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 .
Response to Amendment
The amendment filed 10/13/2025 is acknowledged. Regarding the Office action mailed 07/15/2025, all rejections under 35 USC 112(b), 102 and 103 are withdrawn in view of the amendment. After additional consideration and search, new grounds of rejection are set forth below. This Office action is NON-FINAL.
The “Claim Interpretation” set forth on pages 2-4 of the Office action mailed 07/15/2025 is maintained but is not reiterated here.
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 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.
Claim(s) 1, 2, 3, 13, 14, 16, 17, 19, 28, 32, 39 and 43 is/are rejected under 35 U.S.C. 103 as being unpatentable over Bostick (US 2020/0339978, previously cited) in view of Wende (US 2016/0024563). Claim 43 is generic to claim 1. Therefore, what is discussed for claim 1 applies equally to claim 43.
Regarding claim 1, Bostick disclosed:
A method for paired amplification of alpha and beta T cell receptor (TCR) sequences from a single T cell, the method comprising: placing a single T cell into a cell lysis solution to provide a T cell lysate comprising RNA;
Example 2, paragraphs [0208]-[0211] and Figures 6 and 7. See, e.g., Figure 6: “Single-cell input” and paragraph [0208]: “Single cells were sorted into lysis buffer in individual wells of a 96-well plate.”
generating first strand cDNA from the RNA;
See, e.g., Figure 6: “RT reaction” and paragraph [0210]: “First-strand cDNA synthesis was primed by the TCR dT Primer and performed by an MMLV-derived reverse transcriptase (RT).”
amplifying the first strand cDNA to provide amplified cDNA;
See, e.g., Figure 6: “Pre-amplification PCR” and paragraph [0211]: “A pre-amplification PCR step was then performed in each well to generate double-stranded cDNA, which serves as starting material for PCR amplification and library construction.” As can be seen in Figure 6, the primers used for this amplification were “TCR dT Primer” and “Pre-Amp PCR Primer”.
and amplifying the alpha and beta TCR sequences from the amplified cDNA in a single reaction, wherein the alpha and beta TCR sequences are amplified using three sets of TCR amplification primers.
See, e.g., Figure 6: “TCR-specific amplification” and paragraph [0211]: “Following a cleanup step, cDNA from each column of the 96-well plate was pooled in separate tubes (see FIG. 7), and TCR-specific sequences were amplified by PCR using primers that are complementary to the oligonucleotide-templated sequence (“SMART Primer 1”) and the constant region(s) of TCR-α and/or TCR-β subunits (“TCR a/b Human Primer 1”).”
Note that the “three sets of TCR amplification primers” are shown in Figure 6 as “SMART Primer 1” [the “first set”], and “TCR a/b Human Primer 1” [the “second” and “third set”]. While the figure does not show a separate primer for each of the TCR alpha and beta subunits, it would have been obvious to one of ordinary skill in the art prior to the effective filing date of the application to use two such primers, i.e. one for the TCR alpha subunit and one for the TCR beta subunit. Firstly, paragraph [0211] states (emphasis provided): “…and TCR-specific sequences were amplified by PCR using primers that are complementary to the oligonucleotide-templated sequence (“SMART Primer 1”) and the constant region(s) of TCR-α and/or TCR-β subunits (“TCR a/b Human Primer 1”).” Secondly, paragraph [0208] states (emphasis provided): “Inclusion of the barcodes allowed for demultiplexing of sequencing data for each pool, thereby allowing for single-cell resolution and pairing of sequence information for TCR-α and TCR-β subunits.” Based on the disclosure as to how the process was carried out, the fact that there was only one cell in each well of the 96-well plate used for the RT and Pre-amplification PCR steps shown in Figure 6, and the fact that the wells of each column of the plate were pooled for the TCR-specific amplification shown in Figure 6, it would have been obvious to use a TCR-α-specific primer and a TCR-β-specific primer in the TCR-specific amplification step of Figure 6 to allow for “pairing of sequence information for TCR-α and TCR-β subunits” from single cells. This would also have arrived at the limitations of claim 28.
Regarding claim 2, see Figure 6, where the “first RT primer set” corresponds to the “TCR dT Primer” and the “second RT primer set” corresponds to the “SMART-Seq Indexed Oligonucleotide”, which is a template-switching oligonucleotide. An enzyme having reverse transcriptase activity, a first period of time, and conditions that generate first strand cDNA are implicit. See also paragraph [0210]: “…MMLV-derived reverse transcriptase (RT).”
Regarding claim 3, TCR dT primer would have been understood to comprise a poly-T subsequence, both from the name “dT” and the illustration of the primer annealing to the poly-A portion of the target in Figure 6.
Regarding claims 13, 14, 16, 17 and 19, the “first set of TCR amplification primers” corresponds to “SMART Primer 1” and the “set of TCR alpha chain amplification primers” and the “set of TCR beta chain amplification primers” are represented by TCR a/b Human Primer 1, which is indicated as corresponding to the “constant region”.
Regarding claim 32, see paragraph [0090]: “For example, in some instances, cells of a cellular sample may be sorted based on expressing one or more immune cell markers including e.g., a T cell marker, a B cell marker, or the like, and collected for further downstream processes. In one example, T cells may be selected based on the expression of one or more T cell surface markers (e.g., CD4, CD8, etc.) and the T cells may be collected for further processing.”
Regarding claim 39, Bostick disclosed putting the components for performing the method into kits (paragraph [0186]).
As noted above in discussing the amplification of the alpha and beta TCR sequences in a single reaction using three sets of amplification primers, it would have been obvious to implement this amplification (shown in Fig. 6 as “PCR 1”) using separate primers for the TCR alpha and TCR beta constant regions, thus arriving at three sets of TCR amplification primers in a single reaction (see discussion of claim 1). However, Bostick did not disclose or suggest that in such a case, the ratio of “SMART Primer 1” to TCR alpha primer to TCR beta primer would be in a ratio of 2:1:1.
Wende (at paragraph [0047]) discussed two types of amplification: symmetric amplification, in which “the forward and reverse primers are used in an approx. equimolar concentration”, thereby “predominantly a double-stranded target amplicon is produced”; and asymmetric amplification, in which one primer “is used in excess of the primer”, thereby generating excess copies of one strand. Wende noted: “However, asymmetric PCR reactions generally hold the disadvantage of lower sensitivity and lower amplicon yield as compared to symmetric PCR reactions.” Therefore, according to Wende, performing symmetric amplification, with equal concentrations of the forward and reverse primers, provided an advantage compared to asymmetric amplification, where the two primers were at unequal concentrations.
Translating this principle to the situation in Bostick’s “PCR 1” of Figure 6, where both TCR alpha and TCR beta primers are used in conjunction with “SMART Primer 1” to amplify both TCR alpha and TCR beta targets, it can be seen that “SMART Primer 1” is used as a primer for each target. It follows, then, that in order to have equal primer concentrations for each target (as suggested by Wende), one would need to use twice the amount of “SMART Primer 1” as compared to each of TCR alpha primer and TCR beta primer. In this way, half the available “SMART Primer 1” (equal to the concentration of TCR alpha primer) would be available to amplify TCR alpha, and the remaining half (equal to the concentration of TCR beta primer) would be available to amplify TCR beta. Thus, one would arrive at a ratio of 2:1:1 (“SMART Primer 1”:TCR alpha primer:TCR beta primer).
Claim(s) 4 is/are rejected under 35 U.S.C. 103 as being unpatentable over Bostick (US 2020/0339978, previously cited) in view of Wende (US 2016/0024563) as applied to claims 1, 2, 3, 13, 14, 16, 17, 19, 28, 32, 39 and 43 above, and further in view of Hornung (US 2016/0122818, previously cited).
The disclosures of Bostick and Wende have been discussed. While Bostick’s RT primer comprised a poly-T priming sequence (Figure 6, paragraph [0204], “TCR dT Primer”; paragraph [0143], “poly dT”; ), Bostick did not disclose the presence of a 3’ “VN”. However, the presence of a 3’ “VN” at the end of a poly-T priming sequence for reverse transcription of polyadenylated mRNA has long been a common practice.
Hornung disclosed (paragraph [0091], emphasis provided): “Consequently, in preferred embodiments where the nucleic acid of interest is a eukaryotic mRNA, the primer is a universal RT primer that under condition of a reverse transcriptase enzymatic activity acts as a starting point for template-directed cDNA synthesis. Such a primer may comprise at its 3′-end a target-complementary region comprising or consisting of a poly(dT)-dV-3′OH or poly(dT)-dV-dN-3′OH sequence to facilitate hybridization to the polyadenylation sequence of the mRNA, wherein the poly(dT) sequence is 5 to 50 nucleotides in length and V is A. C or G and N is A, C, G or T. The V or VN sequence at its 3′-end is a degenerate sequence, i.e. the RT primer includes a population of primers that differ in this position in that they have either an A, C or G nucleotide for V and, if present, any one of A, G, C or T for N, This allows recognition of the end of the poly-A tail of the mRNA molecule and recognition of the 3′-end of the gene-specific mRNA region, which in turn ensures that reverse transcription of the gene-specific mRNA sequence is correctly initiated.”
It would have been prima facie obvious to one of ordinary skill in the art prior to the effective filing date of the application to modify the RT primer of Bostick by adding a 3’ VN, as Hornung disclosed this would ensure that the reverse transcription was “correctly initiated”.
Claim(s) 6, 7, 8, 10 and 22 is/are rejected under 35 U.S.C. 103 as being unpatentable over Bostick (US 2020/0339978, previously cited) in view of Wende (US 2016/0024563) as applied to claims 1, 2, 3, 13, 14, 16, 17, 19, 28, 32, 39 and 43 above, and further in view of Zhu (US 2004/0006033, previously cited).
The disclosure of Bostick has been discussed.
With regard to claims 6 and 7, Bostick’s method utilized a single TSO (Figure 6, “SMART-Seq Indexed Oligonucleotide”) as a “second RT primer set”.
Regarding claim 8, the entirety of Bostick’s TCR dT primer (the claimed “first RT primer set”), or any 3’ segment thereof, can be considered a “first amplification priming subsequence”. In addition, the 5’ segment of the SMART-Seq Indexed Oligonucleotide (which is the TSO of the “second RT primer set”) can be considered a “second amplification priming subsequence”.
Regarding claim 10, the “third primer set” corresponds to Bostick’s “Pre-Amp PCR Primer” and “TCR dT Primer”, used in the pre-amplification PCR of Bostick’s Figure 6. The hybridizing portions of these primers constitutes the “third amplification priming subsequences”.
Regarding claim 22, as shown in Bostick Figure 6, SMART Primer 1 (corresponding to the “first set of TCR amplification primers) comprises a 3’ sequence (an anchor subsequence) identical to the 5’ segment of the SMART-Seq Indexed Oligonucleotide (which is the TSO of the second RT primer set).
Bostick did not disclose that the TSO comprised a 3’ terminal ribonucleotide as recited in claim 6.
Zhu disclosed (paragraph [0177]): “A template-switching oligonucleotide is included during reverse transcription, which produces an mRNA-antisense cDNA hybrid. The template-switching oligonucleotide hybridizes to the CAP site at the 5'end of mRNA strand and serves as a short, extended template for CAP-dependent extension of the 3' end of the antisense cDNA strand. Template-switching oligonucleotides typically require a few ribonucleotides at the 3' end to promote CAP-dependent extension. Thus, template-switching oligonucleotides generally contain between about 1 and about 5 ribonucleotides at their 3' ends.”
It would have been prima facie obvious to one of ordinary skill in the art prior to the effective filing date of the application to incorporate between about 1 to 5 ribonucleotides at the 3’ end of Bostick’s TSO, as this was disclosed to be “general” practice as noted by Zhu.
Claim(s) 45 is/are rejected under 35 U.S.C. 103 as being unpatentable over Bostick (US 2020/0339978, previously cited) in view of Wende (US 2016/0024563) and Zhu (US 2004/0006033, previously cited) as applied to claims 6, 7, 8, 10 and 22 above, and further in view of Ramenani (WO 2019/191459, IDS ref).
The disclosures of Bostick, Wende and Zhu have been discussed. None of these references disclosed a template switch oligonucleotide (TSO) comprising SEQ ID NO: 2.
Ramenani disclosed a TSO comprising SEQ ID NO: 2; see page 86, line 4.
It would have been prima facie obvious to one of ordinary skill in the art prior to the effective filing date of the application to substitute the TSO used by Bostick with the TSO taught by Ramenani, as it is obvious to substitute equivalents known for the same purpose (MPEP 2144.06). One of ordinary skill would have understood this would also require modifying the sequences of Bostick’s “Pre-Amp PCR Primer” and “SMART Primer 1” based on the substituted TSO.
Claim(s) 9, 11 and 12 is/are rejected under 35 U.S.C. 103 as being unpatentable over Bostick (US 2020/0339978, previously cited) in view of Wende (US 2016/0024563) and Zhu (US 2004/0006033, previously cited) as applied to claims 6, 7, 8, 10 and 22 above, and further in view of Li (Nature Protocols 14:2571-2594 (2019), previously cited).
The disclosures of Bostick, Wende and Zhu have been discussed. The difference between claims 9, 11 and 12 and the disclosure of Bostick is that Bostick used a “first RT primer” (TCR dT) that was not disclosed to contain an “amplification priming subsequence” that was “substantially identical” to an “amplification priming subsequence” of the “TSO” (SMART-Seq Indexed Oligonucleotide) as recited in claim 9, such that a “single primer” could be used as the “third primer [set]” to amplify the “first strand cDNA” as recited in claim 11, wherein the “amplification priming subsequences” of the “first RT primer”, the “TSO” and the “third primer [set]” were “substantially identical” as recited in claim 12. Rather, Bostick used the same primer (TCR dT) as the first RT primer and as a primer to amplify the first strand cDNA.
Li disclosed a technique similar to that of Bostick, where RNA from a single cell was reverse transcribed to cDNA, the cDNA was amplified, and the amplified cDNA was subsequently amplified with TCR alpha and beta primers; see Figure 1, panel b. In Li’s technique, the reverse transcription primer had an oligo-dT portion (like Bostick’s), but also had a 5’ segment (P1) that was identical to a 5’ segment (P1) of the TSO, such that the first strand cDNA could be amplified by a single primer, which also comprised the P1 sequence.
It would have been prima facie obvious to one of ordinary skill in the art prior to the effective filing date of the application to modify the first RT primer of Bostick to contain the same 5’ sequence as the TSO (SMART-Seq Indexed Oligonucleotide) so that a single primer (Pre-Amp PCR Primer) could be used for amplifying the first strand cDNA. It would have been obvious to do this because Li’s approach represents an art-recognized alternative for achieving the same purpose as Bostick’s, namely synthesizing first strand cDNA and then amplifying it.
Claim(s) 20 is/are rejected under 35 U.S.C. 103 as being unpatentable over Bostick (US 2020/0339978, previously cited) in view of Wende (US 2016/0024563) as applied to claims 1, 2, 3, 13, 14, 16, 17, 19, 28, 32, 39 and 43 above, and further in view of Gaudernack (US 2023/0192804).
The disclosures of Bostick and Wende have been discussed. Bostick did not disclose that the TCR beta primer could amplify both type 1 and type 2 beta constant regions (or even the existence of such subtypes).
Gaudernack disclosed (paragraph [0106]): “There are two human β-chain subtypes, defined by the sequence of their constant region: subtype 1 and subtype 2.”
It would have been prima facie obvious to one of ordinary skill in the art prior to the effective filing date of the application to use a primer capable of amplifying both subtypes of TCR beta chain in order to ensure amplification of the TCR beta chain regardless of which subtype was expressed by a given cell.
Claim(s) 21 is/are rejected under 35 U.S.C. 103 as being unpatentable over Bostick (US 2020/0339978, previously cited) in view of Wende (US 2016/0024563 and Gaudernack (US 2023/0192804) as applied to claim 20 above and further in view of GenBank Accession AF045804 [online] 25 Jul 2016 [retrieved on 24 Jan 2026] retrieved from https://www.ncbi.nlm.nih.gov/nuccore/af045804 and AF071470 [online] 30 Jun 2000 [retrieved on 24 Jan 2026] retrieved from https://www.ncbi.nlm.nih.gov/nuccore/af071470.
The disclosures of Bostick, Wende and Gaudernack have been discussed. None of these references disclosed or suggested using a primer of SEQ ID NO: 98 as a primer that could amplify both type 1 and type 2 TCR beta chains.
GenBank AF045804 disclosed the sequence of the type 2 TCR beta chain, and GenBank AF071470 disclosed the sequence of the type 1 TCR beta chain. The sequence of SEQ ID NO: 98 is found in each of these sequences.
AF045804:
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100
414
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AF071470:
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104
418
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It would have been prima facie obvious to one of ordinary skill in the art prior to the effective filing date of the application to modify the method suggested by the combined disclosures of Bostick, Wende and Gaudernak by consulting the known sequences of the type 1 and 2 TCR beta chain sequences disclosed in GenBank Accession numbers AF071470 and AF045804 and selecting a suitable sequence found in both in order to obtain a primer capable of amplifying both types.
Claim(s) 6, 7, 8, 9, 10, 11, 12, 22 and 46 is/are rejected under 35 U.S.C. 103 as being unpatentable over Bostick (US 2020/0339978, previously cited) in view of Wende (US 2016/0024563) as applied to claims 1, 2, 3, 13, 14, 16, 17, 19, 28, 32, 39 and 43 above, and further in view of Picelli (Nature Protocols 9:171-181 (2014)).
The disclosure of Bostick has been discussed.
With regard to claims 6 and 7, Bostick’s method utilized a single TSO (Figure 6, “SMART-Seq Indexed Oligonucleotide”) as a “second RT primer set”.
Regarding claim 8, the entirety of Bostick’s TCR dT primer (the claimed “first RT primer set”), or any 3’ segment thereof, can be considered a “first amplification priming subsequence”. In addition, the 5’ segment of the SMART-Seq Indexed Oligonucleotide (which is the TSO of the “second RT primer set”) can be considered a “second amplification priming subsequence”.
Regarding claim 22, as shown in Bostick Figure 6, SMART Primer 1 (corresponding to the “first set of TCR amplification primers) comprises a 3’ sequence (an anchor subsequence) identical to the 5’ segment of the SMART-Seq Indexed Oligonucleotide (which is the TSO of the second RT primer set).
Bostick did not disclose that the TSO comprised a 3’ terminal ribonucleotide as recited in claim 6. Bostick did not disclose that the “first amplification priming subsequence” of the “first RT primer set” and the “second amplification priming subsequence” of the “second RT primer set” (i.e. the TSO) have substantially identical nucleotide sequences as recited in claim 9. Bostick did not disclose that the “third primer set” used to amplify the first strand cDNA (corresponding to Bostick’s Pre-Amp PCR Primer and TCR dT primer of figure 6) comprised a single primer as recited in claim 11, more particularly comprising the sequence of SEQ ID NO: 3 as recited in claim 46. Bostick did not disclose that the first amplification priming subsequence of the first RT primer set, the second amplification priming subsequence of the second RT primer set, and the third amplification priming subsequence of the third primer set all have substantially identical nucleotide sequences as recited in claim 12.
Picelli disclosed a process for synthesizing and amplifying cDNA from single cells similar to that of Bostick (see figure 1). Picelli’s technique differed from Bostick’s in that the “first RT primer” (i.e. Bostick’s TCR dT primer) comprised a 5’ segment that was identical to the 5’ segment of the “second RT primer” (i.e. the TSO). This allowed for a single primer, ISPCR, (i.e. a “third primer set” as recited in claim 10) to be used to amplify the first strand of cDNA (Picelli, figure 1, “PCR preamplification of cDNA”). In addition, Picelli’s TSO comprised a 3’ terminal ribonucleotide subsequence (as recited in claim 6). Picelli disclosed the sequence of the ISPCR primer (top of page 174), which is identical to SEQ ID NO: 3 as recited in claim 46. While Picelli performed different steps following the preamplification of the cDNA compared to Bostick, Picelli’s technique up to that point arrives at amplified cDNA from a single cell just like Bostick’s method.
It would have been prima facie obvious to one of ordinary skill in the art prior to the effective filing date of the application to modify Bostick’s method by employing a first RT primer, a second RT primer (i.e., TSO), and a “third primer” as disclosed by Picelli in order to arrive at the same point in Bostick’s method, i.e. amplified first strand cDNA. One of ordinary skill in the art would have viewed this merely as an alternative approach to Bostick for generating amplified first strand cDNA. This would also have arrived at the limitations of claims 8-12 where “first”, “second” and “third” amplification priming subsequences were identical (note Picelli’s disclosed TSO, Oligo-dT and ISPCR oligos at the top of page 174 can be seen to comprise all comprise the same sequence, identical to instant SEQ ID NO: 3. One of ordinary skill in the art would have understood this would also have necessitated designing Bostick’s SMART Primer 1 (see Bostick figure 6) to contain an appropriate sequence for the subsequent amplification step.
Conclusion
Claims 23, 24, 27, 47, 48 and 49 are objected to as being dependent upon a rejected base claim, but would be allowable if rewritten in independent form including all of the limitations of the base claim and any intervening claims. There was no apparent reason from the prior art to arrive at primers with these particular sequences or 3’ termini absent the application of unreasonable hindsight reasoning.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to SAMUEL C WOOLWINE whose telephone number is (571)272-1144. The examiner can normally be reached 9am-5:30pm.
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/SAMUEL C WOOLWINE/ Primary Examiner, Art Unit 1681