragraph 0142) Furthermore, Nicol expressly cites Griffiths as teaching a variety of methods for forming emulsions (i.e. encapsulation) known in the art and “the emulsion may comprise a fluorinated oil” (i.e. a fluorocarbon oil and water mixture – immiscible fluids). (Nicol, paragraph 0157)
Therefore, the ordinary artisan would have been reasonably confident that inclusion of fluorinated oil in the emulsion forming step would have facilitated separation of the single cells into monodisperse droplets because of the teachings of Nicol and Menon that emulsions comprising monodisperse droplets are readily formed by vortexing immiscible fluids comprising an aqueous phase and fluorinated oils.
Regarding Claims 13-14, Nicol teaches that the second fluid (the immiscible fluid for partitioning aqueous target particle and capture template particle complexes) may comprise a fluorinated oil (i.e. a fluorocarbon oil) (Nicol, paragraph 0157).
Regarding Claims 15-18, Nicol teaches capture of B cells (i.e. circulating cells) displaying a specific antibody from patient samples (i.e. a human biological sample) (Nicol, paragraph 0375). In this particular embodiment, Nicol teaches “the cell[s]… come from an HIV infected patient’s sample, such as from patients showing Broadly Neutralizing HIV-1 antibodies in their serum, or patients at different stages of infection.” (i.e. a human body fluid) (Nicol, paragraph 0375)
Regarding claim 96, Menon et al. teaches steps for binding the detectable antibody to cell surface antigens (Menon, page 5, protocol 3; step 3-4), centrifuging the binding mixture (Menon, page 6, protocol 3; step 5.1 (i.e. generating a supernatant and pellet), removing the supernatant (Menon, page 6, protocol 3; step 5.2), and washing and resuspending the pellet in a new buffer (Menon, page 6, protocol 3, step 6-7).
Regarding newly added claim 97, Nicol et al. teach the target-specific capture element (i.e. an antibody or protein “X” in figure 3) (which may also comprise an aptamer as taught by Nicol et al. or Gao et al.; Nicol et al., paragraph 0062; Gao et al., paragraph 0086) is attached to a capture moiety linked to the template particle (see Nicol et al., Figure 3 reproduced below for convenience).
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Regarding newly added claim 100, Nicol et al. teach that “capture moieties” comprise biotin and a capture moiety specific binding agent is avidin or streptavidin (Nicol et al., paragraph 0070). In a particular example, Nicol et al. teach that antibodies (i.e. a target specific capture element) can be biotinylated, and the capture moiety to can be streptavidin (Nicol et al., paragraph 0395).
It would have been prima facie obvious prior to the effective filing date of the claimed invention for one of ordinary skill in the art to have switched the affinity-pair labels on the capture element and capture moiety because the labeling of one member or the other of a pair of molecules of interest with one or the other member of an affinity-pair label is an obvious variation of the other configuration (i.e. a biotinylated capture element and a streptavidin-coupled capture moiety Vs. a biotinylated capture moiety and a streptavidin-coupled capture element). Furthermore, as discussed above, either of Nicol et al. or Gao et al. teach that aptamers are suitable alternative capture moieties to antibodies (Nicol et al., Paragraph 0062 or Gao et al., paragraph 0086).
Response to Arguments
The response asserts: “Nicol in view of, Griffiths, Gao, and Menon fails to teach or suggest all of the features of independent claim 12, at least in view of the claim amendments.”
This argument has been thoroughly considered and is not persuasive. As described in the amended 103 rejection above, Nicol et al. in view of Griffiths, Gao, and Menon do indeed teach the limitations of amended independent claim 12 (See Nicol et al., Figure 3, reproduced here for convenience), including the limitations now incorporated into the independent claim from claims 98-99 and 101, now each canceled. For additional clarity, reference sign “X” corresponds to the presently recited “target-specific capture element” (i.e. “capture protein or antibody or aptamer”) and reference signs “Ai”, “Bi”, “Ci”, and “Di” correspond to a target-specific capture element genetic identifier comprising a unique barcode sequence.
Therefore, the assertion that incorporation of these limitations renders the amended claims non-obvious over the cited combination of references is not persuasive and the claims are/remain rejected under 35 U.S.C. 103.
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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 12-18 and 96 remain/are rejected on the ground of nonstatutory double patenting as being unpatentable over Claims 1, 12 and 18 of U.S. Patent No. 11,866,782 in view of Nicol et al. (US 2018/0112212 A1 (published April 26, 2018), Griffiths et al. (US 2006/0078888 A1 published April 13, 2006), and Menon V et al. (J Vis Exp. 2014 Dec 18;(94):52241). Although the claims at issue are not identical, they are not patentably distinct from each other.
Regarding Claim 12, ‘782 teaches a method for single cell analysis in monodisperse droplets (i.e. target capture and barcoding in monodisperse droplets) comprising contacting cells (i.e. target particles) with nucleic-acid-labelled, target-specific antibodies, washing away unbound antibodies, (i.e. generating a supernatant and pellet, removing the supernatant, washing, and resuspending) and contacting the resulting complexes with template particles comprising: capture sequences/probes that hybridize to nucleic acid targets as well as “to the capture portion of one or more of the nucleic acid labels.” A second fluid is then added, and the resulting mixture is sheared to generate a plurality of monodisperse droplets that contain one template particle and one target cell. (‘782, Claims 1 and 18)
‘782 does not teach the recited steps of: “generating a supernatant and pellet from the mixture”; “removing the supernatant to separate the plurality of capture template particles and associated target particles from the solution of the first mixture; washing and resuspending the pellet in a reaction buffer…” (lines 8 and 11-12 of claim 12); “generating a pellet and a supernatant comprises centrifuging” (claim 96).
However, Menon teaches a protocol for labeling cell surface antigens on live or fixed cells (i.e. target particles) with antibodies conjugated to a detectable label (i.e. a target-specific element) prior to fluorescence activated cell sorting. Menon teaches steps for binding the detectable antibody to cell surface antigens (Menon, page 5, protocol 3; step 3-4), centrifuging the binding mixture (Menon, page 6, protocol 3; step 5.1) (i.e. generating a supernatant and pellet), removing the supernatant (Menon, page 6, protocol 3; step 5.2), and washing and resuspending the pellet in a new buffer (Menon, page 6, protocol 3, step 6-7).
Therefore, it would have been prima facie obvious prior to the effective filing date of the claimed invention for one of ordinary skill in the art to use the routine and explicit centrifugation, washing, and resuspension steps taught by Menon as the wash step taught by ‘782. ‘782 does not explicitly teach generating a supernatant and pellet and removing the supernatant to separate the plurality of capture template particles. Menon provides the clear teachings of wash steps required by the claims. The ordinary artisan would have been motivated use the wash protocol taught by Menon because centrifugation is a convenient and widely used method known to those in the art for exchanging the buffer(s) in which particles (i.e. cells, viruses, target particles, etc.) are suspended. Therefore, the ordinary artisan would have been reasonably confident that steps directed to washing cells (target particles) bound by detectable antibody complexes (i.e. target-specific elements) would have been conveniently and successfully practiced by the centrifugation and resuspension method taught by Menon.
Regarding Claims 13-14, ‘782 teaches that the second fluid comprises an oil. (‘782, Claim 12)
‘782 does not teach that said oil may comprise a fluorocarbon oil.
However, Nicol and Griffiths teach that an emulsion may comprise a fluorinated oil in the context of encapsulating single cells into monodisperse emulsion droplets. (Nicol, paragraph 0157; Griffiths, paragraph 0142)
Therefore, it would have been prima facie obvious prior to the effective filing date of the claimed invention for one of ordinary skill in the art to use fluorocarbon oils as the oil claimed by ‘782 in the method directed to single-cell analysis in monodisperse droplets. The ordinary artisan would have been motivated to use a fluorinated oil to facilitate emulsion formation because Griffiths teaches that non-fluorinated compounds are essentially insoluble in fluorocarbons (fluorocarbon oils) and that this property is advantageous in the context of emulsion formation. (Griffiths, paragraph 0142) Therefore, the ordinary artisan would have been reasonably confident that inclusion of fluorinated oil in the emulsion forming step would have facilitated separation of the single cells into monodisperse droplets.
Regarding Claims 15-18, ‘782 teaches a method of analyzing single cells from a plurality of target cells in a first fluid. (i.e. a biological sample) (‘782, Claim 18)
The Claims of ‘782 do not teach that the first fluid may comprise a (human) body fluid further comprising circulating cells.
However, Nicol teaches analyzing antibody-producing B-cells (i.e. circulating cells) from HIV-positive patient samples (Nicol, paragraph 0368), such as from patients showing Broadly Neutralizing HIV-1 antibodies in their serum, or patients at different stages of infection. (i.e. human body fluids) in a monodisperse emulsion. (Nicol, paragraph 0375)
Therefore, it would have been prima facie obvious prior to the effective filing date of the claimed invention for one of ordinary skill in the art to apply the single-cell analysis method of ‘782 to circulating human cells. The ordinary artisan would have been motivated to analyze single circulating human cells by the teaching of Nicol that individual B cells producing specific neutralizing antibodies against HIV can be isolated and the mRNAs encoding said antibodies can be uniquely barcoded simultaneously. (Nicol, paragraph 0368) Therefore, the ordinary artisan would have been reasonably confident that the method of single-cell analysis in monodisperse droplets claimed in ‘782 would have been applicable to circulating human cells.
Regarding Claims 19-20, ‘782 teaches a method of analyzing single cells wherein template particles comprising capture probes (i.e. capture template particles) hybridize to the capture portion of target-specific capture elements. (nucleic-acid labeled antibodies) (i.e. the association is target-specific via the capture element) (‘782, claim 18)
Claims 12-15 and 96 remain/are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1 and 17 of copending Application No. 17/864,655 in view of Nicol et al. (US 2018/0112212 A1, published April 26, 2018) and Griffiths et al. (US 2006/0078888 A1, published April 13, 2006). Although the claims at issue are not identical, they are not patentably distinct from each other.
Regarding claims 12- 15, ‘655 teaches a method of: screening small molecules coupled to DNA barcodes with single cells (as defined by the instant application, a biological sample comprising target particles), hybridizing said barcodes to capture oligos coupled to template particles (i.e. target capture and barcoding in a first mixture), shearing the resulting capture template-target particle complexes in the presence of an immiscible fluid to partition single complexes into single partitions, lysing said cells, and barcoding released cellular mRNA and the small-molecule coupled DNA barcodes. (‘665, Claim 1) ‘655 additionally teaches that the captured cells are washed after binding (‘655, claim 17). Therefore, ‘655 teaches a method of target capture and barcoding in monodisperse droplets comprising: binding of capture template particles (barcoded small molecules hybridized to template particles), washing cells after binding, combining with an immiscible fluid, and shearing to produce monodisperse droplets containing target-capture template particle complexes.
The claims of ‘655 do not teach that the immiscible fluid comprises a fluorocarbon oil.
However, Nicol teaches: “Emulsion may be achieved by a variety of methods known in the art. (see, for example, [Griffiths et al.], of which paragraphs [0139]-[0143] are incorporated by reference herein)” Nicol teaches the emulsion may comprise a fluorinated oil (i.e. a fluorocarbon oil). (Nicol, paragraph 0157)
Griffiths teaches: “Emulsions may be produced from any suitable combination of immiscible liquids… preferably…an aqueous liquid… and an oil.” (Griffiths, paragraph 0140)
Further, Griffiths teaches that non-fluorinated compounds are essentially insoluble in fluorocarbons (fluorocarbon oils) and that this property is advantageous in the context of emulsion formation. (Griffiths, paragraph 0142)
Therefore, it would have been prima facie obvious prior to the effective filing date of the claimed invention for one of ordinary skill in the art to use fluorocarbon oils as the immiscible fluid claimed by ‘655. The ordinary artisan would have been motivated to use a fluorinated oil to facilitate emulsion formation because Griffiths teaches that non-fluorinated compounds are essentially insoluble in fluorocarbons (fluorocarbon oils) and that this property is advantageous in the context of emulsion formation. (Griffiths, paragraph 0142)
Therefore, the ordinary artisan would have been reasonably confident that inclusion of fluorinated oil in the emulsion forming step would have facilitated separation of the single cells into monodisperse droplets.
The claims of ‘655 do not teach “generating a supernatant and pellet from the first mixture, optionally by centrifuging; removing the supernatant to separate the plurality of capture template particles and associated target particles from the solution of the first mixture; washing and resuspending the pellet in a reaction buffer…” (limitations of steps 3-5)
However, Menon teaches a protocol for labeling cell surface antigens on live or fixed cells (i.e. target particles) with antibodies conjugated to a detectable label (i.e. a target-specific element) prior to fluorescence activated cell sorting. Menon teaches steps for binding the detectable antibody to cell surface antigens (Menon, page 5, protocol 3; step 3-4), centrifuging the binding mixture (Menon, page 6, protocol 3; step 5.1 (i.e. generating a supernatant and pellet), removing the supernatant (Menon, page 6, protocol 3; step 5.2), and washing and resuspending the pellet in a new buffer (Menon, page 6, protocol 3, step 6-7).
Therefore, it would have been prima facie obvious prior to the effective filing date of the claimed invention for one of ordinary skill in the art to use the routine and explicit centrifugation, washing, and resuspension steps taught by Menon as the wash step taught by ‘655. ‘655 does not explicitly teach generating a supernatant and pellet and removing the supernatant to separate the plurality of capture template particles. Menon provides the clear teachings of wash steps required by the claims. The ordinary artisan would have been motivated use the wash protocol taught by Menon because centrifugation is a convenient and widely used method known to those in the art for exchanging the buffer(s) in which particles (i.e. cells, viruses, target particles, etc.) are suspended. Therefore, the ordinary artisan would have been reasonably confident that steps directed to washing cells (target particles) bound by detectable antibody complexes (i.e. target-specific elements) would have been conveniently and successfully practiced by the centrifugation and resuspension method taught by Menon.
Regarding claims 16-18, the claims of ‘655 do not teach that the biological sample (the first fluid comprising cells) may comprise human body fluids further comprising circulating cells.
However, Nicol teaches analyzing antibody-producing B-cells (i.e. circulating cells) from HIV-positive patient samples (Nicol, paragraph 0368), such as from patients showing Broadly Neutralizing HIV-1 antibodies in their serum, or patients at different stages of infection. (i.e. human body fluids) in a monodisperse emulsion. (Nicol, paragraph 0375)
Therefore, it would have been prima facie obvious prior to the effective filing date of the claimed invention for one of ordinary skill in the art to combine the claimed method of ‘655 directed to capturing and barcoding single cells that bind a given small molecule of interest in monodisperse droplets with the teachings of Nicol to capture single circulating human cells that bind a capture-element of interest. The ordinary artisan would have been motivated to analyze single circulating human cells by the teaching of Nicol that individual B cells producing specific neutralizing antibodies against HIV can be isolated and the mRNAs encoding said antibodies can be uniquely barcoded simultaneously. (Nicol, paragraph 0368)
Therefore, the ordinary artisan would have been reasonably confident that the method of ‘655 would be broadly applicable to the capture of individual circulating cells from samples comprising human bodily fluids.
This is a provisional nonstatutory double patenting rejection because the patentably indistinct claims have not in fact been patented.
Claims 12-18 and 96 remain/are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1 and 14 of copending Application No. 17/747,603 in view of Nicol et al. (US 2018/0112212 A1 published April 26, 2018), Griffiths et al. (US 2006/0078888 A1, published April 13, 2006), and Menon V et al. (J Vis Exp. 2014 Dec 18;(94):52241). Although the claims at issue are not identical, they are not patentably distinct from each other.
Regarding Claim 12, ‘603 teaches a method for single cell analysis comprising: preparing a mixture comprising extracellular vesicles (i.e. target particles in a first fluid), target-specific antibodies linked to index oligonucleotides (i.e. target-specific capture elements), and template particles comprising capture oligonucleotides (together, capture template particles), and shearing the mixture (i.e. the second mixture) in the presence of a partitioning oil (i.e. a second fluid comprising an oil) to produce partitions (i.e. monodisperse droplets) containing one target particle – capture template particle complex. (‘603, Claim 1) Furthermore, ‘603 teaches that the target particles “are from a sample from a subject” (the sample comprises a human body fluid). (‘603, Claim 14)
’603 does not teach “generating a supernatant and pellet from the first mixture, optionally by centrifuging ; removing the supernatant to separate the plurality of capture template particles and associated target particles from the solution of the first mixture; washing and resuspending the pellet in a reaction buffer…” (limitations of steps 3-5)
However, Menon teaches a protocol for labeling cell surface antigens on live or fixed cells (i.e. target particles) with antibodies conjugated to a detectable label (i.e. a target-specific element) prior to fluorescence activated cell sorting. Menon teaches steps for binding the detectable antibody to cell surface antigens (Menon, page 5, protocol 3; step 3-4), centrifuging the binding mixture (Menon, page 6, protocol 3; step 5.1 (i.e. generating a supernatant and pellet), removing the supernatant (Menon, page 6, protocol 3; step 5.2), and washing and resuspending the pellet in a new buffer (Menon, page 6, protocol 3, step 6-7).
Therefore, it would have been prima facie obvious prior to the effective filing date of the claimed invention for one of ordinary skill in the art to use the routine and explicit centrifugation, washing, and resuspension steps taught by Menon as the wash step taught by ‘603. ‘603 does not explicitly teach generating a supernatant and pellet and removing the supernatant to separate the plurality of capture template particles. Menon provides the clear teachings of wash steps required by the claims. The ordinary artisan would have been motivated use the wash protocol taught by Menon because centrifugation is a convenient and widely used method known to those in the art for exchanging the buffer(s) in which particles (i.e. cells, viruses, target particles, etc.) are suspended. Therefore, the ordinary artisan would have been reasonably confident that steps directed to washing cells (target particles) bound by detectable antibody complexes (i.e. target-specific elements) would have been conveniently and successfully practiced by the centrifugation and resuspension method taught by Menon.
Regarding Claim 13, ‘603 teaches shearing the mixture (i.e. the second mixture) in the presence of a partitioning oil (i.e. a second fluid comprising an oil) to produce partitions (i.e. monodisperse droplets) containing one target particle – capture template particle complex. (‘603, Claim 1)
Regarding Claim 14, ‘603 does not claim that the partitioning oil comprises a fluorocarbon oil.
However, Nicol teaches: “Emulsion may be achieved by a variety of methods known in the art. (see, for example, [Griffiths et al.], of which paragraphs [0139]-[0143] are incorporated by reference herein)” Nicol teaches the emulsion may comprise a fluorinated oil (i.e. a fluorocarbon oil). (Nicol, paragraph 0157)
Griffiths teaches: “Emulsions may be produced from any suitable combination of immiscible liquids… preferably…an aqueous liquid… and an oil.” (Griffiths, paragraph 0140) Griffiths further teaches that non-fluorinated compounds are essentially insoluble in fluorocarbons (fluorocarbon oils) and that this property is advantageous in the context of emulsion formation. (Griffiths, paragraph 0142)
Therefore, it would have been prima facie obvious prior to the effective filing date of the claimed invention for one of ordinary skill in the art to use fluorocarbon oils as the immiscible fluid claimed by ‘603. The ordinary artisan would have been motivated to use a fluorinated oil to facilitate emulsion formation because Griffiths teaches that non-fluorinated compounds are essentially insoluble in fluorocarbons (fluorocarbon oils) and that this property is advantageous in the context of emulsion formation. (Griffiths, paragraph 0142)
Therefore, the ordinary artisan would have been reasonably confident that inclusion of fluorinated oil in the emulsion forming step would have facilitated separation of the target particles into monodisperse droplets.
Regarding claims 15-18, ‘603 teaches a method for single cell analysis comprising: preparing a mixture comprising extracellular vesicles (i.e. target particles in a first fluid), target-specific antibodies linked to index oligonucleotides (i.e. target-specific capture elements), and template particles comprising capture oligonucleotides (together, capture template particles), and shearing the mixture (i.e. the second mixture) in the presence of a partitioning oil (i.e. a second fluid comprising an oil) to produce partitions (i.e. monodisperse droplets) containing one target particle – capture template particle complex. (‘603, Claim 1) Furthermore, ‘603 teaches that the target particles are extracellular vesicles “from a sample from a subject” (i.e. a human body fluid). (‘603, Claim 14)
‘603 does not claim that the method can be applied to capture circulating cells in addition to extracellular vesicles.
However, Nicol teaches a method comprising capturing single circulating cells from patient samples on capture template particles and partitioning the capture-template particle – target cell complexes into individual partitions in the presence of a fluorinated oil (Nicol, paragraph 0157) in the context of characterizing HIV-neutralizing antibodies. (Nicol, paragraph 0375)
Therefore, it would have been prima facie obvious prior to the effective filing date of the claimed invention for one of ordinary skill in the art to apply the method of ‘603 to capture circulating single cells. The ordinary artisan would have been motivated to apply the method of ‘603 to samples comprising circulating cells because Nicol teaches isolation of anti-gp120-producing cells and characterization of the genes encoding the heavy and light chains of said antibodies from circulating blood of HIV patients (a human body fluid comprising circulating cells). (Nicol, paragraph 0368) Therefore, the ordinary artisan would have been reasonably confident that the method of ‘603 would have enabled capture of circulating cells (such as B-cells) from samples comprising human body fluids.
This is a provisional nonstatutory double patenting rejection because the patentably indistinct claims have not in fact been patented.
Regarding claims 19 and 20, ‘603 claims that the extracellular vesicles (i.e. target particles) are captured by target-specific antibodies linked to index oligonucleotides (i.e. target-specific capture elements) and template particles comprising capture oligonucleotides (together, capture template particles).
Claims 12-14 and 96 remain/are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claim 6 of copending Application No. 17/210,737 in view of Nicol et al. (US 2018/0112212 A1, published April 26, 2018). Although the claims at issue are not identical, they are not patentably distinct from each other.
Regarding Claim 12, ‘737 claims a method of virus detection and barcoding comprising capturing virus particles (i.e. target particles) from an aqueous sample with template particles that comprise antibodies that capture the virus particles, (i.e. capture-template particles) adding an immiscible second fluid to the first fluid, and vortexing (i.e. shearing) the mixture to generate monodisperse emulsion droplets that encapsulate a single virus-template particle complex (target-particle – capture-template particle complex) (‘737, Claim 6).
The claims of ’737 do not teach “generating a supernatant and pellet from the first mixture, optionally by centrifuging; removing the supernatant to separate the plurality of capture template particles and associated target particles from the solution of the first mixture; washing and resuspending the pellet in a reaction buffer…” (limitations of steps 3-5)
However, Menon teaches a protocol for labeling cell surface antigens on live or fixed cells (i.e. target particles) with antibodies conjugated to a detectable label (i.e. a target-specific element) prior to fluorescence activated cell sorting. Menon teaches steps for binding the detectable antibody to cell surface antigens (Menon, page 5, protocol 3; step 3-4), centrifuging the binding mixture (Menon, page 6, protocol 3; step 5.1 (i.e. generating a supernatant and pellet), removing the supernatant (Menon, page 6, protocol 3; step 5.2), and washing and resuspending the pellet in a new buffer (Menon, page 6, protocol 3, step 6-7).
Therefore, it would have been prima facie obvious prior to the effective filing date of the claimed invention for one of ordinary skill in the art to use the routine and explicit centrifugation, washing, and resuspension steps taught by Menon as the wash step taught by ‘603. ‘603 does not explicitly teach generating a supernatant and pellet and removing the supernatant to separate the plurality of capture template particles. Menon provides the clear teachings of wash steps required by the claims. The ordinary artisan would have been motivated use the wash protocol taught by Menon because centrifugation is a convenient and widely used method known to those in the art for exchanging the buffer(s) in which particles (i.e. cells, viruses, target particles, etc.) are suspended. Therefore, the ordinary artisan would have been reasonably confident that steps directed to washing cells (target particles) bound by detectable antibody complexes (i.e. target-specific elements) would have been conveniently and successfully practiced by the centrifugation and resuspension method taught by Menon.
Regarding Claims 13-14, ‘737 claims that the immiscible second fluid comprises an oil, (‘737, Claim 6)
The claims of ‘737 do not teach that the second fluid comprises a fluorocarbon oil. However, Nicol teaches: “Emulsion may be achieved by a variety of methods known in the art. (see, for example, [Griffiths et al.], of which paragraphs [0139]-[0143] are incorporated by reference herein)” Nicol teaches the emulsion may comprise a fluorinated oil (i.e. a fluorocarbon oil). (Nicol, paragraph 0157) Griffiths teaches: “Emulsions may be produced from any suitable combination of immiscible liquids… preferably…an aqueous liquid… and an oil.” (Griffiths, paragraph 0140) Griffiths further teaches that non-fluorinated compounds are essentially insoluble in fluorocarbons (fluorocarbon oils) and that this property is advantageous in the context of emulsion formation. (Griffiths, paragraph 0142)
Therefore, it would have been prima facie obvious prior to the effective filing date of the claimed invention for one of ordinary skill in the art to use the fluorocarbon oils taught by Nicol and Griffiths in the method claimed by ‘737. The ordinary artisan would have been motivated by the suggestion of Griffiths “non-fluorinated compounds are essentially insoluble in fluorocarbons…” and that this property is advantageous in the context of emulsion formation. (Griffiths, paragraph 0142) Therefore, the ordinary artisan would have been reasonably confident that this combination would have provided an advantage for efficient emulsification.
This is a provisional nonstatutory double patenting rejection because the patentably indistinct claims have not in fact been patented.
Claim 12 is provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claim 1 of copending Application No. 18/403,902 in view of Nicol et al. (US 2018/0112212 A1, published April 26, 2018) and Menon and/or Gao et al. Although the claims at issue are not identical, they are not patentably distinct from each other.
Regarding claim 12, the claims of ‘902 render obvious the claimed invention in view of Nicol et al. Briefly, claim 1 of ‘902 recites a method comprising incubating nucleic acid labeled, target specific antibodies with target cells, washing the target cells, combining template particles and washed, labeled cells, adding a second fluid to the first fluid, and shearing the fluids to generate a plurality of monodisperse droplets…
The instant claim 12 differs from claim 1 of ‘902 only in that the capture moiety (the nucleic acid labeled antibodies) are not supplied pre-linked to the capture template particle but are captured after antibody binding to the cells and a wash step that does not explicitly require centrifugation. As described in the 103 rejection above, it would have been prima facie obvious to one of ordinary skill in the art to modify the two-step binding of labeled antibodies to target cells followed by a wash (comprising centrifugation) followed by capture of labeled cells with a capture template particle into a single step wherein the capture template particle comprises the antibody (or other affinity reagent) because Nicol teaches alternative examples using either of these one- or two- step labeling and capture schemes. Further, as described above, it is routine and well known in the art (as taught by Menon and/or Gao et al.) to wash cells after labeling with antibodies or after capture on a bead comprising a specific affinity reagent through which the bead and cell are associated by methods comprising centrifugation and resuspension in a new buffer.
This is a provisional nonstatutory double patenting rejection because the patentably indistinct claims have not in fact been patented.
Response to Arguments
The response requests that the double patenting rejection be held in abeyance until allowable subject matter is reached. This request has been noted, however MPEP 804(I)(b)(1) and 37 C.F.R. 1.111(b), which allows that some objections may be held in abeyance, includes no provision for holding rejections in abeyance. Thus, for the reasons above and those already of record, the rejection is maintained.
Conclusion
No claim is allowed.
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/Z.M.T./Examiner, Art Unit 1682
/WU CHENG W SHEN/Supervisory Patent Examiner, Art Unit 1682
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