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 .
Priority
The current application is a 371 of PCT/JP2020/027123 filed on 07/10/2020, which claims priority to JAPAN 2019-129017 filed 07/11/2019.
Claim Status
Claims 27-51 are pending and under examination. Claims 1-26 have been cancelled. Claims 27 is the only dependent claim.
Claim Rejections - 35 USC § 112
The following is a quotation of the first paragraph of 35 U.S.C. 112(a):
(a) IN GENERAL.—The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor or joint inventor of carrying out the invention.
The following is a quotation of the first paragraph of pre-AIA 35 U.S.C. 112:
The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor of carrying out his invention.
Claims 41-43, and 46-51 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, as failing to comply with the written description requirement. The claim(s) contains subject matter which was not described in the specification in such a way as to reasonably convey to one skilled in the relevant art that the inventor or a joint inventor, or for applications subject to pre-AIA 35 U.S.C. 112, the inventor(s), at the time the application was filed, had possession of the claimed invention. The term “specific binding partner” used in claims 41, and subsequently all of the dependent claims that don’t redefine the term, is a very broad term that is defined by its function as recited in claim 43 “at least one kind of molecule that binds to a cell surface molecule directly, or indirectly through a specific-binding partner molecule,” without providing any structural limitation whatsoever. Read according to its plain language, these claims encompass an extraordinarily broad and structurally diverse genus of molecules, including but not limited to: antibodies of all isotypes and formats (IgG, IgM, IgA, IgE, etc.); antibody fragments (Fab, scFv, Fv, nanobodies, VHH domains etc.); non-antibody protein scaffolds with binding activity (DARPins, affimers, monobodies, etc.); nucleic acid aptamers; peptide aptamers; lectin receptor ligands, and their analogs; small molecule-protein conjugates; and any other molecule capable of binding a cell surface target, as well as a full range of indirect binding configurations achievable through any conceivable specific-binding partner molecule. Small molecules and ligands alone account for thousands of substances. This is a vast, structurally heterogeneous, and functionally undefined genus.
To satisfy the written description requirement of 35 USC 112(a), the specification must “clearly allow persons of ordinary skill in the art to recognize that the inventor invented what is claimed,” Ariad Pharm., Inc. v. Eli Lilly & Co., 598 F.3d 1336, 1340, 94 USPQ2d 1161, 1167 (Fed. Cir. 2010). For genus claims, this requires that the specification disclose either a representative number of species falling within the scope of the genus, or structural features common to the members of the genus such that a skilled artisan can visualize or recognize the members of the claimed genus. A “mere wish or plan” for achieving a claimed genus, or a disclosure limited toa small number of specific embodiments within the vast genus defined purely by function, is insufficient to establish that the inventors possessed the full scope of the claimed genus. Regents of the Univ. of Cal. v. Eli Lilly & Co., 119 F.3d 1559, 1573, 43 USPQ2d 1398, 1410 (Fed. Cir. 1997). Where a claim is directed to a brad functional genus, the specification must provide disclosure commensurate in scope with the full breadth of the claim. AbbVie Deutschland GmbH & Co., KG v. Janssen Biotech, Inc., 759 F.3d 1285, 1300, 111 USPQ2d 1780, 1790 (Fed. Cir. 2014).
The specification discloses only a narrow subset of the claimed genus of “specific-binding molecules.” The disclosed embodiments are limited to: (1) antibodies and antibody fragments against cell surface molecules (corresponding to the species in dependent claim 44); and (2) avidin-type molecules used in combination with biotin-type molecules on the antibody as an indirect binding configuration (corresponding to the species claimed in dependent claim 45). These two specific embodiments represent a small and structurally homogeneous corner of the full genus of claims 41 and 43.
Critically, the specification provides no disclosure, working example, or structural guidance for the vast remainder of the claimed genus. There is no description of non-antibody protein binding scaffolds (affibodies, DARPins, affimers, monobodies, anticalins, or other alternative binding proteins) as specific-binding molecules; nucleic acid aptamers or peptide aptamers capable of binding cell surface molecules; lectins or carbohydrate-binding molecules; small molecule-based binding agents conjugated to the oligonucleic acid; receptor ligands or engineered receptor binding domains; or any indirect binding configuration other than the streptavidin-biotin pair. The specification does not identify any structural feature or property that is common to all members of the claimed genus that would allow a skilled artisan to visualize or recognize the full scope of what is claimed. Instead, the claimed genus is defined solely by the functional property of binding a cell surface molecule, a property shared by an enormous and structurally diverse array of molecules that the specification makes no attempt to describe, characterize, or exemplify beyond two narrow species.
The fact that the dependent claims 44 and 45 recite specific embodiments within the broader genus of claim 41, and 43, does not cure this deficiency. To the contrary, the existence of these narrow dependent claims highlights the disproportionate gap between what the specification actually describes and the vastly broader genus that claims 41 and 43 purport to claim. A disclosure limited to two specific species within a structurally unlimited functional genus is not commensurate in scope with the full breadth of the claimed genus. AbbVie 759 F.3d.
Furthermore, a skilled artisan reading the specification would not be able to identify from the disclosure any common structural feature, property, or design principle that characterizes “specific-binding molecules” as a class beyond their shared functional property of binding a cell surface target. The specification thus fails to provide the “blaze marks” in the art necessary to guide a skilled artisan in identify which molecules fall within the claimed genus and which do not. Fujikawa v. Wattanasin, 93 F.3d 1559, 1571, 39 USPQ2d 1895, 1905 (Fed. Cir. 1996). The inventor may well have possessed antibodies and the streptavidin-biotin binding pair as of the filing date, but there is insufficient evidence in the specification to demonstrate that they possessed the full structurally unlimited functional genus of “any molecule that binds a cell surface molecule directly or indirectly through any binding partner.”
Claim Rejections - 35 USC § 103
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
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.
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 27-39 are rejected under 35 U.S.C. 103 as being unpatentable over Fu et al. (US 2016/0312276 A1, published Oct. 27, 2016, on IDS 02/27/2022) in view of Lazinski et al. (US 2015/0203887 A1, published Jul. 23, 2015).
In regards to claim 27, Fu teaches “a method for nucleic acid amplification using a solid-phase carrier” including “a target nucleic acid-capturing step of capturing a target nucleic acid comprising mRNA, on a solid-phase carrier through a target-capturing nucleic acid bound on the solid carrier” wherein a stochastic barcode comprising an oligo-dT region hybridizes to the poly-A tail of mRNA and can be attached to a solid support (see [0198]) Fu further teaches “a complementary-strand synthesis step of synthesizing a complementary DNA strand that is complementary to the target nucleic acid… from the target nucleic acid captured on the solid-phase carrier” by reverse transcription using a stochastic barcode as a primer (see [0198]) Fu also teaches “an exonuclease treatment step of degrading and removing, target-capturing nucleic acid that has not captured the target nucleic acid” (see [0199]). Fu further teaches homopolymer tailing of cDNA using terminal deoxynucleotidyl transferase (TdT), wherein “the homopolymer tail can comprise a polyA tail, a polyU tail, a polyC tail, and/or a polyG tail” and that the second strand synthesis primer can comprise a region complementary to the homopolymer tail (see [0191]). Fu further teaches amplification of the resulting double-stranded cDNA molecule (see [0251]).
However, Fu does not explicitly teach carrying out the homopolymer addition step “in the presence of dCTP or dGTP, and a chain-terminating nucleotide triphosphate which is a chain-terminating CTP or chain-terminating GTP,” nor does Fu teach “wherein the ratio of addition of a chain-terminating CTP and dCTP or a chain- terminating GTP and dGTP in the homopolymer addition step is 1:15 to 1:40 nor does Fu explicitly teach that “the chain length of the complementary homopolymer portion of the second- strand-synthesizing primer is 6 to 13 bases.”
Lazinski teaches methods for performing homopolymer mediated nucleic acid amplification and teaches performing homopolymer tailing using TdT in the presence of both deoxynucleotide triphosphate and a chain terminator (ddNTP), wherein the chain terminator can be a dideoxynucleotide such as ddCTP or ddGTP (see [0011], [0032], [0049]). Lazinski further taches that “the ratio of dNTP to ddNTP is about 11 to 1 to about 29 to 1,” (see [0033], [0054]), which corresponds toa ddNTP:dNTP ratio of approximately 1:11 to 1:29, overlapping the claimed ratio of 1:15 to 1:40. Lazinski additionally teaches that a second homopolymer may have a length of 4 to 8 nucleotides (see [0029]), which overlaps the claimed 6 to 13 base range.
It would have been prima facie obvious to one of ordinary skill in the art at the time of filing to modify the homopolymer tailing step of Fu to include a chain-terminating nucleotide such as ddCTP or ddGTP in combination with dCTP of dGTP, as taught by Lazinski in order to control the average length of the homopolymer tail added by TdT and thereby improve uniformity and reproducibility of downstream priming and amplification. Lazinski expressly teaches that inclusion of ddNTP during TdT-mediated tailing permits control of homopolymer length through adjustment of the ratio of dNTP to ddNTP (see [0185]). Because homopolymer tail length directly affects annealing efficiency of a complementary homopolymer primer, and because Fu already teaches use of complementary homopolymer primer for second-strand synthesis, it would have ben a predictable and routine optimization to select ddNTP:dNTP ratio within the overlapping range taught by Lazinski to obtain a desired tail length suitable for efficient priming. The claimed ratio of 1:15 to 1:40 represents an optimization of a result-effective variable, namely the concentration ratio of chain terminator to nucleotide triphosphate, which Lazinski teaches controls tail length (see [0185]). Further, selecting a complementary homopolymer primer length within the overlapping range of 6 to 13 bases would have been an obvious design choice in view of Lazinski’s teaching of complementary homopolymer lengths of 4 to 8 bases, as primer length is a known parameter affecting hybridization stability and specificity. Accordingly, claim 27 is unpatentable under over Fu in view of Lazinski.
In regards to claim 28-30, Lazinski expressly teaches that the chain terminator used in the TdT-mediated homopolymer addition step is a dideoxynucleotide (ddNTP), including ddCTP and ddGTP (see [0032], [0054]). A ddNTP is a 3’-deoxy nucleotide triphosphate lacking a 3’-OH group and therefore reads on the limitations of a “ddNTP in which the 3’-position is modified with an atomic group comprising no OH group”.
In regards to claim 31, Fu teaches degradation of RNA following reverse transcription (see [0191], [0204]) and teaches TdT-mediated homopolymer addition to cDNA (see [0184]). Although Fu does not explicitly state that the mRNA degradation step and the homopolymer addition step are carried out simultaneously, it would have been obvious to one of ordinary skill in the art to perform these enzymatic steps in a single reaction mixture in order to reduce handling steps, minimize sample loss, and streamline the workflow, particularly in low-input or single-cell applications. RNase degrades RNA and does not interfere with DNA substrates while TdT acts on DNA 3’ ends; thus combining the reactions would have represented a predictable use of prior art elements according to their established functions. Accordingly, the additional limitation of claim 31 does not render the claimed method nonobvious.
In regards to claims 32-33, Fu expressly teaches that the target-capturing nucleic acid can comprise an oligo-dT region that hybridizes to the poly-A tail on mRNA and can be attached to a solid support (see [0198]). Fu further teaches the oligo-dT comprises sequencing primer sequences and other adapter sequences positioned upstream of the oligo-dT region (see Figs. 5 & 17, [0217]-[0218]). The adapter or sequencing primer sequence located 5’ of the oligo-dT region corresponds to the claimed “first adapter portion on the 5’ side of the poly(T) portion”.
In regards to claim 34, Fu teaches that the solid-phase carrier can be a bead associated with cells (see [0165]) and teaches a target-capturing nucleic acid attached to the bead comprising a sequencing primer region (adapter) a barcode (cell label), and an oligo-dT region for capturing mRNA (see Fig. 5, [0198], [0251]) The barcode portion located between the sequencing primer/adapter region and the oligo-dT region corresponds to the claimed “bead identification barcode portion between the first adapter portion and the poly(T) portion.”
In regards to claim 35, Fu teaches that cells can be associated with beads and/or in microwells (see [00165]), thereby teaching compartmentalized solid-phase formats on plates. Fu further teaches that barcodes can include spatial labels and cellular labels for identifying the origin of captured nucleic acids (see [0251]). Fu also teaches a target-capturing nucleic acid comprising a sequencing primer (adapter) region, a barcode, and an oligo-dT region (see [0198], [0217]). The spatial or compartment-identifying barcode positioned between the adapter and oligo-dT region corresponds to the claimed “compartment identification barcode portion between the first adapter portion and the poly(T) portion.”
In regards to claim 36 and 37, Fu teaches performing TdT-mediated homopolymer tailing of cDNA and contacting the tailed cDNA with a second strand synthesis primer that binds to the homopolymer tail (see [0191]). Fu further teaches that primers and adaptors can comprise sequencing primer sequences or universal labels, barcodes including molecular indices and cell labels incorporated into the oligonucleotide (see [0217], [0251]). Thus, Fu teaches a second-strand-synthesizing primer comprising a homopolymer portion complementary to the tail and an adapter portion positioned 5’ of that complementary region, and incorporation of molecular barcode portions into oligonucleotides used in amplification workflows. It would have been obvious to incorporate a molecular barcode portion between a second adapter portion and a homopolymer-binding region in a second-strand-synthesizing primer in order to enable molecular indexing and downstream identification of amplification products. Such barcodes placement is conventional in sequencing library construction.
In regards to claim 38, Fu teaches that sequencing primer sequences and universal labels are incorporated into adaptor regions and that amplification can be carried out using primers targeting those universal or sequencing primer sequences (see [0217], [0251]). Amplification using a primer targeting a first adapter portion and a primer targeting a second adapter portion corresponds to the dual-adapter PCR strategy expressly taught by Fu.
In regards to claim 39, Fu teaches that cells can be associated with beads and/or in a microwell (see Fig. 5, [0007], [0112], [0117]-[0144], [0165]), thereby teaching bead-based solid-phase carrier and performing capture step in a microwell format. Because claim 39 recites that the capture step is carried out in a microwell or microdroplet, Fu’s express teaching of microwell-based bead capture satisfies the claim. Moreover, at the time of filling, bead-based single-cell nucleic acid capture in microfluidic droplets was a well-known alternative compartmentalization strategy in the art, and microwells and microdroplets were recognized as interchangeable formats for isolating individual cells with barcoded beads in single-cell workflows. Substituting a microdroplet format for a microwell format would have been a predictable variation involving the use of a known alternative compartmentalization technique to achieve the same isolation and capture function.
Claims 27-40 is rejected under 35 U.S.C. 103 as being unpatentable over Fu (US Fu et al. (US 2016/0312276 A1, published Oct. 27, 2016, on IDS 02/27/2022) in view of Lazinski et al. (US 2015/0203887 A1, published Jul. 23, 2015) as applied to claims 27-39 above, included here for reason supra, and further in view of Suzuki et al. (US 2016/0289760 A1, published Oct. 6, 2016).
As set forth in the rejections of claims 27, and 36 above, Fu discloses a method for whole transcriptome amplification from single cells using a solid-phase carrier (bead) bearing immobilized barcoded capture probes (see Abstract, Fig. 1, throughout). Fu teaches capture probes comprising from 5’ to 3’: a first universal label sequence (first adapter portion); a cellular label; a molecular label (molecular barcode); and a target-biding (poly(T)) region (see Fig. 4). Fu further discloses that second-strand synthesis is primed using a primer comprising a second universal label sequence (second adapter portion) on the 5’-side of a complementary homopolymer portion, corresponding directly to the second-strand synthesizing primer of claim 36 (see [0324], [0329] and throughout). To the extent that the controlled-length homopolymer tailing details of the base method require further support, Lazinski supplies the explicitly teaching of TdT-mediated homopolymer addition using dNTP combination with ddNTP chain terminators to produce a defined-length homopolymer tail, with a complementary homopolymer-bearing adapter primer used for second-strand synthesis and subsequent amplification, as set forth in the rejection of claim 1 and incorporated herein by reference (see [0033], [0054], [0185], [0223]).
The combination of Fu and Lazinski thus fully address the limitations inherited from claims 27 and 36. However, neither Fu nor Lazinski discloses performing the nucleic acid amplification step using a primer targeting a T cell receptor (TCR) constant region paired with a primer targeting the second adapter portion for the purpose of amplifying cDNA regions encoding a TCR variable region.
Fu discloses, in addition to its whole-transcriptome amplification embodiment, an alternative approach in which amplification is carried out using a gene-specific primers to target a desired region in cDNA in combination with the universal label primers corresponding to the target amplification (see [0249]-[0251]). However, Fu does not explicitly disclose that the gene-specific primers target the T cell receptor constant region.
Suzuki is directed to a system and method for TCR and B Cell Receptor (BCR) repertoire analysis comprising the unbiased amplification and sequence of all rearranged TCR variable region genes from biological samples including single immune cells. Suzuki teaches that TCR genes comprise V (variable), D (diversity), J (joining), and C (constant) regions, and that antigen specificity is encoded by the hypervariable CDR3 region within the V region generated by somatic VDJ recombination (see [0003]-[0004]). Because the large number of possible VDJ rearrangements makes multiple V-region specific primer amplification inherently biased (see [0011]), Suzuki specifically teaches an unbiased alternative, the adapter ligation PCR (AL-PCR) method, as a preferred approach for complete and unbiased TCR variable region amplification (see Abstract, [0034], and throughout).
Suzuki’s AL-PCR method comprises: (1) synthesis of cDNA from mRNA by reverse transcription using poly(T) priming; (2) synthesis of double-stranded cDNA from the first strand cDNA; (3) ligation of an adapter sequence to the 5’ end of the double stranded cDNA; and (4) PCR amplification using two primers, an adapter primer targeting the ligated adapter sequence, and a TCR constant region-specific primer (see claim 1 and throughout). Suzuki explicitly teaches that this two-primer combination enables complete, unbiased amplification of all TCR variable region sequences regardless of VDJ rearrangement, because the constant region primer anchors to the conserved 3’ region of the TCR transcript and the adapter primer anchors to the 5’ end of the cDNA, with the intervening complete variable region recovered in the amplification product (see [0920], [0932] and throughout).
The two-primer amplification strategy of Suzuki maps directly to the amplification step of the present claim: the TCR constant region-specific primer of Suzuki corresponds precisely to the “primer targeting a T cell receptor constant region”, while the adapter primer of Suzuki, targeting the adapter ligated to the 5’ end of the cDNA corresponds precisely to the “primer targeting the second adapter portion” recited in the present claim (see [0920], [0932] and throughout).
In the context of the Fu/Lazinski base method, the second adapter portion is introduced at the 5’ end of the cDNA via the second-strand synthesis primer of claim 36, and therefore occupies the same structural and functional position as the adapter ligated at the 5’end of the cDNA in Suzuki’s AL-PCR method. The second adapter primer of claim 36 and the adapter primer of Suzuki this perform identical roles in their respective amplification schemes.
The amplification product of Suzuki’s AL-PCR, spanning from the 5’ adapter through the complete VDJ rearrangement and CDR3 region to the constant region primer binding site, directly constitutes “cDNA regions encoding a T cell receptor variable region” as recited in the present claim.
It would have been prima facie obvious to one of ordinary skill in the art at the time of filing to apply the TCR constant region primer amplification strategy of Suzuki to the solid-phase single-cell cDNA platform established by Fu and Lazinski. One would be motivated to combine these teachings as Suzuki teaches that coupling TCR repertoire information with single-cell molecular analysis enables powerful and clinically significant applications in immunology, cancer immunology, infections disease, and autoimmunity. The combination of single-cell cDNA profiling (Fu) with TCR variable region amplification (Suzuki) from the same cell and the same bead-capture cDNA library is a direct and natural extension of both technologies toward this explicitly articulated goal. Additionally, the technical combination is straightforward and requires no inventive step. The cDNA produced on the solid-phase carrier in Fu’s platform, primed poly(T) capture probes and bearing the second adapter at its 5’ end via the second-strand synthesis primer of claim 36, is structurally and functionally identical to the adapter-ligated, poly(T)-primed cDNA used as the input template for Suzuki’s AL-PCR. The second adapter primer already present in Fu’s amplification scheme performs precisely the same 5’-anchor role as the adapter primer in Suzuki’s AL-PCR. The only modification required to arrive at the present claim is the addition of Suzuki’s TCR constant region primer to Fu’s amplification reaction, a single, well-known, fully characterized, and commercially available primer directed to the conserved TCR constant region sequence. This represents a combination of known elements using known methods to achieve a predictable result, fully consistent with the obviousness standard of KSR Int'l Co. v. Teleflex Inc., 550 U.S. 398, 416, 82 USPQ2d 1385, 1395 (2007).
A reasonable expectation of success is firmly established by Suzuki’s own reduction to practice of unbiased TCR variable region amplification using the constant region/adapter primer pair from poly(T)-primed cDNA, precisely the cDNA substrate produced by Fu’s method. There is no technical barrier to combining these teachings. Accordingly, the present claim is unpatentable over the combination of Fu, Lazinski, and Suzuki.
Claims 27-51 are rejected under 35 U.S.C. 103 as being unpatentable over Fu et al. (US 2016/0312276 A1, published Oct. 27, 2016, on IDS 02/27/2022) in view of Lazinski et al. (US 2015/0203887 A1, published Jul. 23, 2015) and Suzuki (US 2016/0289760 A1, published Oct. 6, 2016) as applied to claim 27-40 above, included here for reasons supra, and further in view of Stoeckius et al. (US 2018/0251825 A1, published Sep. 6, 2018, on IDS 02/27/2022).
In regards to claims 41-43, as set forth above the combination of Fu and Lazinski make obvious the limitations of claim 27 for which claim 41 depends. However the combination of Fu an Lazinski does not disclose the additional limitations of claim 41, namely a target nucleic acid that further comprises an oligonucleic acid bound to a specific-binding molecule; a target-capturing nucleic acid comprises both an mRNA-capturing sequence and an oligonucleic acid capturing region; the oligonucleic acid comprising a capture target region complementary to the oligonucleic acid-capturing region; and simultaneous cDNA synthesis from mRNA and complementary strand synthesis from the oligonucleic acid.
Stoeckius discloses methods and compositions for simultaneously identifying or quantifying multiple targets in a biological sample, specifically including both cell surface proteins and intracellular mRNA from single cells (CITE-seq) (see Abstract and throughout). Stoeckius teaches a construct comprising a ligand (antibody or antibody fragment) attached to a polymer construct comprising an oligonucleotide. The oligonucleotide component of the construct comprises: an amplification handle sequence, a ligand barcode sequence identifying the specific-binding molecule , and an anchor sequence (poly(A) or other sequence) configured to hybridize to a complementary capture sequence on a solid support (bead) (see Abstract, Figs. 1-2, 6, [0006], [0016], and throughout). This construct directly constitutes the oligonucleic acid bound to a specific-binding molecule wherein the oligo nucleic acid comprises a capture target region (the anchor/poly(A) sequence) complementary to the oligonucleic acid-capturing region on the bead, as recited in claim 41.
Stoeckius further teaches that the antibody-oligonucleotide construct is incubated with cells prior to lysis, such that the antibody binds to its target cell surface molecules, reading on the limitation of claim 43 (see [0015], [0021], and throughout). Upon cell lysis in the presence of a poly(T) capture bead (of the type disclosed in Fu), both the cellular mRNA (via its poly(A) tail) and the construct oligonucleotide (via its poly(A) anchor) are co-captured on the same bead through hybridization to the poly(T) sequence (reading on the limitations of claim 42)(see Fig. 1, [0015], [0021], [0032] and throughout). The poly(T) capture sequences on the bead thereby function simultaneously as both the mRNA-capturing sequence and the oligonucleic acid capturing region as recited in claim 41. Stoeckius further disclosed that from the co-captured targets, reverse transcription of mRNA and complementary strand synthesis from the construct oligonucleotide are carried out in the same reaction (see Fig. 1, [0015], [0021], [0032], and throughout), directly disclosing the requirement of claim 41 that “cDNA synthesis by reverse transcription reaction from the mRNA and complementary-strand synthesis from the oligonucleic acid are carried out” in the complementary-strand synthesis step.
One of ordinary skill in the art would have been motivated to modify the solid-phase single-cell whole transcriptome amplification method of Fu (as enhanced with the controlled homopolymer tailing of Lazinski) to incorporate the antibody-oligonucleotide capture approach taught by Stoeckius. The motivation is explicitly articulated in Stoeckius, which teaches that simultaneously capturing and sequencing both mRNA transcripts and antibody conjugated oligonucleotides from the same single cell provides substantially more informative multimodal measurements of cell identity and state than transcriptome data alone (see [0448], [0454]-[0457]) and that the technical implementation requires no more than adding the antibody-oligonucleotide constructs (whose poly(A) anchor is designed to co-capture on the existing poly(T) bead) to an otherwise conventional single-cell RNA-seq workflow. A person of ordinary skill in the art would have recognized that the poly(T) bead platform of Fu is directly compatible with the poly(A) anchored construct oligonucleotides of Stoeckius, as both rely on the same poly(A)/poly(T) hybridization chemistry. The combination represents nothing more than the use of a known technique (antibody-oligonucleotide co-capture, Stoeckius) applied toa known solid-phase single-cell sequencing platform (Fu) to achieve predictable and well-motivated results consistent with KSR Int'l Co. v. Teleflex Inc., 550 U.S. 398, 416, 82 USPQ2d 1385, 1395 (2007). A reasonable expectation of success is confirmed by Stoeckius itself, which demonstrates successful reduction to practice of this exact multimodal configuration.
In regards to claim 44, Stoeckius explicitly and repeatedly discloses the use of antibodies and antibody fragments as the ligand (specific-binding molecule) component of the oligonucleotide conjugated constructs (see Figs 1-2, [0015]-[0016], and throughout).
In regards to claim 45, Stoeckius discloses the use of streptavidin (an avidin type molecule) conjugated to the oligonucleotide construct which then bind to a biotinylated antibody (a biotin-type molecule) that is in turn bound to the cell surface molecule (see [0045], [0083], [0107] and throughout). This streptavidin-biotin indirect binding configuration is expressly described in the Stoeckius disclose as one embodiment of how the oligonucleotide-conjugated construct is tether to cell surface targets (see [0108]). Streptavidin is well-recognized in the art as an avidin-type molecule, and biotin as a biotin-type molecule, both being members of the broader avidin/biotin family of high-affinity binding pairs. Accordingly clam 45 is directly taught by the combination of Fu, Lazinski, Suzuki, and Stoeckius.
In regards to claim 46, Stoeckius explicitly discloses this exact workflow sequence as the foundational methodology of CITE-seq. Stoeckius teaches: (1) incubating cells with antibody-oligonucleotide conjugates to allow the antibody to bind its cell surface target; (2) washing away unbound conjugates; (3) co-depositing antibody-labeled cells with barcoded capture beads; and (4) lysing the cells to simultaneously release mRNA and the antibody -tethered oligonucleotides for co-capture on the bead (see Fig. 1, [0449], and throughout). This sequence maps precisely to the pre-capture steps of claim 46: contacting the cell with the specific-binding molecule labeled with the oligonucleic acid, binding it to the cell surface, and then lysing the cell, followed by the capture step of claim 41. Accordingly, the additional limitations of claim 46 are completely disclosed in Stoeckius and the combination of Fu, Lazinski, Suzuki, and Stoeckius renders the claim unpatentable.
In regards to claim 47, Stoeckius discloses that the oligonucleotide component of the antibody-conjugated construct comprises, in addition to the poly(A) anchor (capture target region), a common sequence element, specifically an amplification handle sequence that is shared across all construct oligonucleotides regardless of which antibody they are conjugated to (see Abstract, Fig. 1, [0009], [0013]-[0014], and throughout). This common/shared sequence on the 5’-side of the capture target region corresponds directly to the common sequence recited in claim 47. Fu discloses that the bead-immobilized capture probes comprise a first universal label (adapter) portion on the 5’-side of the oligo-dT (poly(T)) portion, corresponding to the first adapter portion on the 5’-side of the poly(T) portion as recited in claim 47 (and previously incorporated from claim 33) (see Fig 5). The combination of Fu and Stoeckius thus discloses all structural elements added by claim 47.
In regards to claim 48, Stoeckius discloses that the oligonucleotide construct comprises a ligand barcode sequence (also referred to as an antibody barcode or feature barcode) positioned between the amplification handle (common sequence) and the poly(A) anchor (capture target region) (see Abstract, [0009], and throughout). This ligand barcode uniquely identifies which specific-binding molecule (antibody) is conjugated to the oligonucleotide, thereby enabling multiplexed detection of multiple cell surface targets in a single experiment. This barcode positioned between the common sequence and the capture target region directly and completely corresponds to the specific-binding molecule-identification barcode portion between the common sequence and the capture target region recited in claim 48.
In regards to claim 49, as discussed in the rejection of claim 36, Fu discloses a second-strand synthesis primer comprising a second universal label (adapter) portion and a complementary homopolymer portion, corresponding to a primer comprising the second adapter portion on the 5’-side of the complementary homopolymer portion of claim 49 (see [0217], [0251]). Stoeckius discloses that a primer comprising the common/amplification handle sequence of the construct oligonucleotide is used for second-strand synthesis (or priming of the complementary strand) from the captured oligonucleic acid (see [0088]). This common sequence primer corresponds directly to the “primer comprising a common sequence” of claim 49. The use of two distinct second-strand primers, one targeting the adapter-homopolymer junction (for mRNA derived cDNA) and one targeting the common sequence (for oligonucleic acid derived complementary strand), is the natural and predictable implementation when combining a barcoded bead mRNA platform (Fu) with antibody-oligonucleotide co-capture (Stoeckius), as the two distinct captured nucleic acid species each require a distinct priming strategy for second-strand synthesis. A person of ordinary skill would have recognized this and implemented the dual-primer second-strand synthesis approach with a reasonable expectation of success.
In regards to claim 50, Fu discloses PCR amplification of the whole transcriptome amplification library using primers targeting the first and second universal label (adapter) sequences, corresponding to primers targeting the first adapter portion and second adapter portion of claim 50 (see Title, Abstract, [0009], [0010], and throughout). Stoeckius discloses that amplification of the antibody-oligonucleotide derived library components is carried out using a primer comprising the common/amplification handler sequence, corresponding to the primer comprising the common sequence of the present claim (see Abstract, [0015], and throughout). The three-primer amplification is thus a direct and predictable result of combing the dual-adapter amplification strategy of Fu with the common-sequence amplification strategy of Stoeckius in a single multiplex PCR reaction to simultaneously amplify both the mRNA-derived cDNA library and the oligonucleic acid-derived library from the same reaction vessel. This represents a straightforward combination of known primer designs applied to a combined library, well within the ordinary skill in the art.
Stoeckius further discloses following co-amplification of the full-length mRNA derived cDNA library and the antibody-oligonucleotide-derived library in the same reaction, size-based fractionation is performed to separate the two amplified products from one another (see [0032], [0037], [0491], and throughout). Specifically, Stoeckius teaches the use of gel electrophoresis (see [0037], [0402]) and SPRI (Solid-Phase Reversible Immobilization) bead-based size selection (see [0491]), a form of size fractionation well-known in the art, at a ration of 0.6X to exploit the large size difference between the two product populations: the mRNA-derived cDNA library which consist of large heterogeneous fragments (hundred to thousands of base pairs), is retained on the SPRI beads, while the short, uniform antibody derived tags library (approximately 170-180 base pairs), derived from the antibody-conjugated oligonucleic acid, is recovered from the supernatant fraction. This size-based fractionation step is explicitly presented in Stoeckius as a necessary and routine part of the workflow for separately recovering and sequencing the two libraries. Size fractionation of nucleic acid populations of different lengths by SPRI or equivalent means was a routine and ubiquitous technique in molecular biology library preparation at the time of the invention and its application to separate two co-amplified products of vastly different sizes would have required no more than ordinary skill and no inventive step. A person of ordinary skill in the art would have been motivated to perform size fractionation after co-amplification to obtain separate, clean libraries suitable for independent downstream sequencing, precisely as taught by Stoeckius.
In regards to claim 51, as disclosed above, the combination of Fu, Lazinski, and Stoeckius renders obvious the limitations of claim 49 for which claim 51 depends. This includes primers corresponding to a first adapter portion, a primer targeting the second adapter portion, and a primer comprising the common sequence. The instant claim further recites the use of a 4th primer in the amplification scheme, wherein the additional primer targets a desired region of cDNA, specifically the TCR constant region as described in claim 40.
Fu discloses, in addition to its whole-transcriptome amplification embodiment, an alternative approach in which amplification is carried out using a gene-specific primers to target a desired region in cDNA in combination with the universal label primers corresponding to the target amplification (see [0249]-[0251]). However, Fu does not explicitly disclose that the gene-specific primers target the T cell receptor constant region.
As disclosed above in regards to claim 40, Suzuki discloses methods utilizing a TCR constant region-specific primer. Suzuki explicitly teaches that this two-primer combination enables complete, unbiased amplification of all TCR variable region sequences regardless of VDJ rearrangement, because the constant region primer anchors to the conserved 3’ region of the TCR transcript and the adapter primer anchors to the 5’ end of the cDNA, with the intervening complete variable region recovered in the amplification product.
It would have been prima facie obvious to one of ordinary skill in the art at the time of filing to apply the TCR constant region primer amplification strategy of Suzuki to the multimodal amplification strategy outlined by the combination of Fu, Lazinski, and Stoeckius. One would be motivated to combine these teachings as Suzuki teaches that coupling TCR repertoire information with single-cell molecular analysis enables powerful and clinically significant applications in immunology, cancer immunology, infections disease, and autoimmunity.
Conclusion
No claim is allowed.
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/MATTHEW HAROLD RAYMONDA/Examiner, Art Unit 1684
/AARON A PRIEST/Primary Examiner, Art Unit 1681