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 .
Election/Restrictions
Applicant’s election of the particular species that are SEQ ID NO: 62 and SEQ ID NO: 129, in the reply filed on 07/10/2025 is acknowledged.
In light of the Examiner’s search and consideration of the claims, the species election requirement as set forth in the papers of 06/11/2025 is withdrawn.
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.
Claim(s) 1-8, 13-22 and 23 is/are rejected under 35 U.S.C. 103 as being unpatentable over Dziubianau et al (2013) in view of Chovanec et al (2018).
Dziubianau et al provides methods for the analysis of sequences of rearranged adaptive immune response genes (recombined TCRβ locus). Relevant to claim 1 steps a) and b), the reference teaches obtaining CD3+ T-cells from peripheral blood samples (relevant to claims 3-6) and obtaining genomic DNA from the cells (e.g.: p.2843 - Sample collection, ex vivo antigen-specific stimulation and enrichment of antigen-specific T cells, cell line propagation, nucleic acid extraction). Relevant to claim 1 steps c) and), the reference teaches amplification of recombined TCRβ loci including the complementarity determining region 3 comprising V, D and J regions (relevant to claims 7 and 8) using PCR and hybridization and extension of primers specific to the V and J regions of the TCRβ loci (relevant to claims 14-18) (e.g.: p.2843 - Primer design and amplification of the recombined TCRβ locus; Supplemental Table 3). Relevant to step e) of claim 1, the reference teaches sequencing the resulting amplicons to determine the sequence of the rearranged gene (e.g.: p.2843 – Amplicon sequencing; Sequence analysis).
Relevant to claims 22 and 23, the reference teaches sequencing analysis results evident of a plurality of rearranged genomic sequences in the sample, and teaches the analysis of the frequency of different sequences to determine a repertoire (e.g.: Table 1; p.2844 - NGS allows in-depth TCR repertoire analysis and reveals oligoclonal clonotype composition of antigen-specific T cells).
Dziubianau et al does not teach a method of sequential hybridization comprising an isolation or purification of first extended probes followed by hybridization of second probes to the isolated or purified first extended probes and extension of the hybridized second probes (as set forth in parts iii-v of step c) of claim 1). However, the purification of extended first probes, in the context of sequencing rearranged VDJ genes, was known in the prior art and is taught by Chovanec et al.
Relevant to the steps of claim 1, Chovanec et al teaches hybridization and extension of a first primers specific to the J region of rearranged loci, purification and isolation of the extended primers, followed by hybridization and extension of a second primer that is specific for the isolated and purified extended first primers, then amplification by PCR and sequencing (e.g.: Figure 1a).
Further relevant to claim 2, the teachings of Chovanec et al include fragmenting a DNA sample and end-repair of the fragmented DNA, as well as ligating amplification adapters to nucleic acids, relevant to claim 13 (e.g.: Figure 1a). Relevant to claims 19-21, Chovanec et al teaches that the analysis of rearranged VDJ loci can include the addition of unique molecular identifiers and sequencing indices which are used in the identification of clones (e.g.: Fig 1b; Fig. 2; p.1237 - UMI considerations).
It would have been prima facie obvious to someone with ordinary skill in the relevant art before the effective filing date of the rejected claims to combined the teachings of Dziubianau et al with regard to amplification of rearranged VDJ loci using probes specific to the rearranged gene, with the sequential steps of a first probe hybridization and extension, purification of extended probes, and a first probe hybridization and extension, as taught by Chovanec et al. The skilled artisan would have been motivated to include a second set of probes specific for a second portion of a rearranged gene (as taught by Dziubianau et al; as required by parts iv and v of step c) of claim 1) based on the expressed teachings of Dziubianau et al that such reagents, in combination with the next generation sequencing analysis of genomic DNA amplified with partly degenerate primers covering all functional Vβ and Jβ genes, provide an unbiased characterization of the T cell receptor repertoire of a sample. As such the use of a second set of probes/primers as taught by Dziubianau et al in the methods of Chovanec et al would be the simple substitution of one element (i.e.: the degenerate Vβ primers of Dziubianau et al), for the another (i.e.: the adapter specific primer of Chovanec et al) with predictable results.
Claim(s) 9-12 is/are rejected under 35 U.S.C. 103 as being unpatentable over Dziubianau et al (2013) in view of Chovanec et al (2018), as applied to claims 1-8, 13-22 and 23 above, and further in view of Raymond et al WO 2014/093330 (2014; cited on the IDS of 01/31/2025).
Dziubianau et al in view of Chovanec et al renders obvious the method of claim 1, from which instantly rejected claims 9-12 depend.
Dziubianau et al in view of Chovanec et al does not provide for methods that include copying an extended sequence with T4 DNA pol and T4 gene 32 (relevant to claim 9), or the inclusion of PEG8000 at 7.5% (relevant to claims 10-12). However, such methods as applied to processing nucleic acid fragments in methods that include capture and amplification were known in the prior art and art taught by Raymond et al.
Raymond et al teach methods of nucleic acid fragment analysis including capture, amplification and sequencing. Relevant to the instantly rejected claims, Raymond et al teaches that such methods may include copying an extended sequence with T4 DNA pol and T4 gene 32 and also teaches that PEG8000 at 7.5% is useful as an additive in polymerase extension reactions (e.g.: p.168; p.174).
It would have been prima facie obvious to someone with ordinary skill in the relevant art before the effective filing date of the rejected claims to have included the steps of copying a sequence with T4 DNA polymerase and T4 gene 32 protein in the presence of PEG8000 as taught by Raymond et al in the methods rendered obvious by Dziubianau et al in view of Chovanec et al. The skilled artisan would have been motivated to include such methods based on the expressed teachings of Raymond et al that T4 polymerase can copy many and diverse genomic sequences provided it is supplemented with T4 gene 32 protein in the presence of PEG8000 - a molecular
crowding agent, and the exemplifications of Raymond et al which include PEG8000 at 7.5%.
Claim(s) 24 and 25 is/are rejected under 35 U.S.C. 103 as being unpatentable over Dziubianau et al (2013) in view of Chovanec et al (2018), as applied to claims 1-8, 13-22 and 23 above, and further in view of Baum et al (2012).
Dziubianau et al in view of Chovanec et al renders obvious the method of claim 1, from which instantly rejected claims 24 and 25 depend.
Dziubianau et al in view of Chovanec et al does not provide for methods that include profiling a TCR repertoire in a whole blood sample. However, the analysis of whole blood TCR repertoires was known in the prior art and is taught by Baum et al (2012).
Raymond et al teaches (e.g.: Fig 1B, p.3469 – right col) that a “whole-blood TCR repertoire” is the number of of unique TCRs found in a fixed volume of whole blood.
It would have been prima facie obvious to someone with ordinary skill in the relevant art before the effective filing date of the rejected claims to have performed the methods rendered obvious by Dziubianau et al in view of Chovanec et al using genomic material obtained from a whole blood sample. The skilled artisan would have been motivated to analyze such a sample based on the expressed teachings of Baum et al that the analysis of such a whole blood sample reflects the absolute abundance of different T-cell subpopulations in the blood and may define the current state
of the immune system’s ability to recognize a pathogen.
Claim(s) 26 is/are rejected under 35 U.S.C. 103 as being unpatentable over Dziubianau et al (2013) in view of Chovanec et al (2018) and Baum et al (2012) as applied to claims 24 and 25 above, and further in view of Iams et al (2017).
Dziubianau et al in view of Chovanec et al and Baum et al renders obvious the analysis of TCR repertoires in whole blood samples.
Dziubianau et al in view of Chovanec et al and Baum et al does not teach the assessment of both circulating nucleic acids and immune repertoires from a single sample.
However, the analysis of both circulating nucleic acids and immune repertoires from a single sample was known in the prior art and is taught by Iams et al.
Iams et al teaches a blood-based assay for simultaneous sequencing of circulating, cell-free tumor DNA (cfDNA) and determination of the peripheral a and b T cell receptor repertoire in patients with SCLC.
It would have been prima facie obvious to someone with ordinary skill in the relevant art before the effective filing date of the rejected claims to combined the methods rendered obvious by Dziubianau et al in view of Chovanec et al and Baum et al (i.e.: capture-based analysis of immune repertoires in whole blood) with the analysis of multiple analytes (e.g.: cell-free tumor DNA and α and β T cell receptor genes) in a single sample as taught by Iams et al. The skilled artisan would have been motivated to apply the methods rendered obvious by Dziubianau et al in view of Chovanec et al and Baum et al to the combined analytes of Iams et al based on the expressed teachings of Iams et al that coupled determination of T cell repertoire together with cfDNA monitoring will merge into a clinically useful “molecular image” of each patient’s disease status and real-time host immune response.
Claim(s) 27 and 28 is/are rejected under 35 U.S.C. 103 as being unpatentable over Dziubianau et al (2013) in view of Chovanec et al (2018) claims 1-8, 13-22 and 23 above, and further in view of Kurokawa et al (2001) and Zhang et al (1992).
Dziubianau et al in view of Chovanec et al renders obvious the analysis of TCR repertoires in immune cells from a blood sample.
Dziubianau et al in view of Chovanec et al does not teach whole genome amplification of genomic DNA from a single cell (relevant to claim 26) or analysis of paring between alpha and beta chain TCR within a single cell
However, the analysis of alpha and beta chain TCR in single cells, and the use of whole genome amplification for analysis of genetic material from single cells, was known in the prior art.
Kurakowa et al teaches the analysis of T cell receptor genes that is performed by the paired cloning of TCR α/β genes directly from a single T cells (e.g.: p.340 – Single-cell PCR). Kurakowa et al teaches analysis of mRNA extracted from single cells (e.g.: p.344, right col). However, Dziubianau et al teaches that there are situations in which the analysis of genomic DNA may be a more suitable analytical template as compared to mRNA.
In this regard, Zhang et al teaches methods of whole genome amplification to increase the amount of DNA available for an analysis of genomic DNA obtained from a single cell (e.g.: p.5847 - PEP of Single-Sperm DNA). Zhang et al teaches that whole genome amplification extends the possible applications of single cell studies.
It would have been prima facie obvious to someone with ordinary skill in the relevant art before the effective filing date of the rejected claims to used methods rendered obvious by Dziubianau et al in view of Chovanec et al to analyze paired TCR α/β genes directly from a single T cells, as taught by Kurakowa et al. The skilled artisan would have been motivated to analyze gene from a single cell based on the expressed teachings of Kurakowa et al that such methods make it possible to obtain recombinant TCR molecules from a single T cell without cellular cloning and promotes the investigation of T cell antigen specificity. The skilled artisan would recognize that Kurakowa et al exemplifies methods of analysis using mRNA and RT-PCR analysis. In this regard it would have been prima facie obvious to someone with ordinary skill in the relevant art before the effective filing date of the rejected claims to have performed the whole genome amplification methods of Zhang et al on genomic DNA from a single cell to perform the analysis of Kurakowa using the methods of Dziubianau et al in view of Chovanec et al. The skilled artisan would have been motivated to use genomic DNA based on the expressed teachings of Dziubianau et al that genomic DNA may be a superior source of genetic information, and the teachings of Zhang et al that whole genome amplification provides more material from the analysis of gDNA from a single cell.
Conclusion
Claims 1-28 are rejected in view of the teachings of the cited prior art.
Claims 29 and 30 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.
Claims 29 and 30 require particular first or second, respectively, probes that are at least 90% identical to a sequence selected from SEQ ID NOs: 62-128 or SEQ ID NOs: 129-227. The particular probes that are provided in the recited SEQ ID NOs have a particular 45 nucleotide tail sequence, followed by a 4 nucleotide random tag sequence, followed by a probe sequence specific for a J region of the TCR gene (SEQ ID NOs: 62-128) or specific for a v region of the TCR gene (SEQ ID NOs: 129-227). The prior art does not teach or specifically suggest these particular probe sequence for the extension of rearranged adaptive immune response genes in a method of sequential hybridization a capture of the target loci.
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Stephen Kapushoc
Primary Examiner
Art Unit 1683
/STEPHEN T KAPUSHOC/ Primary Examiner, Art Unit 1683