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 .
Claim Status
Claims 1-6,8-9,12,14-16 and 27-30 are pending, of which claims 8-9, 12, 14-16, and 27-28 have been withdrawn. Claim 1 is amended. Claims 29-30 are new. Claim 1 is the only independent claim. Claims 1-6, and 29-30 are currently under examination.
Response to Arguments
Rejections Withdrawn
The rejection of claims 1, 4, and 6 under 35 U.S.C. 102(a)(1)/102(a)(2) as being anticipated by Stoeckius et al. is withdrawn following the applicants’ amendments.
The rejection of claims 1-6 under 35 U.S.C. 103 as being unpatentable over Stoeckius as applied to claims 1, 4, and 6 above, and further in view of Bertozzi et al. is withdrawn following the applicants’ amendments.
As stated in the applicant’s remarks, Stoeckius alone does not teach the sandwich architecture that is required by claim 1 of the instant claims, specifically resulting from immobilizing the and therefore is the rejection is withdrawn.
New Rejections
Claim Objections
Claim 30 is objected to under 37 CFR 1.75 as being a substantial duplicate of claim 29. When two claims in an application are duplicates or else are so close in content that they both cover the same thing, despite a slight difference in wording, it is proper after allowing one claim to object to the other as being a substantial duplicate of the allowed claim. See MPEP § 608.01(m).
The claim limitations of claim 29 “wherein (i) step (b) comprises capturing the target molecule on a solid support using a capture antibody that binds an epitope of the target molecule different from the epitope bound by the antibody-DNA oligonucleotide conjugate, (iii) step (d) comprises in-situ amplification of the DNA sequence while the target molecule remains immobilized on the solid support,” are already present in claim 1, leaving only new limitation “(ii) the SPACER comprises a polythymidine sequence of 12-30 nucleotides” which is claim 30. Since claim 30 is also dependent on claim 1, the duplicate limitations from claim 29 are already part of claim 30 as well, therefore the SPACER comprising a polythymidine sequence of 12-30 nucleotides, is restating the limitation of claim 29.
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.
The text of those sections of Title 35, U.S. Code not included in this action can be found in a prior Office action.
Claims 1-6 are rejected under 35 U.S.C. 103 as being unpatentable over Aghvanyan et al. (US 2014/0272939 A1, published Sep. 18, 2014) in view of Peterson et al. (US 2018/0208975 A1, published Jul. 26, 2018).
Aghvanyan is directed to “improved methods for conducting immunoassays” designed to detect an analyte of interest in a sample (see Aghvanyan [0003]). In regards to claim 1, Aghvanyan explicitly discloses step (a) “conjugating if a DNA oligonucleotide to an antibody to the target molecule to form an antibody-DNA oligonucleotide conjugate,” throughout the disclosure. Example 1 describes in detail conjugating thiol-modified proximity probe oligonucleotides to detection antibodies using sulfo-SMCC, a heterobifunctional NHS-ester/maleimide crosslinker (see Aghvanyan [0235]-[0236]). The disclosed proximity probes (SEQ ID NOs 1 and 2) each carry 5’-thiol group used as the reactive moiety for conjugation to the maleimide-activated antibody. This directly teaches conjugation a DNA oligonucleotide to an antibody, corresponding to the claimed reactive group R = thiol, which is explicitly listed as one of the claimed options.
Further, Aghvanyan discloses a sandwich immunoassay format on a solid support (see Aghvanyan [0237] disclosing (“each binding domain on the plate include a capture antibody and an anchoring moiety”). Claim 1 of Aghvanyan expressly recites “a capture reagent on a surface comprising the capture reagent for the analyte” and “a first detection reagent for the analyte that is linked to a first nucleic acid probe” and “a second detection reagent for the analyte that is linked to a second nucleic acid probe.” The use of a capture antibody and separate detection antibodies in a sandwich format inherently requires recognition of distinct epitopes on the target, the dual-antibody sandwich format is architecturally defined by epitope non-overlap to enable simultaneous binding. This structural requirement is explicitly taught by Aghvanyan’s three-antibody format, one capture plus two detection antibodies (see Aghvanyan [0008], Claim 8 “the capture reagent and the first and second detection reagents are antibodies to the analyte”), reading on step (b) of claim 1, “immobilizing the target molecule,” and “wherein step (b) comprises capturing the target molecule on a solid support using a capture antibody that binds an epitope of the target molecule different from the epitope bound by the antibody-DNA oligonucleotide conjugate.” Aghvanyan further discloses a step (c) “binding the target molecule with the antibody-DNA oligonucleotide conjugate” as outlined above (see Aghvanyan [0237] disclosing “A solution of detection antibodies labeled with proximity probes 1 and 2… was added to each well (25 µL per well), and incubated with shaking for 1-2 hours”).
Aghvanyan further discloses “amplifying and detecting the DNA sequence in the antibody-DNA oligonucleotide conjugate; wherein d) comprises in situ amplification of the DAN sequence while the target molecule remains immobilized on the solid support”. Aghvanyan directly and explicitly teaches this limitation (see Aghvanyan [0011] disclosing “the complex can remain bound to the Surface after the extending step” [0021], [0025], [0030], and throughout).
To the extent that Aghvanyan does not expressly teach the inclusion of oligo nucleotides with the defined structure alternatives of claim 1, Aghvanyan’s proximity probes (SEQ ID NOs 1 and 2) have the structure 5’-SH-Poly-A-Probe Sequence-3’. The SH is the reactive thiol group (R), the poly-A run is explicitly described as a spacer “a poly(A) sequence… used as a linker sequence between the surface and the complementary (hybridizing) region to extent the complementary region away from the surface” (see Aghvanyan [0163]), and the “probe sequence” hybridize with connector/circularization oligonucleotides, reading on the ”bridge left” and “bridge right” portions of the present claim (see Aghvanyan Fig. 4, [0164]). However, Aghvanyan’s probes are designed for proximity ligation/RCA-based detection with electrochemical or photo luminescent read out and do not expressly disclose the inclusion of defined unique identifier sequence for protein tracking after sequencing, or the full upstream adapter + identifier + downstream adapter/bridge architecture of the three structural alternatives recited in claim 1. However, Peterson bridges this knowledge gap and supplies all of these structural elements and in fact discloses oligonucleotide structures that directly and completely map to the structural alternatives recited in claim 1, either individually or in combination with its internal teachings.
Peterson discloses the complete structure of alternative 2 (5' R-SPACER-UPSTADAPTER-DEFINED IDENTIFIER SEQUENCE-BRIDGELEFT 3'). Peterson teaches that “a universal protein linker nucleotide sequence (10-30 bp) is added to the SOMAmer or aptamer sequence to enable binding to the protein detection probe with an identification nucleotide sequence that identifies the single cell or sample, a unique molecular identifier, universal primer sequence, a complementary universal protein linker nucleotide sequence.” This passage directly discloses, in a single conjugated oligonucleotide: a protein linker/spacer region, a unique identification sequence, a universal primer sequence (adapter), and a complementary universal protein linker (bridge) (see Peterson [0027], [0039], Figs 1-4). Peterson further discloses that the reactive group for conjugation is a thiol group used for covalent amine-based crosslinking of the oligonucleotide to the antibody, directly corresponding to the claimed R group.
It would have been prima facie obvious to one of ordinary skill in the art at the time of filing to utilize the oligonucleotide architecture of Peterson with the solid-phase sandwich immunoassay of Aghvanyan in order to enable the protein detection readout to be performed by next-generation sequencing rather than electrochemical or photo luminescence, thereby enabling massively multiplexed protein tracking across many analytes simultaneously.
Both references operate in the same technical field of antibody-DNA conjugate-based protein detection. Both use similar covalent based antibody-DNA conjugation chemistry. Both use PCR-based amplification of the conjugated DNA sequences as part of the detection readout. The sole distinction between the combined art and the claimed method is the substitution of Peterson’s sequencing compatible oligonucleotide architecture (unique identifier + PCR adapters + Bridge sequence) into Aghvanyan’s solid-phase in situ amplification assay format, a straightforward combination that a person of ordinary skill in the art would have been motivated to make to achieve highly sensitive immunoassay with a sequencing based multiplexed capability. No unexpected results would be anticipated from this combination as the structural and functional compatibility of the two systems is apparent from the references themselves and each element of the claimed oligonucleotide structures was individually well characterized in the art prior to the claimed invention.
In regards to claims 2, 3, and 5, Aghvanyan and Peterson each teach using cross-linker sulfo-SMCC and reducing reagent DTT in the conjugation step a) of claim 1 (see Aghvanyan [0235]-[0236], Peterson [0026]) .
In regards to claim 6, Peterson teaches amplification performed using polymerase chain reaction (PCR) and outlines using PCR to add sequencing indexes and universal primers (see Peterson [0032]-[0033])
In regards to claims 29 and 30, as outlined above, Aghvanyan discloses methods “wherein (i) step (b) comprises capturing the target molecule on a solid support using a capture antibody that binds an epitope of the target molecule different from the epitope bound by the antibody-DNA oligonucleotide conjugate,” “and (iii) step (d) comprises in-situ amplification of the DNA sequence while the target molecule remains immobilized on the solid support,” as these are limitations also recited in claim 1. Aghvanyan teaches that “the anchoring oligonucleotide may also comprise a non-complementary region (for example a poly (A) sequence) that is used as a linker sequence between the Surface and the complementary (hybridizing) region to extend the complementary region away from the Surface” (see Aghvanyan [0163]). Thus, Aghvanyan expressly teaches a homopolymeric nucleotide spacer sequence functioning to physically sperate one functional region of the oligonucleotide from another region. Peterson further teaches the used of poly(dT) nucleotide sequences having lengths of 10-40 nucleotides (see Peterson [0017]). It would have been obvious to a person of ordinary skill in the art at the time of the invention to substitute the poly(A) linker sequence taught by Aghvanyan with a polythymidine sequence having a length of 10-40 nucleotides as taught by Peterson because both poly(A) and poly(T) sequences were known homopolymeric nucleotide linker sequences suitable for physically separating functional oligonucleotide domains while minimizing interference with adjacent functional regions. Such substitutions would merely represent the predictable use of one known nucleotide spacer sequence in place of another to achieve the same known purpose of spatial separation within the oligonucleotide construct.
Conclusion
No claim is allowed.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to Matthew H Raymonda whose telephone number is (703)756-5807. The examiner can normally be reached Monday - Friday 10:00 am - 4:00 pm.
Examiner interviews are available via telephone, in-person, and video conferencing using a USPTO supplied web-based collaboration tool. To schedule an interview, applicant is encouraged to use the USPTO Automated Interview Request (AIR) at http://www.uspto.gov/interviewpractice.
If attempts to reach the examiner by telephone are unsuccessful, the examiner’s supervisor, Heather Calamita can be reached at 571-272-2876. The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300.
Information regarding the status of published or unpublished applications may be obtained from Patent Center. Unpublished application information in Patent Center is available to registered users. To file and manage patent submissions in Patent Center, visit: https://patentcenter.uspto.gov. Visit https://www.uspto.gov/patents/apply/patent-center for more information about Patent Center and https://www.uspto.gov/patents/docx for information about filing in DOCX format. For additional questions, contact the Electronic Business Center (EBC) at 866-217-9197 (toll-free). If you would like assistance from a USPTO Customer Service Representative, call 800-786-9199 (IN USA OR CANADA) or 571-272-1000.
/MATTHEW HAROLD RAYMONDA/Examiner, Art Unit 1684 /AARON A PRIEST/Primary Examiner, Art Unit 1681
Prosecution Insights