WO2021142437A1 - Surveillance d'adn acellulaire - Google Patents

Surveillance d'adn acellulaire Download PDF

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WO2021142437A1
WO2021142437A1 PCT/US2021/012945 US2021012945W WO2021142437A1 WO 2021142437 A1 WO2021142437 A1 WO 2021142437A1 US 2021012945 W US2021012945 W US 2021012945W WO 2021142437 A1 WO2021142437 A1 WO 2021142437A1
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cfdna
tumor
therapy
cancer
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PCT/US2021/012945
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James Xin SUN
Matthew Joseph DAVIS
Desiree SCHENK
Daniel NAVARRO GOMEZ
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Gritstone Bio, Inc.
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Priority to EP21738150.8A priority Critical patent/EP4087940A4/fr
Publication of WO2021142437A1 publication Critical patent/WO2021142437A1/fr
Priority to US17/861,088 priority patent/US20230104840A1/en

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    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B20/00—ICT specially adapted for functional genomics or proteomics, e.g. genotype-phenotype associations
    • G16B20/20—Allele or variant detection, e.g. single nucleotide polymorphism [SNP] detection
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6869—Methods for sequencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • cancers with a high mutational burden such as non-small cell lung cancer (NSCLC) and melanoma
  • NSCLC non-small cell lung cancer
  • melanoma melanoma
  • NSCLC non-small cell lung cancer
  • neoantigen-based vaccination can elicit T- cell responses and that neoantigen targeted cell-therapy can cause tumor regression under certain circumstances in selected patients.
  • neoantigen vaccine design is which of the many coding mutations present in subject tumors can generate the “best” therapeutic neoantigens, e.g., antigens that can elicit anti-tumor immunity and cause tumor regression.
  • Targeting antigens that are shared among patients with cancer hold great promise as a vaccine strategy, including targeting both neoantigens with a mutation as well as tumor antigens without a mutation (e.g., tumors antigens that are improperly expressed).
  • Challenges with shared antigen vaccine strategies include at least monitoring cancer status and/or efficacy of a vaccine prior to or following administration of a cancer vaccine to a subject.
  • many standard methods to monitor disease that are invasive or burdensome such as radiological assessments (e.g., CT scans) or tumor biopsies.
  • certain existing cell-free DNA monitoring methods suffer from reduced monitoring capability of cancer status and burden, such as reduced monitoring sensitivity, as they only monitor a small fraction of mutations (e.g., less than 50) associated with a tumor exome.
  • certain existing cell-free DNA monitoring methods e.g., Wan et al. ⁇ , Science Translational Medicine 17 Jun 2020:Vol. 12, Issue 548) suffer from reduced accuracy and reliability as they only monitor greater numbers of mutations at low-sequencing depth.
  • cancer monitoring methods such as cell-free DNA sequencing methods that offer broad target coverage (e.g., at least 95% of mutations present in a cancer exome) at high sequencing read depth (e.g., at least 1000X).
  • the method comprises the steps of: a. obtaining or having obtained sequencing data of cell-free DNA (cfDNA) from a sample from the subject, and wherein the sequencing data comprises a target coverage of at least 50% of all polynucleotide regions of interest corresponding to mutations present in an exome of the cancer and wherein the sequenced polynucleotide regions of interest have a mean read depth of at least 1000X, optionally wherein the polynucleotide regions of interest comprise at least 50 mutations, optionally wherein the mean read depth is mean duplex read depth, optionally wherein the cfDNA has been enriched prior to sequencing using a library of subject- specific and cancer- specific polynucleotide probes configured to capture the polynucleotide regions of interest, and optionally wherein obtaining the sequencing data comprises collecting or having collected the sample from the subject, isolating or having isolated the cfDNA, enriching or having enriched the cfDNA, and/or sequencing or having sequenced the
  • the method comprises the steps of: a. obtaining or having obtained sequencing data of cell-free DNA (cfDNA) from a sample from the subject, and wherein the sequencing data comprises a target coverage of at least 95% of all polynucleotide regions of interest corresponding to mutations present in an exome of the cancer, wherein the polynucleotide regions of interest comprise at least 50 mutations , and wherein the sequenced polynucleotide regions of interest have a mean duplex read depth of at least 1000X, wherein the cfDNA has been enriched prior to sequencing using a library of subject-specific and cancer-specific polynucleotide probes configured to capture the polynucleotide regions of interest, and optionally wherein obtaining the sequencing data comprises collecting or having collected the sample from the subject, isolating or having isolated the cfDNA, enriching or having enriched the cfDNA, and/or sequencing or having sequenced the cfDNA; and b.
  • cfDNA cell-free DNA
  • determining or having determined a frequency of the at least 50 mutations present in the exome to assess the status of the cancer optionally wherein assessment of the status comprises assessment of presence and/or cancer burden.
  • assessment of the status comprises assessment of presence and/or cancer burden.
  • the method comprises the steps of: a. obtaining or having obtained sequencing data of cell-free DNA (cfDNA) from a pre-therapy sample from the subject, and wherein the sequencing data comprises a target coverage of at least 50% of all polynucleotide regions of interest corresponding to mutations present in an exome of the cancer and wherein the sequenced polynucleotide regions of interest have a mean read depth of at least 1000X, optionally wherein the polynucleotide regions of interest comprise at least 50 mutations, optionally wherein the mean read coverage is mean duplex read coverage, and optionally wherein obtaining the sequencing data comprises collecting or having collected the pretherapy sample from the subject, isolating or having isolated the pre-therapy cfDNA, enriching or having enriched the pre-therapy cfDNA, and/or sequencing or having sequenced the pre-therapy cfDNA; b.
  • cfDNA cell-free DNA
  • sequencing data comprises a target coverage of at least 50% of all polynucleotide regions of interest corresponding to mutations present in an exome of the cancer and wherein the sequenced polynucleotide regions of interest have a mean read depth of at least 1000X, optionally wherein the polynucleotide regions of interest comprise at least 50 mutations, optionally wherein the mean read coverage is mean duplex read coverage, and optionally wherein obtaining the sequencing data comprises collecting or having collected the post-therapy sample from the subject, isolating or having isolated the post-therapy cfDNA, enriching or having enriched the post-therapy cfDNA, and/or sequencing or having sequenced the post-therapy cfDNA; and c.
  • determining or having determined the frequency the mutations present in the exome of the pre-therapy cfDNA relative to the post-therapy cfDNA to assess the efficacy of the therapy optionally wherein an increase in the frequency of the mutations in the post-therapy cfDNA relative to the pre-therapy cfDNA indicates an increased likelihood that tumor burden of the subject is increasing, and optionally wherein a decrease or maintenance of the frequency of the mutations in the post-therapy cfDNA relative to the pre-therapy cfDNA indicates an increased likelihood that tumor burden of the subject is decreasing or stable.
  • the method comprises the steps of: a. obtaining or having obtained sequencing data of tumor-derived DNA from a cancer-diseased tissue from the subject, optionally wherein obtaining the sequencing data comprises collecting or having collected the cancer-diseased tissue, isolating or having isolated the tumor-derived DNA, and sequencing or having sequenced the tumor-derived DNA; b.
  • sequencing data comprises a target coverage of at least 50% of all polynucleotide regions of interest corresponding to the tumor-associated mutations and wherein the sequenced polynucleotide regions of interest have a mean read depth of at least 1000X, optionally wherein the mean read coverage is mean duplex read coverage, and optionally wherein obtaining the sequencing data comprises collecting or having collected the pre-therapy sample from the subject, isolating or having isolated the pre-therapy cfDNA, enriching or having enriched the pre-therapy cfDNA, and/or sequencing or having sequenced the pretherapy cfDNA; e.
  • cfDNA cell-free DNA
  • sequencing data comprises cell-free DNA (cfDNA) from a post-therapy sample from the subject, optionally wherein the therapy comprises a cancer vaccine comprising the neoantigen or expression system encoding the same, wherein the post-therapy cfDNA was enriched prior to sequencing using the subject- specific and tumor- specific polynucleotide probes, and wherein the sequencing data comprises a target coverage of at least 50% of all polynucleotide regions of interest corresponding to the tumor- associated mutations and wherein the sequenced polynucleotide regions of interest have a mean read depth of at least 1000X, optionally wherein the mean read coverage is mean duplex read coverage, and optionally wherein obtaining the sequencing data comprises collecting or having collected the post-therapy sample from the subject, isolating or having isolated the post-therapy cfDNA, enriching or having enriched the post-therapy cfDNA, and/or sequencing or having sequenced the post-therapy cfDNA; and f.
  • the therapy comprises a cancer vaccine comprising the
  • determining or having determined the frequency of the tumor-associated mutations of the pre-therapy cfDNA relative to the post-therapy cfDNA to assess the efficacy of the therapy optionally wherein at least the one or more tumor-associated mutations associated with the neoantigen is determined, optionally wherein an increase in the frequency of the mutations in the posttherapy cfDNA relative to the pre-therapy cfDNA indicates an increased likelihood that tumor burden of the subject is increasing, and optionally wherein a decrease or maintenance of the frequency of the mutations in the post-therapy cfDNA relative to the pre-therapy cfDNA indicates an increased likelihood that tumor burden of the subject is decreasing or stable.
  • the method comprises one or more of the steps of: a. collecting or having collected the sample from the subject; b. isolating or having isolated the cfDNA; c. enriching or having enriched the cfDNA; or d. sequencing or having sequenced the cfDNA.
  • the method comprises each of the steps of: a. collecting or having collected the sample from the subject; b. isolating or having isolated the cfDNA; c. enriching or having enriched the cfDNA; and d. sequencing or having sequenced the cfDNA.
  • the mean read depth comprises at least 1500X, at least 2000X, at least 2500X, 3000X, at least 3500X, at least 4000X, at least 4500X, or at least 5000X mean read coverage. In some aspects, the mean read depth comprises a range from 1000X to 5000X mean read coverage. In some aspects, the mean read depth comprises a range from 1000X to 4000X, 1000X to 3000X, 1000X to 2000X, 2000X to 5000X, 2000X to 4000X, 2000X to 3000X, 3000X to 5000X, 3000X to 4000X, or 4000X to 5000X mean read coverage. In some aspects, the mean read depth comprises mean read duplex depth.
  • each of the polynucleotide regions of interest corresponding to the mutations present in the exome comprise a read depth of at least 1000X. In some aspects, each of the polynucleotide regions of interest corresponding to the mutations present in the exome comprise a read depth of at least 1000X, at least 1500X, at least 2000X, at least 2500X, 3000X, at least 3500X, at least 4000X, at least 4500X, or at least 5000X.
  • the target coverage comprises at least 60%, at least 70%, at least 80%, or at least 90% of polynucleotide regions of interest corresponding to the mutations present in the exome of the cancer. In some aspects, the target coverage comprises at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or 100% of polynucleotide regions of interest corresponding to the mutations present in the exome of the cancer. In some aspects, the target coverage comprises at least 95% of polynucleotide regions of interest corresponding to the mutations present in the exome of the cancer.
  • the polynucleotide regions of interest comprise at least 50, at least 60, at least 70, at least 80, or at least 90 mutations. In some aspects, the polynucleotide regions of interest comprise at least 50 mutations. In some aspects, the polynucleotide regions of interest comprise at least 100, at least 150, at least 200, at least 250, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, or at least 1000 mutations.
  • the method comprises the steps of: a. obtaining or having obtained sequencing data of tumor-derived DNA from a cancer-diseased tissue from the subject, optionally wherein obtaining the sequencing data comprises collecting or having collected the cancer-diseased tissue, isolating or having isolated the tumor-derived DNA, and sequencing or having sequenced the tumor-derived DNA; b.
  • the cancer is selected from the group consisting of: lung cancer, melanoma, breast cancer, ovarian cancer, prostate cancer, kidney cancer, gastric cancer, colon cancer, testicular cancer, head and neck cancer, pancreatic cancer, brain cancer, B-cell lymphoma, acute myelogenous leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia, T cell lymphocytic leukemia, non- small cell lung cancer, and small cell lung cancer.
  • the subject has been administered a therapy.
  • the therapy comprises a cancer vaccine.
  • the cancer vaccine comprises an epitope-encoding nucleic acid sequence encoding at least one of the mutations present in the exome of the cancer.
  • the cancer vaccine comprises a self-amplifying alphavims-based expression system.
  • the cancer vaccine comprises a chimpanzee adenovirus (ChAdV)-based expression system.
  • the method comprises obtaining sequencing data of cfDNA from two or more samples from the subject. In some aspects, the two or more samples are collected at different time points. In some aspects, the two or more samples are collected at different time points relative to administration of a therapy.
  • a pre-therapy sample is collected prior to administration of the therapy and a post-therapy cfDNA is collected subsequent to administration of the therapy.
  • the determining step comprises determining or having determined the frequency of the mutations of the pre-therapy cfDNA relative to the post-therapy cfDNA to assess the efficacy of the therapy, optionally wherein at least the one or more tumor-associated mutations associated with the neoantigen is determined, optionally wherein an increase in the frequency of the mutations in the posttherapy cfDNA relative to the pre-therapy cfDNA indicates an increased likelihood that tumor burden of the subject is increasing, and optionally wherein a decrease or maintenance of the frequency of the mutations in the post-therapy cfDNA relative to the pre-therapy cfDNA indicates an increased likelihood that tumor burden of the subject is decreasing or stable.
  • an increase in the frequency of the mutations in the post-therapy cfDNA relative to the pre-therapy cfDNA indicates an increased likelihood that tumor burden of the subject is increasing.
  • a decrease or maintenance of the frequency of the mutations in the post-therapy cfDNA relative to the pre-therapy cfDNA indicates an increased likelihood that tumor burden of the subject is decreasing or stable.
  • the decrease comprises a Complete Response (CR) or a Partial Response (PR).
  • the method further comprises administering a therapy to the subject following the assessment of the status of the cancer.
  • the assessment of the frequency of the mutations in the cfDNA indicates a likelihood the subject has cancer.
  • the therapy comprises a cancer vaccine.
  • the cancer vaccine comprises an epitope-encoding nucleic acid sequence encoding at least one of the mutations present in the exome.
  • the cancer vaccine comprises a self-amplifying alphavims-based expression system.
  • the cancer vaccine comprises a chimpanzee adenovirus (ChAdV)-based expression system.
  • the collecting step comprises collecting a blood sample.
  • the isolation step comprises centrifugation to separate cfDNA from cells and/or cellular debris. In some aspects, the isolation step comprises isolating cfDNA from whole blood. In some aspects, isolating cfDNA from whole blood comprises separating the plasma layer, huffy coat, and red blood cells. In some aspects, the cfDNA is isolated from the plasma layer.
  • the sequencing step comprises next generation sequencing (NGS) or Sanger sequencing. In some aspects, NGS comprises duplex sequencing, whole-exome sequencing, whole-genome sequencing, de novo sequencing, phased sequencing, targeted amplicon sequencing, or shotgun sequencing. In some aspects, NGS comprises duplex sequencing. In some aspects, NGS comprises whole-exome sequencing.
  • the enrichment step comprises enriching the cfDNA for the polynucleotide regions of interest corresponding to the mutations present in the exome prior to sequencing. In some aspects, the enrichment comprises using subject- specific and tumor- specific polynucleotide probes. In some aspects, the subject- specific and tumor- specific polynucleotide probes comprises each of the polynucleotide regions of interest corresponding to the mutations present in the exome.
  • the subject-specific and tumor-specific polynucleotide probes comprises at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or 100% of polynucleotide regions of interest corresponding to the mutations present in the exome of the cancer.
  • the subject-specific and tumor-specific polynucleotide probes comprises at least 50, at least 60, at least 70, at least 80, at least 90 mutations, at least 100, at least 150, at least 200, at least 250, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, or at least 1000 mutations, optionally the mutations present in the exome of the cancer.
  • the enrichment step comprises hybridizing one or more polynucleotide probes to the one or more polynucleotide regions of interest.
  • the polynucleotide probes are 80 to 150 base pairs (bp) in length.
  • the polynucleotide probes are 80 to 140, 80 to 130, 80 to 120, 80 to 110, 80 to 100, 80 to 90, 90 to 150, 90 to 140, 90 to 130, 90 to 120, 90 to 110, 90 to 100, 100 to 150, 100 to 140, 100 to 130, 100 to 120, 100 to 110, 110 to 150, 110 to 140, 110 to 130, 110 to 120, 120 to 150, 120 to 140, 120 to 130, 130 to 150, 130 to 140, 140 to 150 bp in length.
  • the one or more polynucleotide probes is biotinylated.
  • the polynucleotide probes are designed or selected following sequencing of a tumor of the subject. In some aspects, the polynucleotide probes are designed or selected following exome sequencing of the tumor of the subject. In some aspects, the polynucleotide probes are designed or selected to target all mutations of the sequenced tumor. [0027] In some aspects, the sequencing step comprises ligating sequencing adaptors to the cfDNA. In some aspects, the sequencing adaptors are configured for duplex sequencing.
  • one or more of the mutations comprises a point mutation, a frameshift mutation, a non-frameshift mutation, a deletion mutation, an insertion mutation, a splice variant, a genomic rearrangement, a proteasome-generated spliced antigen, or combinations thereof.
  • one or more of the mutations comprises at least one alteration that makes a peptide sequence encoded by the cfDNA distinct from the corresponding peptide sequence encoded by the wild-type germline nucleic acid sequence of the subject.
  • the one or more mutations consists of coding mutations comprising at least one alteration that makes a peptide sequence encoded by the cfDNA distinct from the corresponding peptide sequence encoded by the wild-type germline nucleic acid sequence of the subject.
  • Fig. 1 shows a detailed pipeline for the isolation and processing of ctDNA from patient. Briefly, tumor-specific DNA variant alleles are identified from biopsied tumor tissue (point 1). Blood is drawn from patients at specific points of their dosing schedules, and ctDNA is isolated and used to generate a UMI library (points 2 and 4). Baits designed based on variants identified in patient tumor DNA (point 3) are used to purify ctDNA containing identified variants (point 5).
  • Fig. 2 shows a detailed pipeline following the isolation and processing of ctDNA for analysis from patient following isolation and processing as outlined in Fig. 1.
  • Purified ctDNA is sequenced (point 6) to quantify prevalence of specific identified variants. Repeated testing of ctDNA over the course of treatment allows monitoring of tumor progression or response to therapy.
  • Fig. 3A-F exemplify the isolation and sequencing of circulating tumor DNA (ctDNA) in two patients receiving GRANITE therapy.
  • Fig. 3A-B are graphs showing the absolute (Fig. 3A) and normalized (Fig. 3B) duplex read coverage of identified DNA variants in ctDNA isolated from Patient #1 (identified as pt0009).
  • Fig. 3C is a graph showing the monitoring of tumor- specific DNA variant alleles in Patient #1 over the course of treatment, with TP52 R175H, APC T1556fs, and CDKN2A W110* highlighted.
  • Fig. 3D-F are graphs showing the absolute (Fig. 3C) and normalized (Fig.
  • Fig. 3F is a graph showing the monitoring of tumor- specific DNA variant alleles in Patient #2 over the course of treatment, including TRABD2B A385T, ADAR G751R, VILL L273fs, SURF2 P146L, TP53 P153fs, CSH2 A156V, and MAP2K2 E66K.
  • Fig. 4A-C are graphs showing the monitoring of variant allele frequency (VAF) in Patient #1 (pt0009) over the course of GRANITE therapy.
  • Fig. 4A shows the frequency of 11 identified tumor- specific variant alleles over the course of treatment.
  • Fig. 4B shows the trend in VAF of all variant alleles in isolated ctDNA over the course of treatment.
  • Fig. 4C shows the average percent change in VAF between consecutive dosages over the course of treatment.
  • Fig. 5A-B are graphs that exemplify the monitoring of ctDNA in additional patients receiving GRANITE therapy.
  • Fig. 5A shows the monitoring of ctDNA in a patient with non-small cell lung cancer (NSCLC) who received GRANITE therapy.
  • Fig. 5B shows the tracking of ctDNA in a patient with micro satellite- stable colorectal cancer (MSS-CRC).
  • Fig. 6A-C are graphs that show the monitoring of ctDNA in a patient receiving SLATE therapy (identified as ptOlOl).
  • Fig. 6A-B show the absolute (Fig. 6A) and normalized (Fig. 6B) duplex read coverage of specified KRAS allele variants in ctDNA isolated from patient plasma.
  • Fig. 6C shows the changes in KRAS variant allele duplexes between consecutive doses.
  • Fig. 7 is a graph that shows the monitoring of ctDNA associated with the KRAS G12C mutation in a patient with NSCLC.
  • an antigen is a substance that induces an immune response.
  • An antigen can be a neoantigen.
  • An antigen can be a “shared antigen” that is an antigen found among a specific population, e.g., a specific population of cancer patients.
  • neoantigen is an antigen that has at least one alteration that makes it distinct from the corresponding wild-type antigen, e.g., via mutation in a tumor cell or post- translational modification specific to a tumor cell.
  • a neoantigen can include a polypeptide sequence or a nucleotide sequence.
  • a mutation can include a frameshift or non-frameshift indel, missense or nonsense substitution, splice site alteration, genomic rearrangement or gene fusion, or any genomic or expression alteration giving rise to a neoORF.
  • a mutations can also include a splice variant. Post-translational modifications specific to a tumor cell can include aberrant phosphorylation.
  • Post-translational modifications specific to a tumor cell can also include a proteasome-generated spliced antigen. See Liepe et al., A large fraction of HLA class I ligands are proteasome-generated spliced peptides; Science. 2016 Oct 21;354(6310):354-358. Such shared neoantigens are useful for inducing an immune response in a subject via administration. The subject can be identified for administration through the use of various diagnostic methods, e.g., patient selection methods described further below.
  • tumor antigen is an antigen present in a subject’s tumor cell or tissue but not in the subject’s corresponding normal cell or tissue, or derived from a polypeptide known to or have been found to have altered expression in a tumor cell or cancerous tissue in comparison to a normal cell or tissue.
  • the term “antigen-based vaccine” is a vaccine composition based on one or more antigens, e.g., a plurality of antigens.
  • the vaccines can be nucleotide-based (e.g., virally based, RNA based, or DNA based), protein-based (e.g., peptide based), or a combination thereof.
  • cancer antigen is a mutation or other aberration giving rise to a sequence that may represent an antigen.
  • coding region is the portion(s) of a gene that encode protein.
  • coding mutation is a mutation occurring in a coding region.
  • ORF means open reading frame.
  • NEO-ORF is a tumor- specific ORF arising from a mutation or other aberration such as splicing.
  • missense mutation is a mutation causing a substitution from one amino acid to another.
  • nonsense mutation is a mutation causing a substitution from an amino acid to a stop codon or causing removal of a canonical start codon.
  • frameshift mutation is a mutation causing a change in the frame of the protein.
  • the term “indel” is an insertion or deletion of one or more nucleic acids.
  • the term percent "identity,” in the context of two or more nucleic acid or polypeptide sequences, refer to two or more sequences or subsequences that have a specified percentage of nucleotides or amino acid residues that are the same, when compared and aligned for maximum correspondence, as measured using one of the sequence comparison algorithms described below (e.g., BLASTP and BLASTN or other algorithms available to persons of skill) or by visual inspection. Depending on the application, the percent "identity" can exist over a region of the sequence being compared, e.g., over a functional domain, or, alternatively, exist over the full length of the two sequences to be compared.
  • sequence comparison typically one sequence acts as a reference sequence to which test sequences are compared.
  • test and reference sequences are input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated.
  • sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters.
  • sequence similarity or dissimilarity can be established by the combined presence or absence of particular nucleotides, or, for translated sequences, amino acids at selected sequence positions (e.g., sequence motifs).
  • Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by visual inspection (see generally Ausubel et ah, infra).
  • BLAST algorithm One example of an algorithm that is suitable for determining percent sequence identity and sequence similarity is the BLAST algorithm, which is described in Altschul et ah, J. Mol. Biol. 215:403-410 (1990). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information.
  • non-stop or read-through is a mutation causing the removal of the natural stop codon.
  • epitope is the specific portion of an antigen typically bound by an antibody or T cell receptor.
  • immunogenic is the ability to elicit an immune response, e.g., via T cells, B cells, or both.
  • HLA binding affinity means affinity of binding between a specific antigen and a specific MHC allele.
  • the term “bait” is a nucleic acid probe used to enrich a specific sequence of DNA or RNA from a sample.
  • variant is a difference between a subject’s nucleic acids and the reference human genome used as a control.
  • variant call is an algorithmic determination of the presence of a variant, typically from sequencing.
  • polymorphism is a germline variant, i.e., a variant found in all DNA-bearing cells of an individual.
  • somatic variant is a variant arising in non-germline cells of an individual.
  • allele is a version of a gene or a version of a genetic sequence or a version of a protein.
  • HLA type is the complement of HLA gene alleles.
  • nonsense-mediated decay or “NMD” is a degradation of an mRNA by a cell due to a premature stop codon.
  • truncal mutation is a mutation originating early in the development of a tumor and present in a substantial portion of the tumor’s cells.
  • subclonal mutation is a mutation originating later in the development of a tumor and present in only a subset of the tumor’s cells.
  • exome is a subset of the genome that codes for proteins.
  • An exome can be the collective exons of a genome.
  • logistic regression is a regression model for binary data from statistics where the logit of the probability that the dependent variable is equal to one is modeled as a linear function of the dependent variables.
  • neural network is a machine learning model for classification or regression consisting of multiple layers of linear transformations followed by element-wise nonlinearities typically trained via stochastic gradient descent and back- propagation.
  • the term “proteome” is the set of all proteins expressed and/or translated by a cell, group of cells, or individual.
  • the term “peptidome” is the set of all peptides presented by MHC-I or MHC-II on the cell surface. The peptidome may refer to a property of a cell or a collection of cells (e.g., the tumor peptidome, meaning the union of the peptidomes of all cells that comprise the tumor).
  • the term “ELISpot” means Enzyme-linked immunosorbent spot assay - which is a common method for monitoring immune responses in humans and animals.
  • the term “tolerance or immune tolerance” is a state of immune nonresponsiveness to one or more antigens, e.g. self-antigens.
  • central tolerance is a tolerance affected in the thymus, either by deleting self-reactive T-cell clones or by promoting self-reactive T-cell clones to differentiate into immunosuppressive regulatory T-cells (Tregs).
  • peripheral tolerance is a tolerance affected in the periphery by downregulating or anergizing self-reactive T-cells that survive central tolerance or promoting these T cells to differentiate into Tregs.
  • sample can include a single cell or multiple cells or fragments of cells or an aliquot of body fluid, taken from a subject, by means including venipuncture, excretion, ejaculation, massage, biopsy, needle aspirate, lavage sample, scraping, surgical incision, or intervention or other means known in the art.
  • subject encompasses a cell, tissue, or organism, human or non-human, whether in vivo, ex vivo, or in vitro, male or female.
  • subject is inclusive of mammals including humans.
  • mammal encompasses both humans and non-humans and includes but is not limited to humans, non-human primates, canines, felines, murines, bovines, equines, and porcines.
  • Clinical factor refers to a measure of a condition of a subject, e.g., disease activity or severity.
  • “Clinical factor” encompasses all markers of a subject’s health status, including non-sample markers, and/or other characteristics of a subject, such as, without limitation, age and gender.
  • a clinical factor can be a score, a value, or a set of values that can be obtained from evaluation of a sample (or population of samples) from a subject or a subject under a determined condition.
  • a clinical factor can also be predicted by markers and/or other parameters such as gene expression surrogates.
  • Clinical factors can include tumor type, tumor sub-type, and smoking history.
  • alphavirus refers to members of the family Togaviridae, and are positive- sense single-stranded RNA viruses. Alphaviruses are typically classified as either Old World, such as Sindbis, Ross River, Mayaro, Chikungunya, and Semliki Forest viruses, or New World, such as eastern equine encephalitis, Aura, Fort Morgan, or Venezuelan equine encephalitis and its derivative strain TC-83. Alphaviruses are typically self-replicating RNA viruses.
  • alphavirus backbone refers to minimal sequence(s) of an alphavirus that allow for self-replication of the viral genome. Minimal sequences can include conserved sequences for nonstructural protein-mediated amplification, a nonstructural protein 1 (nsPl) gene, a nsP2 gene, a nsP3 gene, a nsP4 gene, and a polyA sequence, as well as sequences for expression of subgenomic viral RNA including a 26S promoter element.
  • nsPl nonstructural protein 1
  • sequences for nonstructural protein-mediated amplification includes alphavirus conserved sequence elements (CSE) well known to those in the art.
  • CSEs include, but are not limited to, an alphavirus 5’ UTR, a 51-nt CSE, a 24-nt CSE, or other 26S subgenomic promoter sequence, a 19-nt CSE, and an alphavirus 3’ UTR.
  • RNA polymerase includes polymerases that catalyze the production of RNA polynucleotides from a DNA template.
  • RNA polymerases include, but are not limited to, bacteriophage derived polymerases including T3, T7, and SP6.
  • lipid includes hydrophobic and/or amphiphilic molecules.
  • Lipids can be cationic, anionic, or neutral.
  • Lipids can be synthetic or naturally derived, and in some instances biodegradable.
  • Lipids can include cholesterol, phospholipids, lipid conjugates including, but not limited to, polyethyleneglycol (PEG) conjugates (PEGylated lipids), waxes, oils, glycerides, fats, and fat-soluble vitamins.
  • PEG polyethyleneglycol
  • Lipids can also include dilinoleylmethyl- 4-dimethylaminobutyrate (MC3) and MC3-like molecules.
  • lipid nanoparticle includes vesicle like structures formed using a lipid containing membrane surrounding an aqueous interior, also referred to as liposomes.
  • Lipid nanoparticles includes lipid-based compositions with a solid lipid core stabilized by a surfactant.
  • the core lipids can be fatty acids, acylglycerols, waxes, and mixtures of these surfactants.
  • Biological membrane lipids such as phospholipids, sphingomyelins, bile salts (sodium taurocholate), and sterols (cholesterol) can be utilized as stabilizers.
  • Lipid nanoparticles can be formed using defined ratios of different lipid molecules, including, but not limited to, defined ratios of one or more cationic, anionic, or neutral lipids.
  • Lipid nanoparticles can encapsulate molecules within an outer-membrane shell and subsequently can be contacted with target cells to deliver the encapsulated molecules to the host cell cytosol.
  • Lipid nanoparticles can be modified or functionalized with non-lipid molecules, including on their surface.
  • Lipid nanoparticles can be single-layered (unilamellar) or multi- layered (multilamellar). Lipid nanoparticles can be complexed with nucleic acid.
  • Unilamellar lipid nanoparticles can be complexed with nucleic acid, wherein the nucleic acid is in the aqueous interior.
  • Multilamellar lipid nanoparticles can be complexed with nucleic acid, wherein the nucleic acid is in the aqueous interior, or to form or sandwiched between.
  • pharmaceutically effective amount is an amount of a vaccine component (such as a peptide, engineered vector, and/or adjuvant) that is effective in a route of administration to provide a cell with sufficient levels of protein, protein expression, and/or cell-signaling activity (e.g., adjuvant-mediated activation) to provide a vaccinal benefit, i.e., some measurable level of immunity.
  • a vaccine component such as a peptide, engineered vector, and/or adjuvant
  • cell-signaling activity e.g., adjuvant-mediated activation
  • Terms such as “obtaining,” “isolating,” “enriching,” “sequencing,” “acquiring,” “collecting,” and “determining” as used herein refers to directly performing a process (e.g., directly performing a method) to acquire a result, such as directly acquiring a product, including, but not limited to, directly sequencing cfDNA to acquire cfDNA sequencing data, directly isolating cfDNA to acquire isolated cfDNA, directly enriching cfDNA to acquire enriched cfDNA samples including cfDNA, etc..
  • Terms such as “having obtained,” “having isolated,” “having enriched,” “having sequenced,” “having acquired,” “having collected,” and “having determined” as used herein refers to indirectly receiving information or receiving a product without directly performing a process (e.g., without directly performing a method), such as by receiving the knowledge or product from another party or source (e.g., from a third party laboratory that itself directly acquired the cfDNA sequencing data, isolated cfDNA, enriched cfDNA, and/or collect a sample including cfDNA, etc.).
  • the other party or source is directed to directly perform a process (e.g., a third party laboratory directed to acquire cfDNA sequencing data, isolate cfDNA, enrich cfDNA, and/or collect a sample including cfDNA, etc.).
  • a process e.g., a third party laboratory directed to acquire cfDNA sequencing data, isolate cfDNA, enrich cfDNA, and/or collect a sample including cfDNA, etc.
  • the knowledge or product is purchased from another party or source that directly performed a process (e.g., purchasing cfDNA sequencing data, isolated cfDNA, enriched cfDNA, and/or a collected sample including cfDNA, etc.).
  • MHC major histocompatibility complex
  • HLA human leukocyte antigen, or the human MHC gene locus
  • NGS next-generation sequencing
  • PPV positive predictive value
  • TSNA tumor- specific neoantigen
  • FFPE formalin-fixed, paraffin- embedded
  • NMD nonsense-mediated decay
  • NSCLC non- small-cell lung cancer
  • DC dendritic cell
  • cfDNA cell-free DNA
  • monitoring mutation frequency e.g ., tumor associated mutations associated with a cancer
  • cfDNA can be used to monitor the progression of disease in patients receiving therapy.
  • the methods of cfDNA analysis described herein provide a non-invasive manner of assessing and/or monitoring disease, in particular relative to the more invasive procedures such as tumor biopsies.
  • the methods of cfDNA analysis described herein are particularly useful for analyzing large numbers of mutations, such as analyzing all or the majority of a tumor’s exome.
  • the monitoring is performed through sequencing of cfDNA with both broad target coverage (e.g ., at least 50% of all polynucleotide regions of interest corresponding to mutations present in a cancer exome of a subject) and a high read depth of sequencing (“deep sequenced,” e.g., a mean read depth of at least 1000X).
  • broad target coverage e.g ., at least 50% of all polynucleotide regions of interest corresponding to mutations present in a cancer exome of a subject
  • a high read depth of sequencing (“deep sequenced,” e.g., a mean read depth of at least 1000X).
  • methods for monitoring cancer status in a subject includes the steps of: a. obtaining or having obtained sequencing data of cfDNA from a sample from a subject, and wherein the sequencing data includes a target coverage of at least 50% of all polynucleotide regions of interest corresponding to mutations present in an exome of the cancer and wherein the sequenced polynucleotide regions of interest have a mean read depth of at least 1000X; and b. determining or having determined a frequency of the mutations present in the exome to assess the status of the cancer.
  • methods for monitoring cancer status in a subject includes the steps of: a. obtaining or having obtained sequencing data of cfDNA from a first sample from the subject, and wherein the sequencing data includes a target coverage of at least 50% of all polynucleotide regions of interest corresponding to mutations present in an exome of the cancer and wherein the sequenced polynucleotide regions of interest have a mean read depth of at least 1000X; b.
  • sequencing data includes a target coverage of at least 50% of all polynucleotide regions of interest corresponding to mutations present in an exome of the cancer and wherein the sequenced polynucleotide regions of interest have a mean read depth of at least 1000X; and c. determining or having determined the frequency the mutations present in the exome of the first cfDNA relative to the second cfDNA to assess the status of the cancer.
  • Time points can be selected to monitor disease status as specific intervals.
  • time points can be selected based on therapy dosing schedule.
  • Time points based on dosing schedules can include the same day as administration of a therapy.
  • Time points based on dosing schedules can include, but are not limited to, one day, two days, three days, four days, five days, six days after a dose.
  • Time points based on dosing schedules can include, but are not limited to, one week, two weeks, three weeks, four weeks, five weeks, six weeks, eight weeks, ten weeks, twelve weeks after a dose.
  • Time points based on dosing schedules can include, but are not limited to, one month, two months, three months, six months, and twelve months after a dose.
  • Time points can be at regular time intervals, such as regular time intervals over the course of therapy, including, but not limited to, every day, every two days, every three days, every four days, every five days, every six days.
  • Time points based on regular time intervals can include, but are not limited to, once every week, once every two weeks, once every three weeks, once every four weeks, once every five weeks, once every six weeks, every eight weeks, every ten weeks, every twelve weeks. Time points can also be selected base on regular time intervals including, but not limited to, once every month, once every two months, once every three months, once every six months, and once every twelve months. Combinations of one or more of the above mentioned time intervals may also be used.
  • Analysis of cfDNA can be used to monitor the progression of disease in patients receiving a therapy.
  • longitudinal samples can be collected over the course of therapy to monitor cancer status (e.g ., tumor burden over time).
  • cancer status e.g ., tumor burden over time.
  • Increases in the frequency of monitored mutations over longitudinal samples can indicate an increased likelihood that tumor burden of the subject is increasing.
  • Decreases or maintenance of the frequency of the mutations in of monitored mutations over longitudinal samples can indicate an increased likelihood that tumor burden of the subject is decreasing or stable.
  • methods for assessing efficacy of a therapy in a subject having cancer includes the steps of: a. obtaining or having obtained sequencing data of cfDNA from a pre-therapy sample from the subject, and wherein the sequencing data includes a target coverage of at least 50% of all polynucleotide regions of interest corresponding to mutations present in an exome of the cancer and wherein the sequenced polynucleotide regions of interest have a mean read depth of at least 1000X; b.
  • sequencing data includes a target coverage of at least 50% of all polynucleotide regions of interest corresponding to mutations present in an exome of the cancer and wherein the sequenced polynucleotide regions of interest have a mean read depth of at least 1000X; and c. determining or having determined the frequency the mutations present in the exome of the pre-therapy cfDNA relative to the post-therapy cfDNA to assess the efficacy of the therapy.
  • Samples having cfDNA can be collected at different time points relative to administration of a therapy.
  • Samples having cfDNA can be collected prior to administration of a therapy.
  • Samples having cfDNA can be collected subsequent to administration of a therapy.
  • Samples having cfDNA can be collected concurrently with administration of a therapy.
  • Samples having cfDNA can be collected both prior to and subsequent to administration of a therapy.
  • a first sample having cfDNA can be collected prior to administration of a therapy to a subject and a second sample having cfDNA can be collected subsequent to administration of the therapy.
  • Samples having cfDNA can be collected both concurrently with and subsequent to administration of a therapy.
  • a first sample having cfDNA can be collected concurrently with administration of a therapy to a subject and a second sample having cfDNA can be collected subsequent to administration of the therapy.
  • Multiple samples (e.g ., longitudinal samples) having cfDNA can be collected subsequent to administration of a therapy.
  • Obtaining the sequencing data can include one or more of the following steps: collecting or having collected a sample from a subject; isolating or having isolated cfDNA; enriching or having enriched cfDNA, and/or sequencing or having sequenced cfDNA.
  • Obtaining the sequencing data can include each of the following steps: collecting or having collected a sample from a subject; isolating or having isolated cfDNA; enriching or having enriched cfDNA, and/or sequencing or having sequenced cfDNA.
  • An intermediate can be acquired for performing any of the above steps.
  • isolated cfDNA can be acquired from a third-party source and used for performing one or more of the remaining steps, such as enrichment and sequencing.
  • An intermediate can be produced and a third-party directed to perform any of the above steps.
  • enriched cfDNA can be produced and provided to a third-party source for performing one or more of the remaining steps, such as sequencing.
  • Methods described herein can be used to monitor cancer status, such as tumor burden.
  • a subject’s disease can include cancer.
  • Cancer cells can release their genomic DNA into the circulation upon cell death, referred to as circulating tumor DNA (ctDNA) or as cfDNA from a cancer cell.
  • ctDNA circulating tumor DNA
  • cfDNA circulating tumor DNA
  • a variety of cancers can be monitored.
  • cancers that can be monitored include but are not limited to, a carcinoma, a sarcoma, a lymphoma or leukemia, a germ cell tumor, a blastoma, or other cancers.
  • Carcinomas include without limitation epithelial neoplasms, squamous cell neoplasms squamous cell carcinoma, basal cell neoplasms basal cell carcinoma, transitional cell papillomas and carcinomas, adenomas and adenocarcinomas (glands), adenoma, adenocarcinoma, linitis plastica insulinoma, glucagonoma, gastrinoma, vipoma, cholangiocarcinoma, hepatocellular carcinoma, adenoid cystic carcinoma, carcinoid tumor of appendix, prolactinoma, oncocytoma, Hurthle cell adenoma, renal cell carcinoma, Grawitz tumor, multiple endocrine adenomas, endometrioid adenoma, adnexal and skin appendage neoplasms, mucoepidermoid neoplasms, cystic, mucinous and serous
  • Sarcoma includes without limitation Askin's tumor, botryodies, chondrosarcoma, Ewing's sarcoma, malignant hemangio endothelioma, malignant schwannoma, osteosarcoma, soft tissue sarcomas including: alveolar soft part sarcoma, angiosarcoma, cystosarcoma phyllodes, dermatofibro sarcoma, desmoid tumor, desmoplastic small round cell tumor, epithelioid sarcoma, extraskeletal chondrosarcoma, extraskeletal osteosarcoma, fibrosarcoma, hemangiopericytoma, hemangio sarcoma, kaposi's sarcoma, leiomyosarcoma, liposarcoma, lymphangiosarcoma, lymphosarcoma, malignant fibrous histiocytoma, neurofibrosarcoma, rhabdomyosarcoma
  • Lymphoma and leukemia include without limitation chronic lymphocytic leukemia/small lymphocytic lymphoma, B-cell prolymphocytic leukemia, lymphoplasmacytic lymphoma (such as Waldenstrom macroglobulinemia), splenic marginal zone lymphoma, plasma cell myeloma, plasmacytoma, monoclonal immunoglobulin deposition diseases, heavy chain diseases, extranodal marginal zone B cell lymphoma, also called malt lymphoma, nodal marginal zone B cell lymphoma, follicular lymphoma, mantle cell lymphoma, diffuse large B cell lymphoma, mediastinal (thymic) large B cell lymphoma, intravascular large B cell lymphoma, primary effusion lymphoma, Burkitt lymphoma/leukemia, T cell prolymphocytic leukemia, T cell large granular lymphocytic leukemia, aggressive NK cell leukemia, adult T cell
  • Germ cell tumors include without limitation germinoma, dysgerminoma, seminoma, nongerminomatous germ cell tumor, embryonal carcinoma, endodermal sinus tumor, choriocarcinoma, teratoma, polyembryoma, and gonadoblastoma.
  • Blastoma includes without limitation nephroblastoma, medulloblastoma, and retinoblastoma.
  • cancers include without limitation labial carcinoma, larynx carcinoma, hypopharynx carcinoma, tongue carcinoma, salivary gland carcinoma, gastric carcinoma, adenocarcinoma, thyroid cancer (medullary and papillary thyroid carcinoma), renal carcinoma, kidney parenchyma carcinoma, cervix carcinoma, uterine corpus carcinoma, endometrium carcinoma, chorion carcinoma, testis carcinoma, urinary carcinoma, melanoma, brain tumors such as glioblastoma, astrocytoma, meningioma, medulloblastoma and peripheral neuroectodermal tumors, gall bladder carcinoma, bronchial carcinoma, multiple myeloma, basalioma, teratoma, retinoblastoma, choroidea melanoma, seminoma, rhabdomyosarcoma, craniopharyngeoma, osteosarcoma, chondrosarcoma, myosarcoma, liposarcoma
  • Cancers that can be monitored include but are not limited to lung cancer, melanoma, breast cancer, ovarian cancer, prostate cancer, kidney cancer, gastric cancer, colon cancer, testicular cancer, head and neck cancer, pancreatic cancer, brain cancer, B-cell lymphoma, acute myelogenous leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia, T cell lymphocytic leukemia, nonsmall cell lung cancer, and small cell lung cancer.
  • Tumor specific mutations can include previously identified tumor specific mutations, for example found at the Catalogue of Somatic Mutations in Cancer (COSMIC) database.
  • these mutations can be present in the genome, transcriptome, proteome, or exome of cancer cells of a subject having cancer but not in normal tissue from the subject.
  • Specific methods for identifying neoantigens, including shared neoantigens, that are specific to tumors are known to those skilled in the art, for example the methods described in more detail in international patent application publications WO/2017/106638, WO/2018/195357, and WO/2018/208856, each of which are herein incorporated by reference, in their entirety, for all purposes.
  • Genetic mutations in tumors can be considered useful for the immunological targeting of tumors and/or monitoring tumor burden (e.g., disease status) if they lead to changes in the amino acid sequence of a protein exclusively in the tumor.
  • Useful mutations include: (1) non-synonymous mutations leading to different amino acids in the protein; (2) read-through mutations in which a stop codon is modified or deleted, leading to translation of a longer protein with a novel tumor- specific sequence at the C-terminus; (3) splice site mutations that lead to the inclusion of an intron in the mature mRNA and thus a unique tumor- specific protein sequence; (4) chromosomal rearrangements that give rise to a chimeric protein with tumor- specific sequences at the junction of 2 proteins (i.e., gene fusion); (5) frameshift mutations or deletions that lead to a new open reading frame with a novel tumor- specific protein sequence. Mutations can also include one or more of non-frameshift indel, missense or nonsense substitution, splice
  • Peptides with mutations or mutated polypeptides arising from for example, splice- site, frameshift, readthrough, or gene fusion mutations in tumor cells can be identified by sequencing DNA, RNA, or protein in tumor versus normal cells.
  • a variety of methods are available for detecting the presence of a particular mutation or allele in an individual's DNA or RNA. Any of the sequencing methods described herein can be used to determine tumor specific mutations. Advancements in this field have provided accurate, easy, and inexpensive large-scale SNP genotyping. For example, several techniques have been described including dynamic allele- specific hybridization (DASH), microplate array diagonal gel electrophoresis (MADGE), pyrosequencing, oligonucleotide- specific ligation, the TaqMan system as well as various DNA "chip” technologies such as the Affymetrix SNP chips. These methods utilize amplification of a target genetic region, typically by PCR. Still other methods, based on the generation of small signal molecules by invasive cleavage followed by mass spectrometry or immobilized padlock probes and rolling- circle amplification. Several of the methods known in the art for detecting specific mutations are summarized below.
  • PCR based detection means can include multiplex amplification of a plurality of markers simultaneously. For example, it is well known in the art to select PCR primers to generate PCR products that do not overlap in size and can be analyzed simultaneously. Alternatively, it is possible to amplify different markers with primers that are differentially labeled and thus can each be differentially detected. Of course, hybridization based detection means allow the differential detection of multiple PCR products in a sample. Other techniques are known in the art to allow multiplex analyses of a plurality of markers.
  • RNA molecules can be detected by using a specialized exonuclease-resistant nucleotide, as disclosed, e.g., in Mundy, C. R. (U.S. Pat. No. 4,656,127).
  • a primer complementary to the allelic sequence immediately 3' to the polymorphic site is permitted to hybridize to a target molecule obtained from a particular animal or human.
  • the polymorphic site on the target molecule contains a nucleotide that is complementary to the particular exonuclease- resistant nucleotide derivative present, then that derivative will be incorporated onto the end of the hybridized primer. Such incorporation renders the primer resistant to exonuclease, and thereby permits its detection. Since the identity of the exonuclease-resistant derivative of the sample is known, a finding that the primer has become resistant to exonucleases reveals that the nucleotide(s) present in the polymorphic site of the target molecule is complementary to that of the nucleotide derivative used in the reaction. This method has the advantage that it does not require the determination of large amounts of extraneous sequence data.
  • a solution-based method can be used for determining the identity of a nucleotide of a polymorphic site.
  • a primer is employed that is complementary to allelic sequences immediately 3' to a polymorphic site. The method determines the identity of the nucleotide of that site using labeled dideoxynucleotide derivatives, which, if complementary to the nucleotide of the polymorphic site will become incorporated onto the terminus of the primer.
  • Goelet, P. et al. An alternative method, known as Genetic Bit Analysis or GBA is described by Goelet, P. et al. (PCT Appln. No. 92/15712).
  • the method of Goelet, P. et al. uses mixtures of labeled terminators and a primer that is complementary to the sequence 3' to a polymorphic site.
  • the labeled terminator that is incorporated is thus determined by, and complementary to, the nucleotide present in the polymorphic site of the target molecule being evaluated.
  • Cohen et al. Fernch Patent 2,650,840; PCT Appln. No.
  • the method of Goelet, P. et al. can be a heterogeneous phase assay, in which the primer or the target molecule is immobilized to a solid phase.
  • oligonucleotides 30-50 bases in length are covalently anchored at the 5' end to glass cover slips. These anchored strands perform two functions. First, they act as capture sites for the target template strands if the templates are configured with capture tails complementary to the surface-bound oligonucleotides. They also act as primers for the template directed primer extension that forms the basis of the sequence reading.
  • Capture primers function as a fixed position site for sequence determination using multiple cycles of synthesis, detection, and chemical cleavage of the dye-linker to remove the dye. Each cycle adds the polymerase/labeled nucleotide mixture, rinsing, imaging and cleavage of dye.
  • polymerase is modified with a fluorescent donor molecule and immobilized on a glass slide, while each nucleotide is color-coded with an acceptor fluorescent moiety attached to a gamma-phosphate. The system detects the interaction between a fluorescently-tagged polymerase and a fluorescently modified nucleotide as the nucleotide becomes incorporated into the de novo chain.
  • Other sequencing-by- synthesis technologies also exist.
  • any suitable sequencing-by-synthesis platform can be used to identify mutations.
  • four major sequencing-by-synthesis platforms are currently available: the Genome Sequencers from Roche/454 Life Sciences, the 1G Analyzer from Illumina/Solexa, the SOLiD system from Applied BioSystems, and the Heliscope system from Helicos Biosciences. Sequencing-by-synthesis platforms have also been described by Pacific BioSciences and VisiGen Biotechnologies.
  • a plurality of nucleic acid molecules being sequenced is bound to a support (e.g., solid support).
  • a capture sequence/universal priming site can be added at the 3' and/or 5' end of the template.
  • the nucleic acids can be bound to the support by hybridizing the capture sequence to a complementary sequence covalently attached to the support.
  • the capture sequence also referred to as a universal capture sequence
  • the capture sequence is a nucleic acid sequence complementary to a sequence attached to a support that may dually serve as a universal primer.
  • a member of a coupling pair (such as, e.g., antibody/antigen, receptor/ligand, or the avidin-biotin pair as described in, e.g., US Patent Application No. 2006/0252077) can be linked to each fragment to be captured on a surface coated with a respective second member of that coupling pair.
  • sequence can be analyzed, for example, by single molecule detection/sequencing, e.g., as described in U.S. Pat. No. 7,283,337, including template-dependent sequencing-by-synthesis.
  • sequencing-by-synthesis the surface-bound molecule is exposed to a plurality of labeled nucleotide triphosphates in the presence of polymerase.
  • the sequence of the template is determined by the order of labeled nucleotides incorporated into the 3' end of the growing chain. This can be done in real time or can be done in a step-and-repeat mode. For real-time analysis, different optical labels to each nucleotide can be incorporated and multiple lasers can be utilized for stimulation of incorporated nucleotides.
  • Sequencing can also include other massively parallel sequencing or next generation sequencing (NGS) techniques and platforms. Additional examples of massively parallel sequencing techniques and platforms are the Illumina HiSeq or MiSeq, Thermo PGM or Proton, the Pac Bio RS II or Sequel, Qiagen’s Gene Reader, and the Oxford Nanopore MinlON. Additional similar current massively parallel sequencing technologies can be used, as well as future generations of these technologies.
  • NGS next generation sequencing
  • any cell type or tissue can be utilized to isolate nucleic acid samples for use in methods of identifying tumor specific mutations described herein.
  • a DNA or RNA sample can be isolated from a tumor or a bodily fluid, e.g., blood, collected by known techniques (e.g. venipuncture) or saliva.
  • nucleic acid tests can be performed on dry samples (e.g. hair or skin).
  • a sample can be collected for sequencing from a tumor and another sample can be collected from normal tissue for sequencing where the normal tissue is of the same tissue type as the tumor.
  • a sample can be collected for sequencing from a tumor and another sample can be obtained from normal tissue for sequencing where the normal tissue is of a distinct tissue type relative to the tumor.
  • Tumors from which tumor specific mutations can be identified include, but are not limited to, any of the tumors described herein, such as lung cancer, melanoma, breast cancer, ovarian cancer, prostate cancer, kidney cancer, gastric cancer, colon cancer, testicular cancer, head and neck cancer, pancreatic cancer, brain cancer, B-cell lymphoma, acute myelogenous leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia, and T cell lymphocytic leukemia, non-small cell lung cancer, and small cell lung cancer.
  • protein mass spectrometry can be used to identify or validate the presence of mutated peptides bound to MHC proteins on tumor cells.
  • Peptides can be acid-eluted from tumor cells or from HLA molecules that are immunoprecipitated from tumor, and then identified using mass spectrometry.
  • Methods for processing cfDNA are generally known to those skilled in the art.
  • general methods for isolating cfDNA are described in US-2020/0277667-A1, which is herein incorporated by reference for all purposes. See also, e.g., Current Protocols in Molecular Biology, latest edition.
  • Exemplary methods for isolating cfDNA are also described in US-10,385,369-B2 and US- 2020/0277667-A1, Cell-Free Plasma DNA as a Predictor of Outcome in Severe Sepsis and Septic Shock. Clin. Chem. 2008, v. 54, p. 1000- Diagnostics. Clin.
  • kits for isolation and purification of cfDNA are known to those skilled in the art including, but not limited to, the QIAamp circulating nucleic acid kit and the alle MiniMax cfDNA Isolation Kit (Beckman Coulter; Indianapolis, IN).
  • Blood/plasma samples can be collected from a subject and cfDNA can be isolated from the blood/plasma samples. Samples having cfDNA other than blood can be collected (e.g., stool, mucus) for cfDNA isolation and purification.
  • Isolation of cfDNA can occur, for example, through centrifugation to separate cfDNA from cells or cellular debris or from whole blood by separation of the plasma layer, which can contain cfDNA, from the buffy coat and red blood cells.
  • Whole blood can be collected in cell-free DNA BCT tubes, centrifuged at an appropriate speed to separate the plasma layer, buffy coat, and red bloods.
  • the plasma layer can then be removed and spun again to remove any residual cellular material.
  • the supernatant can then be collected and stored at -80°C until extraction.
  • whole blood can be collected in lOmL Streck cell-free DNA BCT tubes (Streck; La Vista, NE, USA), spun at 1600xg for 10 minutes at ambient temperature to separate the plasma layer, buffy coat, and red bloods.
  • the plasma layer can then be removed and spun again at 5000Xg for 10 minutes to remove any residual cellular material.
  • the supernatant can then collected and stored at -80°C until extraction.
  • the cfDNA is generally fragmented, for example, sheared or enzymatically prepared (e.g., fragmented using a NEBNext Ultra II FS DNA Module; NEB, Ipswich, MA), to produce a library of polynucleotide regions of interest.
  • Isolated nucleic acid e.g., isolated cfDNA
  • DNA can be fragmented by physical shearing methods, enzymatic cleavage methods, chemical cleavage methods, and other methods well known to those skilled in the art.
  • Fragment length can be at least 100, at least 150, at least 200, at least 250, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, or at least 1000 bp in length. Fragment length can be 100-250, 150-350, 200-450, 300-700, or 500-1000 bp in length.
  • Fragment length can average at least 100, at least 150, at least 200, at least 250, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, or at least 1000 bp in length. Fragment length can average 100-250, 150-350, 200-450, 300-700, or 500-1000 bp in length.
  • cfDNA can be enriched to improve detection and measurement of specific polynucleotide regions of interest. Typically, enrichment is performed on a library of fragmented cfDNA (e.g., a library of polynucleotide regions of interest). Regions of interest can comprise polynucleotides known or suspected to encode one or more mutations. Regions of interest can also comprise gene translocations (e.g., Bcr-Abl fusion).
  • Regions of interest can comprise polynucleotides encoding a gene coding region or a fragment of a gene coding region, which can include tumor exome polynucleotides, such as tumor exome polynucleotides known or suspected of having subject and/or tumor specific mutations. Enrichment of polynucleotide regions of interest in general can improve targeted measurement of DNA regions of interest (e.g., increasing sensitivity) through subtracting noise from sequencing results.
  • enrichment refers to a partial purification of analytes that have a certain feature (e.g., nucleic acids that are known or suspected to have tumor- specific mutations) from analytes that do not have the feature (e.g., nucleic acids that do not contain tumor- specific mutations).
  • Enrichment typically increases the concentration of the analytes that have the feature (e.g., nucleic acids that contain tumor- specific mutations) by at least 2-fold, at least 5-fold or at least 10-fold relative to the analytes that do not have the feature.
  • at least 10%, at least 20%, at least 50%, at least 80% or at least 90% of the analytes in a sample may have the feature used for enrichment.
  • Enriching cfDNA can comprise hybridizing one or more polynucleotide probes (also referred to herein as “baits”) to the one or more polynucleotide regions of interest.
  • Bait sequences can be based on tumor- specific mutations derived from genomic sequencing, such as sequencing of a tumor exome of a biopsy. Baits can comprise a single polynucleotide sequence or a library of polynucleotide sequences derived from tumor sequencing.
  • Bait sequences derived from tumor sequencing can be subject- specific.
  • a subject’s tumor can be biopsied and sequenced to determine mutations associated with the subject’s tumor, following which the subject and tumor- specific sequences can be used to design subject-specific baits for enriching regions of interest of the tumor exome, including baits capable of enriching all regions of interest having patient specific-tumor variants.
  • Hybridization typically refers to the process by which a strand of nucleic acid joins with a complementary strand through base pairing as known in the art.
  • a nucleic acid is generally considered to selectively hybridize to a reference nucleic acid sequence if the two sequences specifically hybridize to one another under moderate to high stringency hybridization and wash conditions.
  • a hybridization protocol can occur at about 42° C.
  • a hybridization buffer can include, but is not limited to, formamide, SSC, Denhardf s solution, SDS and/or denatured carrier DNA.
  • a hybridization protocol can include washing steps in a buffer that can include SSC and SDS at 42° C.
  • An illustrative, non-limiting hybridization protocol involves hybridization at about 42° C in 50% formamide, 5xSSC, 5xDenhardf s solution, 0.5% SDS and 100 ⁇ g/ml denatured carrier DNA followed by washing two times in 2xSSC and 0.5% SDS at room temperature and two additional times in O.lxSSC and 0.5% SDS at 42° C.
  • Another illustrative, non-limiting example of high-stringency conditions includes hybridization overnight using custom-designed xGen Lockdown Probes and the xGen Hybridization and Wash kit( IDT), which involves hybridizing in xGen Hybridization Buffer, plus Hybridization Buffer Enhancer in a thermocycler at 95°C for 30 seconds, followed by 65°C for 4-16 hours; then washing once in xGen wash buffer once at room temperature; then washing twice in xGen Stringent Wash Buffer at 65°C; and finally washing three times at room temperature in Wash Buffer 1, Wash Buffer 2, and Wash Buffer 3, respectively (per the manufacturer’s instructions).
  • xGen Hybridization Buffer Enhancer in a thermocycler at 95°C for 30 seconds, followed by 65°C for 4-16 hours
  • washing once in xGen wash buffer once at room temperature then washing twice in xGen Stringent Wash Buffer at 65°C
  • washing three times at room temperature in Wash Buffer 1, Wash Buffer 2, and Wash Buffer 3, respectively (per the manufacturer’s instructions
  • Baits can be 80 to 150 base pairs (bp) in length, including 80 to 140, 80 to 130, 80 to 120, 80 to 110, 80 to 100, 80 to 90, 90 to 150, 90 to 140, 90 to 130, 90 to 120, 90 to 110,
  • Baits can be 80 to 150 bp in length. Baits can be 80 to 140 bp in length. Baits can be 80 to 130 bp in length. Baits can be 80 to 120 bp in length. Baits can be 80 to 110 bp in length. Baits can be 80 to 100 bp in length. Baits can be 80 to 90 bp in length. Baits can be 90 to 150 bp in length.
  • Baits can be 90 to 140 bp in length. Baits can be 90 to 130 bp in length. Baits can be 90 to 120 bp in length. Baits can be 90 to 110 bp in length. Baits can be 90 to 100 bp in length. Baits can be 100 to 150 bp in length. Baits can be 100 to 140 bp in length. Baits can be 100 to 130 bp in length. Baits can be 100 to 120 bp in length. Baits can be 100 to 110 bp in length. Baits can be 110 to 150 bp in length. Baits can be 110 to 140 bp in length. Baits can be 110 to 130 bp in length.
  • Baits can be 110 to 120 bp in length., Baits can be 120 to 150 bp in length. Baits can be 120 to 140 bp in length. Baits can be 120 to 130 bp in length. Baits can be 130 to 150 bp in length. Baits can be 130 to 140 bp in length. Baits can be 140 to 150 bp in length.
  • Polynucleotide probes can include an affinity tag.
  • Affinity tags are typically molecules that are capable of covalent linkage to a substrate molecule (e.g ., a hybridization probe) and used for subsequent purification by binding of the tag to another surface or material with (e.g., a biotin tag binding to streptavidin resin). Enrichment of polynucleotides can occur by affinity purification or any other suitable method based on the affinity tag used. In some embodiments, an affinity tag is added to polynucleotide probes, enriching for the DNA molecules that hybridize with probes tagged with the affinity tag; and sequencing the enriched DNA molecules.
  • Polynucleotide probes (“baits”) can be biotinylated.
  • Biotinylation refers to the covalent addition of a biotin moiety to the polynucleotide probes.
  • a biotin moiety can include biotin or a biotin analogue, such as desthiobiotin, oxybiotin, 2-iminobiotin, diaminobiotin, biotin sulfoxide, biocytin, etc.
  • Biotin moieties typically bind to streptavidin with an affinity of at least 10-8 M. Enrichment steps using biotinylated polynucleotide probes may be done using magnetic streptavidin beads, although other supports could be used including but not limited to microparticles, fibers, beads, and supports.
  • enrichment can comprise steps of: (a) linking a biotin moiety to the oligonucleotide probes; (b) hybridizing biotinylated probes to cfDNA; (c) enriching for biotinylated DNA molecules by binding to a support that binds to biotin (e.g., streptavidin beads); (d) amplifying the enriched DNA using polymerase chain reaction; and (f) sequencing the amplified DNA to produce a plurality of sequence reads.
  • multiple polynucleotide regions of interest can be selected for enrichment based on the specific disease or therapy being monitored. In cancer patients for example, sequence analysis of tumor genomic DNA can be used to identify tumor- specific mutations, which can be used to select regions of interest for disease monitoring.
  • Regions of interest can be enriched from cfDNA prior to sequencing. Regions of interest can also comprise polynucleotides encoding a coding region, which can include tumor exome polynucleotides. Sequencing of cfDNA
  • Sequencing of isolated cfDNA can comprise next-generation sequencing (NGS) or Sanger sequencing.
  • NGS next-generation sequencing
  • the terms “next-generation sequencing” or “high-throughput sequencing”, as used herein, refer to the so-called parallelized sequencing-by-synthesis or sequencing-by-ligation platforms.
  • NGS methods may also include nanopore sequencing methods or electronic -detection based methods
  • NGS can comprise duplex sequencing, whole-exome sequencing, whole-genome sequencing, de novo sequencing, phased sequencing, targeted amplicon sequencing, or shotgun sequencing.
  • NGS can be performed on platforms such as NovaSeq using 2x15 lbp and 8bp index reads.
  • NGS platforms include but are not limited to Illumina HiSeq or MiSeq, Thermo PGM or Proton, the Pac Bio RS II or Sequel, Qiagen’s Gene Reader, and the Oxford Nanopore MinlON, or any other appropriate platform. Examples of such methods are described in Margulies et al. (Nature 2005437:376-80); Ronaghi et al. (Analytical Biochemistry 1996242:84-9); Shendure (Science 2005 309:1728); Imelfort et al. (Brief Bioinform. 2009 10:609-18); Fox et al. (Methods Mol Biol. 2009; 55379-108 ); Appleby et al.
  • NGS can result in at least 10,000, at least 50,000, at least 100,000, at least 500,000, at least 1M, at least 10M, at least 100M, or at least IB sequence reads.
  • NGS can result in at least 10,000 sequence reads.
  • NGS can result in at least 50,000 sequence reads.
  • NGS can result in at least 100,000 sequence reads.
  • NGS can result in at least 500,000 sequence reads.
  • NGS can result in at least 1M sequence reads.
  • NGS can result in at least 10M sequence reads.
  • NGS can result in at least 100M sequence reads.
  • NGS can result in at least IB sequence reads.
  • Sequence reads can be analyzed by a computer and, thus instructions for performing the steps can be set forth as programming that may be recorded in a suitable physical computer readable storage medium.
  • Whole library amplification can be performed on cfDNA, including enriched cfDNA, using kits such as KAPA HiFi HotStart ReadyMix and NEBNext Multiple Oligos for Illumina.
  • whole blood can be collected for a given subject or collected from a subject with cancer undergoing therapy and cfDNA can be isolated from the whole blood.
  • Sequencing of DNA from a diseased tissue e.g., a cancer-disease tissue, such as from a tumor biopsy
  • Subject- specific and/or tumor- specific mutations can be used to design a library of biotinylated polynucleotide probes and/or guide selection of biotinylated polynucleotide probes to enrich polynucleotide regions of interest from subject cfDNA.
  • Duplex sequencing adaptors can be ligated to the cfDNA, which can then be analyzed by duplex sequencing to measure the frequency of all variant alleles probed.
  • adaptors are ligated to the cfDNA to facilitate sequencing.
  • sequence adaptor or “adaptor” refer to oligonucleotides that are ligated onto the ends of polynucleotides from prepared libraries prior to sequencing (e.g ., a fragmented cfDNA library of polynucleotide regions of interest).
  • Adaptor ligation can be performed on fragmented, end-repaired DNA using 5-mer non- random unique molecular identifiers (IDT, Coralville, Iowa).
  • Sequencing adaptors can be configured for duplex sequencing.
  • duplex sequencing allows for independent tracking during sequencing of both strands of individual DNA molecules. The paired sequences can be compared to reduce sequencing errors by excluding variations that do not occur on both DNA strands.
  • Adaptors configured for duplex sequencing can include xGen UMI adaptors (IDT). General descriptions of sequencing adaptors for duplex sequencing and uses thereof are described in US 2017/0211140 Al, which is hereby incorporated by reference for all purposes.
  • Sequencing read depth (presented as X-fold, e.g., 1000X, read depth and referred to in some instances as sequencing read coverage) as used herein refers to the level of coverage of reads (e.g., number of unique reads), after detection and removal of duplicate reads (e.g., PCR duplicate reads).
  • greater sequencing read depth correlates with greater variant detection reliability. For example, reliable detection of a variant, e.g., a point mutation, that appears at a frequency of greater than 5% and up to 10, 15 or 20% can typically need >200X sequencing depth to ensure high detection reliability.
  • Sequencing read depth can be the read depth for an individual mutation. Sequencing read depth for an individual mutation can be at least 1000X. Sequencing read depth for an individual mutation can be at least 1500X, at least 2000X, at least 2500X, at least 3000X, at least 3500X, at least 4000X, at least 4500X, or at least 5000X. Sequencing read depth for an individual mutation can be an at least 1500X. Sequencing read depth for an individual mutation can be at least 2000X. Sequencing read depth for an individual mutation can be at least 2500X. Sequencing read depth for an individual mutation can be at least 3000X. Sequencing read depth for an individual mutation can be at least 3500X.
  • Sequencing read depth for an individual mutation can be at least 4000X. Sequencing read depth for an individual mutation can be at least 4500X. Sequencing read depth for an individual mutation can be at least 5000X. Sequencing read depth for an individual mutation can range from 1000X to 5000X, including 1000X to 4000X, 1000X to 3000X, 1000X to 2000X, 2000X to 5000X, 2000X to 4000X, 2000X to 3000X, 3000X to 5000X, 3000X to 4000X, and 4000X to 5000X. Sequencing read depth for an individual mutation can range from 1000X to 5000X. Sequencing read depth for an individual mutation can range from 1000X to 4000X.
  • Sequencing read depth for an individual mutation can range from 1000X to 3000X. Sequencing read depth for an individual mutation can range from 1000X to 2000X. Sequencing read depth for an individual mutation can range from 2000X to 5000X. Sequencing read depth for an individual mutation can range from 2000X to 4000X. Sequencing read depth for an individual mutation can range from 2000X to 3000X. Sequencing read depth for an individual mutation can range from 3000X to 5000X. Sequencing read depth for an individual mutation can range from 3000X to 4000X. Sequencing read depth for an individual mutation can range from 4000X to 5000X. Sequencing read depth for an individual mutation can range from at least 100X to 1000X. [00140] Sequencing read depth can be duplex read depth.
  • Sequencing read depth can be duplex read depth for an individual mutation.
  • Duplex read depth for an individual mutation can be at least 1000X.
  • Duplex read depth for an individual mutation can be at least 1500X, at least 2000X, at least 2500X, at least 3000X, at least 3500X, at least 4000X, at least 4500X, or at least 5000X.
  • Duplex read depth for an individual mutation can be at least 1500X.
  • Duplex read depth for an individual mutation can be at least 2000X.
  • Duplex read depth for an individual mutation can be at least 2500X.
  • Duplex read depth for an individual mutation can be at least 3000X.
  • Duplex read depth for an individual mutation can be at least 3500X.
  • Duplex read depth for an individual mutation can be at least 4000X.
  • Duplex read depth for an individual mutation can be at least 4500X.
  • Duplex read depth for an individual mutation can be at least 5000X.
  • Duplex read depth for an individual mutation can range from 1000X to 5000X, including 1000X to 4000X, 1000X to 3000X, 1000X to 2000X, 2000X to 5000X, 2000X to 4000X, 2000X to 3000X, 3000X to 5000X, 3000X to 4000X, and 4000X to 5000X.
  • Duplex read depth for an individual mutation can range from 1000X to 5000X.
  • Duplex read depth for an individual mutation can range from 1000X to 4000X.
  • Duplex read depth for an individual mutation can range from 1000X to 3000X.
  • Duplex read depth for an individual mutation can range from 1000X to 2000X.
  • Duplex read depth for an individual mutation can range from 2000X to 5000X.
  • Duplex read depth for an individual mutation can range from 2000X to 4000X.
  • Duplex read depth for an individual mutation can range from 2000X to 3000X.
  • Duplex read depth for an individual mutation can range from 3000X to 5000X.
  • Duplex read depth for an individual mutation can range from 3000X to 4000X.
  • Duplex read depth for an individual mutation can range from 4000X to 5000X.
  • Duplex read depth for an individual mutation can range from at least 100X to 1000X.
  • Sequencing read depth can be the mean read depth.
  • Mean read depth refers to the mean sequencing depth of a plurality of polynucleotide regions of interest (e.g., a cancer exome and/or regions of interest targeted for enrichment, such as by baits for regions having subject-specific and tumor- specific variants).
  • Mean read depth can be the mean read depth of a cancer exome.
  • Mean read depth can be the mean read depth of previously identified regions of interest having subject- specific and/or tumor-specific mutations.
  • Mean read depth can be the mean read depth of enriched cfDNA.
  • Mean read depth can be the mean read depth of cfDNA enriched by baits for regions having subject-specific and tumor- specific variants.
  • Mean read depth can be at least 1000X.
  • Mean read depth can be at least 1500X, at least 2000X, at least 2500X, at least 3000X, at least 3500X, at least 4000X, at least 4500X, or at least 5000X.
  • Mean read depth can be at least 1500X.
  • Mean read depth can be at least 2000X.
  • Mean read depth can be at least 2500X.
  • Mean read depth can be at least 3000X.
  • Mean read depth can be at least 3500X. Mean read depth can be at least 4000X. Mean read depth can be at least 4500X. Mean read depth can be at least 5000X. Mean read depth can range from 1000X to 5000X, including 1000X to 4000X, 1000X to 3000X, 1000X to 2000X, 2000X to 5000X, 2000X to 4000X, 2000X to 3000X, 3000X to 5000X, 3000X to 4000X, and 4000X to 5000X. Mean read depth can range from 1000X to 5000X. Mean read depth can range from 1000X to 4000X. Mean read depth can range from 1000X to 3000X. Mean read depth can range from 1000X to 2000X. Mean read depth can range from 2000X to 5000X.
  • Mean read depth can range from 2000X to 4000X.
  • Mean read depth can range from 2000X to 3000X.
  • Mean read depth can range from 3000X to 5000X.
  • Mean read depth can range from 3000X to 4000X.
  • Mean read depth can range from 4000X to 5000X.
  • Mean read depth can range from at least 100X to 1000X.
  • Mean read depth can be mean duplex read depth.
  • Mean duplex read depth can be at least 1000X.
  • Mean duplex read depth can be at least 1500X, at least 2000X, at least 2500X, at least 3000X, at least 3500X, at least 4000X, at least 4500X, or at least 5000X.
  • Mean duplex read depth can be at least 1500X.
  • Mean duplex read depth can be at least 2000X.
  • Mean duplex read depth can be at least 2500X.
  • Mean duplex read depth can be at least 3000X.
  • Mean duplex read depth can be at least 3500X.
  • Mean duplex read depth can be at least 4000X.
  • Mean duplex read depth can be at least 4500X.
  • Mean duplex read depth can be at least 5000X.
  • Mean duplex read depth can range from 1000X to 5000X, including 1000X to 4000X, 1000X to 3000X, 1000X to 2000X, 2000X to 5000X, 2000X to 4000X, 2000X to 3000X, 3000X to 5000X, 3000X to 4000X, and 4000X to 5000X.
  • Mean duplex read depth can range from 1000X to 5000X.
  • Mean duplex read depth can range from 1000X to 4000X.
  • Mean duplex read depth can range from 1000X to 3000X.
  • Mean duplex read depth can range from 1000X to 2000X.
  • Mean duplex read depth can range from 2000X to 5000X.
  • Mean duplex read depth can range from 2000X to 4000X.
  • Mean duplex read depth can range from 2000X to 3000X.
  • Mean duplex read depth can range from 3000X to 5000X.
  • Mean duplex read depth can range from 3000X to 4000X.
  • Mean duplex read depth can range from 4000X to 5000X.
  • Mean duplex read depth can range from at least 100X to 1000X.
  • Methods described herein include multiplex arrays that can sequence (“detect”) multiple polynucleotide regions of interest from a cfDNA sample.
  • a cfDNA sample can comprise ctDNA containing one or more mutant alleles encoding genes in the tumor exome.
  • One or more polynucleotide regions of interest can be selectively enriched through designing baits to target the one or more polynucleotide regions of interest.
  • One or more polynucleotide regions of interest can be selectively enriched through designing baits to target the one or more polynucleotide regions of interest from a tumor exome.
  • One or more polynucleotide regions of interest can be selectively enriched through designing baits to target the one or more polynucleotide regions of interest from a tumor exome known or suspected of having subject and tumor- specific mutations.
  • One or more polynucleotide regions or interest can comprise 10 or more polynucleotide regions of interest.
  • One or more polynucleotide regions or interest can comprise 20 polynucleotide regions of interest.
  • One or more polynucleotide regions or interest can comprise 30 or more polynucleotide regions of interest.
  • One or more polynucleotide regions or interest can comprise 40 or more polynucleotide regions of interest.
  • One or more polynucleotide regions or interest can comprise 50 or more polynucleotide regions of interest.
  • One or more polynucleotide regions or interest can comprise 60 or more polynucleotide regions of interest.
  • One or more polynucleotide regions or interest can comprise 70 or more polynucleotide regions of interest.
  • One or more polynucleotide regions or interest can comprise 80 or more polynucleotide regions of interest.
  • One or more polynucleotide regions or interest can comprise 90 or more polynucleotide regions of interest.
  • One or more polynucleotide regions of interest can comprise 100 or more polynucleotide regions of interest.
  • One or more polynucleotide regions of interest can comprise 150 or more polynucleotide regions of interest.
  • One or more polynucleotide regions of interest can comprise 200 or more polynucleotide regions of interest.
  • One or more polynucleotide regions of interest can comprise 250 or more polynucleotide regions of interest.
  • One or more polynucleotide regions of interest can comprise 300 or more polynucleotide regions of interest.
  • One or more polynucleotide regions of interest can comprise 400 or more polynucleotide regions of interest.
  • One or more polynucleotide regions of interest can comprise 500 or more polynucleotide regions of interest.
  • One or more polynucleotide regions of interest can comprise 600 or more polynucleotide regions of interest.
  • One or more polynucleotide regions of interest can comprise 700 or more polynucleotide regions of interest.
  • One or more polynucleotide regions of interest can comprise 800 or more polynucleotide regions of interest, or One or more polynucleotide regions of interest can comprise 900 or more polynucleotide regions of interest.
  • One or more polynucleotide regions of interest can comprise at least 10% of polynucleotide regions of interest corresponding to mutations present in a tumor exome of the subject (in other words, at least 10% of all subject and tumor- specific mutations associated with a tumor exome).
  • One or more polynucleotide regions of interest can comprise at least 20% of polynucleotide regions of interest corresponding to mutations present in a tumor exome of the subject.
  • One or more polynucleotide regions of interest can comprise at least 30% of polynucleotide regions of interest corresponding to mutations present in a tumor exome of the subject.
  • One or more polynucleotide regions of interest can comprise at least 40% of polynucleotide regions of interest corresponding to mutations present in a tumor exome of the subject.
  • One or more polynucleotide regions of interest can comprise at least 50% of polynucleotide regions of interest corresponding to mutations present in a tumor exome of the subject.
  • One or more polynucleotide regions of interest can comprise at least 60% of polynucleotide regions of interest corresponding to mutations present in a tumor exome of the subject.
  • One or more polynucleotide regions of interest can comprise at least 70% of polynucleotide regions of interest corresponding to mutations present in a tumor exome of the subject.
  • One or more polynucleotide regions of interest can comprise at least 80% of polynucleotide regions of interest corresponding to mutations present in a tumor exome of the subject.
  • One or more polynucleotide regions of interest can comprise at least 90% of polynucleotide regions of interest corresponding to mutations present in a tumor exome of the subject.
  • the one or more polynucleotide regions of interest can comprise at least 95% of polynucleotide regions of interest corresponding to mutations present in a tumor exome of the subject.
  • the one or more polynucleotide regions of interest can comprise at least 96% of polynucleotide regions of interest corresponding to mutations present in a tumor exome of the subject.
  • the one or more polynucleotide regions of interest can comprise at least 97% of polynucleotide regions of interest corresponding to mutations present in a tumor exome of the subject.
  • the one or more polynucleotide regions of interest can comprise at least 98% of polynucleotide regions of interest corresponding to mutations present in a tumor exome of the subject.
  • the one or more polynucleotide regions of interest can comprise at least 99% of polynucleotide regions of interest corresponding to mutations present in a tumor exome of the subject.
  • the one or more polynucleotide regions of interest can comprise at least 99.5% of polynucleotide regions of interest corresponding to mutations present in a tumor exome of the subject.
  • the one or more polynucleotide regions of interest can comprise at least 99.9% of polynucleotide regions of interest corresponding to mutations present in a tumor exome of the subject.
  • the one or more polynucleotide regions of interest can comprise 100% of polynucleotide regions of interest corresponding to mutations present in a tumor exome of the subject.
  • Mutations can comprise but are not limited to a point mutation, a frameshift mutation, a non-frameshift mutation, a deletion mutation, an insertion mutation, a splice variant, a genomic rearrangement, a proteasome-generated spliced antigen, or combinations thereof.
  • Mutations can comprise at least one alteration that makes a peptide sequence encoded by the cfDNA distinct from the corresponding peptide sequence encoded by the wild-type germline nucleic acid sequence of the subject.
  • Mutations can consist of coding mutations comprising at least one alteration that makes a peptide sequence encoded by the cfDNA distinct from the corresponding peptide sequence encoded by the wild-type germline nucleic acid sequence of the subject.
  • One or more mutations can include 10 or more, 20 or more, 30 or more, 40 or more, 50 or more, 60 or more, 70 or more, 80 or more, or 90 or more mutations.
  • One or more mutations can include 100 or more, 150 or more, 200 or more, 250 or more, 300 or more, 400 or more, 500 or more, 600 or more, 700 or more, 800 or more, or 900 or more mutations.
  • Mutations can be associated with a tumor exome.
  • One or more mutations can include at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of mutations present in a tumor exome of the subject.
  • One or more mutations can include at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, 100% of mutations present in a tumor exome of the subject.
  • Target coverage refers to the proportion of a polynucleotide region or plurality of regions that is sequenced (e.g ., regions represented in a sequencing data set to at least some read depth).
  • target coverage is described as a proportion of a desired region or plurality of regions to be covered (e.g., a plurality of polynucleotide regions of interest).
  • target coverage can be the proportion of a whole genome, an exome, a cancer genome, a cancer exome, and/or an enriched region (e.g., regions of interest targeted for enrichment, such as by baits for regions having subject-specific and tumor- specific variants).
  • Target coverage can be the proportion of a tumor and/or cancer exome of a subject that is sequenced. Target coverage can be at least 10% of a tumor and/or cancer exome.
  • Target coverage can be at least 20% of a tumor and/or cancer exome.
  • Target coverage can be at least 30% of a tumor and/or cancer exome.
  • Target coverage can be at least 40% of a tumor and/or cancer exome.
  • Target coverage can be at least 50% of a tumor and/or cancer exome.
  • Target coverage can be at least 60% of a tumor and/or cancer exome.
  • Target coverage can be at least 70% of a tumor and/or cancer exome.
  • Target coverage can be at least 80% of a tumor and/or cancer exome.
  • Target coverage can be at least 90% of a tumor and/or cancer exome.
  • Target coverage can be at least 95% of a tumor and/or cancer exome.
  • Target coverage can be at least 96% of a tumor and/or cancer exome.
  • Target coverage can be at least 97% of a tumor and/or cancer exome.
  • Target coverage can be at least 98% of a tumor and/or cancer exome.
  • Target coverage can be at least 99% of a tumor and/or cancer exome.
  • Target coverage can be at least 99.5% of a tumor and/or cancer exome.
  • Target coverage can be at least 99.9% of a tumor and/or cancer exome.
  • Target coverage can be 100% of a tumor and/or cancer exome.
  • Target coverage can be the proportion of polynucleotide regions of interest that is sequenced.
  • Target coverage can be at least 10% of polynucleotide regions of interest.
  • Target coverage can be at least 20% of polynucleotide regions of interest.
  • Target coverage can be at least 30% of polynucleotide regions of interest. Target coverage can be at least 40% of polynucleotide regions of interest. Target coverage can be at least 50% of polynucleotide regions of interest. Target coverage can be at least 60% of polynucleotide regions of interest. Target coverage can be at least 70% of polynucleotide regions of interest. Target coverage can be at least 80% of polynucleotide regions of interest. Target coverage can be at least 90% of polynucleotide regions of interest. Target coverage can be at least 95% of polynucleotide regions of interest. Target coverage can be at least 96% of polynucleotide regions of interest.
  • Target coverage can be at least 97% of polynucleotide regions of interest.
  • Target coverage can be at least 98% of polynucleotide regions of interest.
  • Target coverage can be at least 99% of polynucleotide regions of interest.
  • Target coverage can be at least 99.5% of polynucleotide regions of interest.
  • Target coverage can be at least 99.9% of polynucleotide regions of interest.
  • Target coverage can be 100% of polynucleotide regions of interest.
  • Target coverage can be the proportion of polynucleotide regions of interest targeted for enrichment that is sequenced (e.g., regions of interest targeted for enrichment by baits for regions having subject-specific and tumor-specific variants).
  • Target coverage can be at least 10% of polynucleotide regions of interest targeted for enrichment.
  • Target coverage can be at least 20% of polynucleotide regions of interest targeted for enrichment.
  • Target coverage can be at least 30% of polynucleotide regions of interest targeted for enrichment.
  • Target coverage can be at least 40% of polynucleotide regions of interest targeted for enrichment.
  • Target coverage can be at least 50% of polynucleotide regions of interest targeted for enrichment.
  • Target coverage can be at least 60% of polynucleotide regions of interest targeted for enrichment.
  • Target coverage can be at least 70% of polynucleotide regions of interest targeted for enrichment.
  • Target coverage can be at least 80% of polynucleotide regions of interest targeted for enrichment.
  • Target coverage can be at least 90% of polynucleotide regions of interest targeted for enrichment.
  • Target coverage can be at least 95% of polynucleotide regions of interest targeted for enrichment.
  • Target coverage can be at least 96% of polynucleotide regions of interest targeted for enrichment.
  • Target coverage can be at least 97% of polynucleotide regions of interest targeted for enrichment.
  • Target coverage can be at least 98% of polynucleotide regions of interest targeted for enrichment.
  • Target coverage can be at least 99% of polynucleotide regions of interest targeted for enrichment.
  • Target coverage can be at least 99.5% of polynucleotide regions of interest targeted for enrichment.
  • Target coverage can be at least 99.9% of polynucleotide regions of interest targeted for enrichment.
  • Target coverage can be 100% of polynucleotide regions of interest targeted for enrichment.
  • Target coverage can be the proportion of polynucleotide regions of interest that is sequenced that corresponds to mutations present in a tumor and/or cancer exome of a subject.
  • Target coverage can be at least 10% of all polynucleotide regions of interest corresponding to mutations present in a tumor and/or cancer exome of a subject (e.g., coverage is at least 10% of all subject-specific and tumor- specific mutations associated with a tumor and/or cancer exome).
  • Target coverage can be at least 20% of all polynucleotide regions of interest corresponding to mutations present in a tumor and/or cancer exome of a subject.
  • Target coverage can be at least 30% of all polynucleotide regions of interest corresponding to mutations present in a tumor and/or cancer exome of a subject.
  • Target coverage can be at least 40% of all polynucleotide regions of interest corresponding to mutations present in a tumor and/or cancer exome of a subject.
  • Target coverage can be at least 50% of all polynucleotide regions of interest corresponding to mutations present in a tumor and/or cancer exome of a subject.
  • Target coverage can be at least 60% of all polynucleotide regions of interest corresponding to mutations present in a tumor and/or cancer exome of a subject.
  • Target coverage can be at least 70% of all polynucleotide regions of interest corresponding to mutations present in a tumor and/or cancer exome of a subject.
  • Target coverage can be at least 80% of all polynucleotide regions of interest corresponding to mutations present in a tumor and/or cancer exome of a subject.
  • Target coverage can be at least 90% of all polynucleotide regions of interest corresponding to mutations present in a tumor and/or cancer exome of a subject.
  • Target coverage can be at least 95% of all polynucleotide regions of interest corresponding to mutations present in a tumor and/or cancer exome of a subject.
  • Target coverage can be at least 96% of all polynucleotide regions of interest corresponding to mutations present in a tumor and/or cancer exome of a subject.
  • Target coverage can be at least 97% of all polynucleotide regions of interest corresponding to mutations present in a tumor and/or cancer exome of a subject.
  • Target coverage can be at least 98% of all polynucleotide regions of interest corresponding to mutations present in a tumor and/or cancer exome of a subject.
  • Target coverage can be at least 99% of all polynucleotide regions of interest corresponding to mutations present in a tumor and/or cancer exome of a subject.
  • Target coverage can be at least 99.5% of all polynucleotide regions of interest corresponding to mutations present in a tumor and/or cancer exome of a subject.
  • Target coverage can be at least 99.9% of all polynucleotide regions of interest corresponding to mutations present in a tumor and/or cancer exome of a subject.
  • Target coverage can be 100% of all polynucleotide regions of interest corresponding to mutations present in a tumor and/or cancer exome of a subject.
  • Target coverage can be the proportion of a tumor and/or cancer genome of a subject that is sequenced.
  • Target coverage can be at least 10% of a tumor and/or cancer genome.
  • Target coverage can be at least 20% of a tumor and/or cancer genome.
  • Target coverage can be at least 30% of a tumor and/or cancer genome.
  • Target coverage can be at least 40% of a tumor and/or cancer genome.
  • Target coverage can be at least 50% of a tumor and/or cancer genome.
  • Target coverage can be at least 60% of a tumor and/or cancer genome.
  • Target coverage can be at least 70% of a tumor and/or cancer genome.
  • Target coverage can be at least 80% of a tumor and/or cancer genome.
  • Target coverage can be at least 90% of a tumor and/or cancer genome.
  • Target coverage can be at least 95% of a tumor and/or cancer genome.
  • Target coverage can be at least 96% of a tumor and/or cancer genome.
  • Target coverage can be at least 97% of a tumor and/or cancer genome.
  • Target coverage can be at least 98% of a tumor and/or cancer genome.
  • Target coverage can be at least 99% of a tumor and/or cancer genome.
  • Target coverage can be at least 99.5% of a tumor and/or cancer genome.
  • Target coverage can be at least 99.9% of a tumor and/or cancer genome.
  • Target coverage can be 100% of a tumor and/or cancer genome.
  • Target coverage can be the percentage of regions of interest that are sequenced and where the sequenced regions of interest exceed a particular read depth.
  • Target coverage can be the percentage of regions of interest that are sequenced and where the sequenced regions of interest have a read depth of at least 1000X.
  • Target coverage can be the percentage of regions of interest that are sequenced and where the sequenced regions of interest have a read depth of at least 1500X, at least 2000X, at least 2500X, at least 3000X, at least 3500X, at least 4000X, at least 4500X, or at least 5000X.
  • Target coverage can be the percentage of regions of interest that are sequenced and where the sequenced regions of interest have a read depth of at least 1500X.
  • Target coverage can be the percentage of regions of interest that are sequenced and where the sequenced regions of interest have a read depth of at least 2000X.
  • Target coverage can be the percentage of regions of interest that are sequenced and where the sequenced regions of interest have a read depth of at least 2500X.
  • Target coverage can be the percentage of regions of interest that are sequenced and where the sequenced regions of interest have a read depth of at least 3000X.
  • Target coverage can be the percentage of regions of interest that are sequenced and where the sequenced regions of interest have a read depth of at least 3500X.
  • Target coverage can be the percentage of regions of interest that are sequenced and where the sequenced regions of interest have a read depth of at least 4000X.
  • Target coverage can be the percentage of regions of interest that are sequenced and where the sequenced regions of interest have a read depth of at least 4500X.
  • Target coverage can be the percentage of regions of interest that are sequenced and where the sequenced regions of interest have a read depth of at least 5000X.
  • Target coverage can be the percentage of regions of interest that are sequenced and where the sequenced regions of interest exceed a particular mean read depth.
  • Target coverage can be the percentage of regions of interest that are sequenced and where the sequenced regions of interest have a mean read depth of at least 1000X.
  • Target coverage can be the percentage of regions of interest that are sequenced and where the sequenced regions of interest have a mean read depth of at least 1500X, at least 2000X, at least 2500X, at least 3000X, at least 3500X, at least 4000X, at least 4500X, or at least 5000X.
  • Target coverage can be the percentage of regions of interest that are sequenced and where the sequenced regions of interest have a mean read depth of at least 1500X.
  • Target coverage can be the percentage of regions of interest that are sequenced and where the sequenced regions of interest have a mean read depth of at least 2000X.
  • Target coverage can be the percentage of regions of interest that are sequenced and where the sequenced regions of interest have a mean read depth of at least 2500X.
  • Target coverage can be the percentage of regions of interest that are sequenced and where the sequenced regions of interest have a mean read depth of at least 3000X.
  • Target coverage can be the percentage of regions of interest that are sequenced and where the sequenced regions of interest have a mean read depth of at least 3500X.
  • Target coverage can be the percentage of regions of interest that are sequenced and where the sequenced regions of interest have a mean read depth of at least 4000X.
  • Target coverage can be the percentage of regions of interest that are sequenced and where the sequenced regions of interest have a mean read depth of at least 4500X.
  • Target coverage can be the percentage of regions of interest that are sequenced and where the sequenced regions of interest have a mean read depth of at least 5000X.
  • Target coverage can be the percentage of regions of interest that are sequenced and where the sequenced regions of interest exceed a particular duplex read depth.
  • Target coverage can be the percentage of regions of interest that are sequenced and where the sequenced regions of interest have a duplex read depth of at least 1000X.
  • Target coverage can be the percentage of regions of interest that are sequenced and where the sequenced regions of interest have a duplex read depth of at least 1500X, at least 2000X, at least 2500X, at least 3000X, at least 3500X, at least 4000X, at least 4500X, or at least 5000X.
  • Target coverage can be the percentage of regions of interest that are sequenced and where the sequenced regions of interest have a duplex read depth of at least 1500X.
  • Target coverage can be the percentage of regions of interest that are sequenced and where the sequenced regions of interest have a duplex read depth of at least 2000X.
  • Target coverage can be the percentage of regions of interest that are sequenced and where the sequenced regions of interest have a duplex read depth of at least 2500X.
  • Target coverage can be the percentage of regions of interest that are sequenced and where the sequenced regions of interest have a duplex read depth of at least 3000X.
  • Target coverage can be the percentage of regions of interest that are sequenced and where the sequenced regions of interest have a duplex read depth of at least 3500X.
  • Target coverage can be the percentage of regions of interest that are sequenced and where the sequenced regions of interest have a duplex read depth of at least 4000X.
  • Target coverage can be the percentage of regions of interest that are sequenced and where the sequenced regions of interest have a duplex read depth of at least 4500X.
  • Target coverage can be the percentage of regions of interest that are sequenced and where the sequenced regions of interest have a duplex read depth of at least 5000X.
  • Target coverage can be the percentage of regions of interest that are sequenced and where the sequenced regions of interest exceed a particular mean duplex read depth.
  • Target coverage can be the percentage of regions of interest that are sequenced and where the sequenced regions of interest have a mean duplex read depth of at least 1000X.
  • Target coverage can be the percentage of regions of interest that are sequenced and where the sequenced regions of interest have a mean duplex read depth of at least 1500X, at least 2000X, at least 2500X, at least 3000X, at least 3500X, at least 4000X, at least 4500X, or at least 5000X.
  • Target coverage can be at least 10% of regions of interest and where the sequenced regions of interest have a read depth or mean read depth of at least 1000X, at least 1500X, at least 2000X, at least 2500X, at least 3000X, at least 3500X, at least 4000X, at least 4500X, or at least 5000X.
  • Target coverage can be at least 20% of regions of interest and where the sequenced regions of interest have a read depth or mean read depth of at least 1000X, at least 1500X, at least 2000X, at least 2500X, at least 3000X, at least 3500X, at least 4000X, at least 4500X, or at least 5000X.
  • Target coverage can be at least 30% of regions of interest and where the sequenced regions of interest have a read depth or mean read depth of at least 1000X, at least 1500X, at least 2000X, at least 2500X, at least 3000X, at least 3500X, at least 4000X, at least 4500X, or at least 5000X.
  • Target coverage can be at least 40% of regions of interest and where the sequenced regions of interest have a read depth or mean read depth of at least 1000X, at least 1500X, at least 2000X, at least 2500X, at least 3000X, at least 3500X, at least 4000X, at least 4500X, or at least 5000X.
  • Target coverage can be at least 50% of regions of interest and where the sequenced regions of interest have a read depth or mean read depth of at least 1000X, at least 1500X, at least 2000X, at least 2500X, at least 3000X, at least 3500X, at least 4000X, at least 4500X, or at least 5000X.
  • Target coverage can be at least 60% of regions of interest and where the sequenced regions of interest have a read depth or mean read depth of at least 1000X, at least 1500X, at least 2000X, at least 2500X, at least 3000X, at least 3500X, at least 4000X, at least 4500X, or at least 5000X.
  • Target coverage can be at least 70% of regions of interest and where the sequenced regions of interest have a read depth or mean read depth of at least 1000X, at least 1500X, at least 2000X, at least 2500X, at least 3000X, at least 3500X, at least 4000X, at least 4500X, or at least 5000X.
  • Target coverage can be at least 80% of regions of interest and where the sequenced regions of interest have a read depth or mean read depth of at least 1000X, at least 1500X, at least 2000X, at least 2500X, at least 3000X, at least 3500X, at least 4000X, at least 4500X, or at least 5000X.
  • Target coverage can be at least 90% of regions of interest and where the sequenced regions of interest have a read depth or mean read depth of at least 1000X, at least 1500X, at least 2000X, at least 2500X, at least 3000X, at least 3500X, at least 4000X, at least 4500X, or at least 5000X.
  • Target coverage can be at least 95% of regions of interest and where the sequenced regions of interest have a read depth or mean read depth of at least 1000X, at least 1500X, at least 2000X, at least 2500X, at least 3000X, at least 3500X, at least 4000X, at least 4500X, or at least 5000X.
  • Target coverage can be at least 96% of regions of interest and where the sequenced regions of interest have a read depth or mean read depth of at least 1000X, at least 1500X, at least 2000X, at least 2500X, at least 3000X, at least 3500X, at least 4000X, at least 4500X, or at least 5000X.
  • Target coverage can be at least 97% of regions of interest and where the sequenced regions of interest have a read depth or mean read depth of at least 1000X, at least 1500X, at least 2000X, at least 2500X, at least 3000X, at least 3500X, at least 4000X, at least 4500X, or at least 5000X.
  • Target coverage can be at least 98% of regions of interest and where the sequenced regions of interest have a read depth or mean read depth of at least 1000X, at least 1500X, at least 2000X, at least 2500X, at least 3000X, at least 3500X, at least 4000X, at least 4500X, or at least 5000X.
  • Target coverage can be at least 99% of regions of interest and where the sequenced regions of interest have a read depth or mean read depth of at least 1000X, at least 1500X, at least 2000X, at least 2500X, at least 3000X, at least 3500X, at least 4000X, at least 4500X, or at least 5000X.
  • Target coverage can be at least 99.5% of regions of interest and where the sequenced regions of interest have a read depth or mean read depth of at least 1000X, at least 1500X, at least 2000X, at least 2500X, at least 3000X, at least 3500X, at least 4000X, at least 4500X, or at least 5000X.
  • Target coverage can be at least 99.9% of regions of interest and where the sequenced regions of interest have a read depth or mean read depth of at least 1000X, at least 1500X, at least 2000X, at least 2500X, at least 3000X, at least 3500X, at least 4000X, at least 4500X, or at least 5000X.
  • Target coverage can be 100% of regions of interest and where the sequenced regions of interest have a read depth or mean read depth of at least 1000X, at least 1500X, at least 2000X, at least 2500X, at least 3000X, at least 3500X, at least 4000X, at least 4500X, or at least 5000X.
  • Target coverage can be at least 10% of regions of interest and where the sequenced regions of interest have a duplex read depth or mean duplex read depth of at least 1000X, at least 1500X, at least 2000X, at least 2500X, at least 3000X, at least 3500X, at least 4000X, at least 4500X, or at least 5000X.
  • Target coverage can be at least 20% of regions of interest and where the sequenced regions of interest have a duplex read depth or mean duplex read depth of at least 1000X, at least 1500X, at least 2000X, at least 2500X, at least 3000X, at least 3500X, at least 4000X, at least 4500X, or at least 5000X.
  • Target coverage can be at least 30% of regions of interest and where the sequenced regions of interest have a duplex read depth or mean duplex read depth of at least 1000X, at least 1500X, at least 2000X, at least 2500X, at least 3000X, at least 3500X, at least 4000X, at least 4500X, or at least 5000X.
  • Target coverage can be at least 40% of regions of interest and where the sequenced regions of interest have a duplex read depth or mean duplex read depth of at least 1000X, at least 1500X, at least 2000X, at least 2500X, at least 3000X, at least 3500X, at least 4000X, at least 4500X, or at least 5000X.
  • Target coverage can be at least 50% of regions of interest and where the sequenced regions of interest have a duplex read depth or mean duplex read depth of at least 1000X, at least 1500X, at least 2000X, at least 2500X, at least 3000X, at least 3500X, at least 4000X, at least 4500X, or at least 5000X.
  • Target coverage can be at least 60% of regions of interest and where the sequenced regions of interest have a duplex read depth or mean duplex read depth of at least 1000X, at least 1500X, at least 2000X, at least 2500X, at least 3000X, at least 3500X, at least 4000X, at least 4500X, or at least 5000X.
  • Target coverage can be at least 70% of regions of interest and where the sequenced regions of interest have a duplex read depth or mean duplex read depth of at least 1000X, at least 1500X, at least 2000X, at least 2500X, at least 3000X, at least 3500X, at least 4000X, at least 4500X, or at least 5000X.
  • Target coverage can be at least 80% of regions of interest and where the sequenced regions of interest have a duplex read depth or mean duplex read depth of at least 1000X, at least 1500X, at least 2000X, at least 2500X, at least 3000X, at least 3500X, at least 4000X, at least 4500X, or at least 5000X.
  • Target coverage can be at least 90% of regions of interest and where the sequenced regions of interest have a duplex read depth or mean duplex read depth of at least 1000X, at least 1500X, at least 2000X, at least 2500X, at least 3000X, at least 3500X, at least 4000X, at least 4500X, or at least 5000X.
  • Target coverage can be at least 95% of regions of interest and where the sequenced regions of interest have a duplex read depth or mean duplex read depth of at least 1000X, at least 1500X, at least 2000X, at least 2500X, at least 3000X, at least 3500X, at least 4000X, at least 4500X, or at least 5000X.
  • Target coverage can be at least 96% of regions of interest and where the sequenced regions of interest have a duplex read depth or mean duplex read depth of at least 1000X, at least 1500X, at least 2000X, at least 2500X, at least 3000X, at least 3500X, at least 4000X, at least 4500X, or at least 5000X.
  • Target coverage can be at least 97% of regions of interest and where the sequenced regions of interest have a duplex read depth or mean duplex read depth of at least 1000X, at least 1500X, at least 2000X, at least 2500X, at least 3000X, at least 3500X, at least 4000X, at least 4500X, or at least 5000X.
  • Target coverage can be at least 98% of regions of interest and where the sequenced regions of interest have a duplex read depth or mean duplex read depth of at least 1000X, at least 1500X, at least 2000X, at least 2500X, at least 3000X, at least 3500X, at least 4000X, at least 4500X, or at least 5000X.
  • Target coverage can be at least 99% of regions of interest and where the sequenced regions of interest have a duplex read depth or mean duplex read depth of at least 1000X, at least 1500X, at least 2000X, at least 2500X, at least 3000X, at least 3500X, at least 4000X, at least 4500X, or at least 5000X.
  • Target coverage can be at least 99.5% of regions of interest and where the sequenced regions of interest have a duplex read depth or mean duplex read depth of at least 1000X, at least 1500X, at least 2000X, at least 2500X, at least 3000X, at least 3500X, at least 4000X, at least 4500X, or at least 5000X.
  • Target coverage can be at least 99.9% of regions of interest and where the sequenced regions of interest have a duplex read depth or mean duplex read depth of at least 1000X, at least 1500X, at least 2000X, at least 2500X, at least 3000X, at least 3500X, at least 4000X, at least 4500X, or at least 5000X.
  • Target coverage can be 100% of regions of interest and where the sequenced regions of interest have a duplex read depth or mean duplex read depth of at least 1000X, at least 1500X, at least 2000X, at least 2500X, at least 3000X, at least 3500X, at least 4000X, at least 4500X, or at least 5000X.
  • Target coverage can be at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of regions of interest and where the sequenced regions of interest have a read depth or mean read depth of at least 1000X.
  • Target coverage can be at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of regions of interest and where the sequenced regions of interest have a read depth or mean read depth of at least 1500X.
  • Target coverage can be at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of regions of interest and where the sequenced regions of interest have a read depth or mean read depth of at least 2000X.
  • Target coverage can be at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of regions of interest and where the sequenced regions of interest have a read depth or mean read depth of at least 2500X.
  • Target coverage can be at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of regions of interest and where the sequenced regions of interest have a read depth or mean read depth of at least 3000X.
  • Target coverage can be at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of regions of interest and where the sequenced regions of interest have a read depth or mean read depth of at least 3500X.
  • Target coverage can be at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of regions of interest and where the sequenced regions of interest have a read depth or mean read depth of at least 4000X.
  • Target coverage can be at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of regions of interest and where the sequenced regions of interest have a read depth or mean read depth of at least 4500X.
  • Target coverage can be at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of regions of interest and where the sequenced regions of interest have a read depth or mean read depth of at least 5000X.
  • Target coverage can be at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of regions of interest and where the sequenced regions of interest have a duplex read depth or mean duplex read depth of at least 1000X.
  • Target coverage can be at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of regions of interest and where the sequenced regions of interest have a duplex read depth or mean duplex read depth of at least 1500X.
  • Target coverage can be at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of regions of interest and where the sequenced regions of interest have a duplex read depth or mean duplex read depth of at least 2000X.
  • Target coverage can be at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of regions of interest and where the sequenced regions of interest have a duplex read depth or mean duplex read depth of at least 2500X.
  • Target coverage can be at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of regions of interest and where the sequenced regions of interest have a duplex read depth or mean duplex read depth of at least 3000X.
  • Target coverage can be at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of regions of interest and where the sequenced regions of interest have a duplex read depth or mean duplex read depth of at least 3500X.
  • Target coverage can be at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of regions of interest and where the sequenced regions of interest have a duplex read depth or mean duplex read depth of at least 4000X.
  • Target coverage can be at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of regions of interest and where the sequenced regions of interest have a duplex read depth or mean duplex read depth of at least 4500X.
  • Target coverage can be at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of regions of interest and where the sequenced regions of interest have a duplex read depth or mean duplex read depth of at least 5000X.
  • sequence reads can be analyzed to provide a quantitative determination of the frequency of variant alleles (also referred to as mutant allele frequency) within the cfDNA of a subject.
  • Methods for quantifying sequencing reads and variant allele frequencies are known to those skilled in the art.
  • Computational programs for sequencing analysis and VAF include, but are not limited to, BWA-MEM (Durbin et al, Bioinformatics, 2010), fgbio toolkit (Fulcrum Genomics), and freebayes (Marth et al, arXiv 2012), each of which is herein incorporated by reference for all purposes.
  • cfDNA e.g ., VAF
  • mutational frequency can be determined by counting the reads of a specific variant allele in comparison to total cfDNA counts for samples taken from a subject.
  • VAF assessments can be combined with cfDNA concentration in plasma (e.g., ng/ml) to estimate tumor genome concentrations in plasma (see Bos, et al Molecular Oncology (2020) doi: 10.1002/1878-0261.12827 and Reinert et al, JAMA Oncol. 2019;5(8): 1124-1131. doi:10.1001/jamaoncol.2019.0528, each herein incorporated by reference for all purposes).
  • mutational frequency or estimated tumor genome content can then be assessed to characterize various disease or subject attributes, such as a status of a disease of a subject, efficacy of a therapy, or combinations thereof, mutational frequency
  • Assessment can be done, for example, to assess disease status of a subject, such as assessing tumor burden of a subject.
  • Assessment of tumor burden can be used in various applications, such as part of disease diagnosis, disease prognosis, disease prediction, and/or monitoring of disease progression.
  • Assessment of disease progression can be done by comparing mutational frequency in samples taken from a subject at various timepoints. Changes in mutational frequency can be relative to a fixed timepoint, e.g., a baseline mutational frequency such as the mutational frequency determined on the first day of a therapy regimen.
  • An increase in mutational frequency from cfDNA mutation analysis of a first sample collected (e.g., an earlier longitudinal sample) relative to mutational frequency from cfDNA mutation analysis of a second sample (e.g., an later longitudinal sample) can be assessed as disease progression, unresponsiveness to therapy, and/or disease recurrence.
  • a decrease in mutational frequency from cfDNA mutation analysis of a first sample collected (e.g., an earlier longitudinal sample) relative to mutational frequency from cfDNA mutation analysis of a second sample (e.g., an later longitudinal sample) can be assessed as a response.
  • a response can be either a complete response (CR) or a partial response (PR).
  • An increase in frequency of mutations in post-therapy cfDNA relative to pre-therapy cfDNA can indicate an increased likelihood that tumor burden of the subject is increasing.
  • a decrease or maintenance of frequency of mutations in post-therapy cfDNA relative to pre-therapy cfDNA can indicate an increased likelihood that tumor burden of the subject is decreasing or stable.
  • An increase in mutational frequency (or alternatively estimated tumor genomes per ml of plasma) over time can be assessed as disease progression and/or recurrence.
  • An increase in mutational frequency can be an at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100% relative increase in mutational frequency between timepoints to be assessed as progression and/or recurrence.
  • An increase in mutational frequency can be an at least 2-fold, at least 3- fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9- fold, or at least 10-fold relative increase in mutational frequency between timepoints to be assessed as progression and/or recurrence.
  • An increase in mutational frequency can be an at least 20-fold, at least 30-fold, at least 40-fold, at least 50-fold, at least 60-fold, at least 70- fold, at least 80-fold, at least 90-fold, or at least 100-fold relative increase in mutational frequency between timepoints to be assessed as progression and/or recurrence.
  • a decrease in mutational frequency (or alternatively estimated tumor genomes per ml of plasma) over time can be assessed as disease remission.
  • a decrease in mutational frequency can be an at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100% relative increase in mutational frequency between timepoints to be assessed as remission.
  • a decrease in mutational frequency can be an at least 2-fold, at least 3 -fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, or at least 10-fold relative increase in mutational frequency between timepoints to be assessed as remission.
  • a decrease in mutational frequency can be an at least 20-fold, at least 30-fold, at least 40-fold, at least 50- fold, at least 60-fold, at least 70-fold, at least 80-fold, at least 90-fold, or at least 100-fold relative increase in mutational frequency between timepoints to be assessed as remission.
  • a decrease in mutational frequency can be to an undetectable level of mutations in the cfDNA to be assessed as remission, e.g., assessed as a complete remission.
  • the frequency of mutations (or alternatively estimated tumor genomes per ml of plasma) in cfDNA can be compared between a sample collected prior to therapy and a sample collected subsequent to therapy.
  • An increase in mutational frequency from cfDNA mutation analysis of a sample collected prior to therapy relative to mutational frequency from cfDNA mutation analysis of a sample collected subsequent to therapy can be assessed as disease progression, unresponsiveness to therapy, and/or disease recurrence.
  • a decrease in mutational frequency from cfDNA mutation analysis of a sample collected prior to therapy relative to mutational frequency from cfDNA mutation analysis of a sample collected subsequent to therapy can be assessed as a response.
  • a response can be either a complete response (CR) or a partial response (PR).
  • An increase in frequency of mutations in post-therapy cfDNA relative to pre-therapy cfDNA can indicate an increased likelihood that tumor burden of the subject is increasing.
  • a decrease or maintenance of frequency of mutations in post-therapy cfDNA relative to pre-therapy cfDNA can indicate an increased likelihood that tumor burden of the subject is decreasing or stable.
  • Further therapy can be administered to a subject following an assessment step. For example, an initial measurement can be obtained from a patient before beginning a multi- dose anti-cancer therapy regimen. Subsequent measurements can be taken prior to administration of each dose. Analysis of variant-allele frequency in cfDNA at each stage can allow assessment of patient response to each dose of the therapy regimen. Assessment can further guide clinical decisions including dosages, therapy choices, etc.
  • a therapy can comprise a cancer vaccine.
  • a therapy can include targeted radiation therapy (e.g external beam radiation, brachy therapy).
  • a therapy can include an immune checkpoint inhibitor, including but not limited to a PD-1 inhibitor (e.g., nivolumab, pembrolizumab), a PD-L1 inhibitor (e.g., avelumab, durvalumab), or a CTLA-4 inhibitor (e.g., ipilimumab).
  • a PD-1 inhibitor e.g., nivolumab, pembrolizumab
  • a PD-L1 inhibitor e.g., avelumab, durvalumab
  • CTLA-4 inhibitor e.g., ipilimumab
  • a therapy can include targeted therapy technologies, such as monoclonal antibody therapies (e.g., trastuzumab, bevacizumab), retinoids (e.g., ATRA, bexarotene), selective steroid hormone receptor modulators (e.g., tamoxifen, toremifene), or inhibitors of oncoprotein such as tyrosine kinases (TK) (e.g., imatinib, erlotinib), mammalian target of rapamyciun (mTOR) (e.g., everolimus, temsirolimus), or histone deacetylase (HD AC) (e.g., valproate, vorinostat).
  • monoclonal antibody therapies e.g., trastuzumab, bevacizumab
  • retinoids e.g., ATRA, bexarotene
  • selective steroid hormone receptor modulators e.g., tamoxi
  • a therapy can include cytotoxic chemotherapy.
  • cytotoxic chemotherapeutic agents include cisplatin, carboplatin, oxaliplatin, nedaplatin, azacytidine, capecitabine, carmofur, cladribine, clofarabine, cytarabine, decitabine, florouracil, floxuridine, fludaramine, mercaptopurine, nelarabine, pentostatin, tegafur, tioguanine, methotrexate, pemetrexed, raltitrexed, hydroxycarbamide, irinotecan, topotecan, danorubicin, doxorubicin, epirubicin, idarubicin, mitoxantrone, valmbicin, etoposide, teniposide, docetaxel, paclitaxel, vinblastine, vincristine, vindesine, vinflunine, vinore
  • a subject has been diagnosed with cancer or is at risk of developing cancer.
  • a subject can be a human, dog, cat, horse or any animal in which a tumor specific immune response is desired.
  • a tumor can be any solid tumor such as breast, ovarian, prostate, lung, kidney, gastric, colon, testicular, head and neck, pancreas, brain, melanoma, and other tumors of tissue organs and hematological tumors, such as lymphomas and leukemias, including acute myelogenous leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia, T cell lymphocytic leukemia, and B cell lymphomas.
  • An antigen can be administered in an amount sufficient to induce a CTL response.
  • An antigen can be administered in an amount sufficient to induce a T cell response.
  • An antigen can be administered in an amount sufficient to induce a B cell response.
  • An antigen can be administered alone or in combination with other therapeutic agents, e.g., a chemotherapeutic therapy, immune checkpoint blockade, and/or other immunotherapy .
  • other therapeutic agents e.g., a chemotherapeutic therapy, immune checkpoint blockade, and/or other immunotherapy .
  • an antigen or its variant can be prepared for intravenous (i.v.) injection, sub-cutaneous (s.c.) injection, intradermal (i.d.) injection, intraperitoneal (i.p.) injection, intramuscular (i.m.) injection.
  • Methods of injection include s.c., i.d., i.p., i.m., and i.v.
  • Methods of DNA or RNA injection include i.d., i.m., s.c., i.p. and i.v.
  • Other methods of administration of the vaccine composition are known to those skilled in the art.
  • a vaccine can be compiled so that the selection, number and/or amount of antigens present in the composition is/are tissue, cancer, and/or subject-specific. For instance, the exact selection of peptides can be guided by expression patterns of the parent proteins in a given tissue or guided by mutation or disease status of a patient. The selection can be dependent on the specific type of cancer, the status of the disease, the goal of the vaccination (e.g., preventative or targeting an ongoing disease), earlier treatment regimens, the immune status of the patient, and, of course, the HLA-haplotype of the patient. Furthermore, a vaccine can contain individualized components, according to personal needs of the particular patient. Examples include varying the selection of antigens according to the expression of the antigen in the particular patient or adjustments for secondary treatments following a first round or scheme of treatment.
  • a patient can be identified for administration of an antigen vaccine through the use of various diagnostic methods, e.g., patient selection methods described further below.
  • Patient selection can involve identifying mutations in, or expression patterns of, one or more genes.
  • patient selection involves identifying the haplotype of the patient.
  • the various patient selection methods can be performed in parallel, e.g., a sequencing diagnostic can identify both the mutations and the haplotype of a patient.
  • the various patient selection methods can be performed sequentially, e.g., one diagnostic test identifies the mutations and separate diagnostic test identifies the haplotype of a patient, and where each test can be the same (e.g., both high-throughput sequencing) or different (e.g., one high-throughput sequencing and the other Sanger sequencing) diagnostic methods.
  • compositions to be used as a vaccine for cancer antigens with similar normal self-peptides that are expressed in high amounts in normal tissues can be avoided or be present in low amounts in a composition described herein.
  • the respective pharmaceutical composition for treatment of a cancer can be present in high amounts and/or more than one antigen specific for this particularly antigen or pathway of this antigen can be included.
  • compositions comprising an antigen can be administered to an individual already suffering from cancer.
  • compositions are administered to a patient in an amount sufficient to elicit a therapeutically effective response, e.g., in an amount sufficient to stimulate an effective CTL response to the tumor antigen and to cure or at least partially arrest symptoms and/or complications.
  • An amount adequate to accomplish this is defined as "therapeutically effective dose.” Amounts effective for this use will depend on, e.g., the composition, the manner of administration, the stage and severity of the disease being treated, the weight and general state of health of the patient, and the judgment of the prescribing physician.
  • compositions can generally be employed in serious disease states, that is, life-threatening or potentially life threatening situations, especially when the cancer has metastasized. In such cases, in view of the minimization of extraneous substances and the relative nontoxic nature of an antigen, it is possible and can be felt desirable by the treating physician to administer substantial excesses of these compositions.
  • administration can begin at the detection or surgical removal of tumors. This can be followed by boosting doses until at least symptoms are substantially abated and for a period thereafter.
  • compositions for therapeutic treatment are intended for parenteral, topical, nasal, oral or local administration.
  • a pharmaceutical compositions can be administered parenterally, e.g., intravenously, subcutaneously, intradermally, or intramuscularly.
  • Compositions can be administered at the site of surgical excision to induce a local immune response to the tumor.
  • Compositions can be administered to target specific diseased tissues and/or cells of a subject.
  • compositions for parenteral administration which comprise a solution of the antigen and vaccine compositions are dissolved or suspended in an acceptable carrier, e.g., an aqueous carrier.
  • aqueous carriers can be used, e.g., water, buffered water, 0.9% saline, 0.3% glycine, hyaluronic acid and the like. These compositions can be sterilized by conventional, well known sterilization techniques, or can be sterile filtered. Resulting aqueous solutions can be packaged for use as is, or lyophilized, the lyophilized preparation being combined with a sterile solution prior to administration.
  • compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, etc.
  • auxiliary substances such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, etc.
  • Antigens can also be administered via liposomes, which target them to a particular cells tissue, such as lymphoid tissue. Liposomes are also useful in increasing half-life. Liposomes include emulsions, foams, micelles, insoluble monolayers, liquid crystals, phospholipid dispersions, lamellar layers and the like. In these preparations the antigen to be delivered is incorporated as part of a liposome, alone or in conjunction with a molecule which binds to, e.g., a receptor prevalent among lymphoid cells, such as monoclonal antibodies which bind to the CD45 antigen, or with other therapeutic or immunogenic compositions.
  • a receptor prevalent among lymphoid cells such as monoclonal antibodies which bind to the CD45 antigen, or with other therapeutic or immunogenic compositions.
  • liposomes filled with a desired antigen can be directed to the site of lymphoid cells, where the liposomes then deliver the selected therapeutic/immunogenic compositions.
  • Liposomes can be formed from standard vesicle-forming lipids, which generally include neutral and negatively charged phospholipids and a sterol, such as cholesterol. The selection of lipids is generally guided by consideration of, e.g., liposome size, acid lability and stability of the liposomes in the blood stream. A variety of methods are available for preparing liposomes, as described in, e.g., Szoka et al., Ann. Rev. Biophys. Bioeng. 9; 467 (1980), U.S. Pat. Nos. 4,235,871, 4,501,728, 4,501,728, 4,837,028, and 5,019,369.
  • a ligand to be incorporated into a liposome can include, e.g., antibodies or fragments thereof specific for cell surface determinants of the desired immune system cells.
  • a liposome suspension can be administered intravenously, locally, topically, etc. in a dose which varies according to, inter alia, the manner of administration, the peptide being delivered, and the stage of the disease being treated.
  • nucleic acids encoding a peptide and optionally one or more of the peptides described herein can also be administered to the patient.
  • a number of methods are conveniently used to deliver the nucleic acids to the patient.
  • a nucleic acid can be delivered directly, as "naked DNA". This approach is described, for instance, in Wolff et al., Science 247: 1465-1468 (1990) as well as U.S. Pat. Nos. 5,580,859 and 5,589,466.
  • Nucleic acids can also be administered using ballistic delivery as described, for instance, in U.S. Pat. No. 5,204,253. Particles comprised solely of DNA can be administered. Alternatively, DNA can be adhered to particles, such as gold particles.
  • Approaches for delivering nucleic acid sequences can include viral vectors, mRNA vectors, and DNA vectors with or without electroporation.
  • Nucleic acids can also be delivered complexed to cationic compounds, such as cationic lipids.
  • Lipid-mediated gene delivery methods are described, for instance, in 9618372WOAWO 96/18372; 9324640WOAWO 93/24640; Mannino & Gould-Fogerite, BioTechniques 6(7): 682-691 (1988); U.S. Pat. No. 5,279,833 Rose U.S. Pat. No. 5,279,833; 9106309WOAWO 91/06309; and Feigner et al., Proc. Natl. Acad. Sci. USA 84: 7413-7414 (1987).
  • Antigens can also be included in viral vector-based vaccine platforms, such as vaccinia, fowlpox, self-replicating alphavirus, marabavirus, adenovirus (See, e.g., Tatsis et al., Adenoviruses, Molecular Therapy (2004) 10, 616 — 629), or lentivims, including but not limited to second, third or hybrid second/third generation lentivims and recombinant lentivims of any generation designed to target specific cell types or receptors (See, e.g., Hu et al., Immunization Delivered by Lentiviral Vectors for Cancer and Infectious Diseases, Immunol Rev.
  • this approach can deliver one or more nucleotide sequences that encode one or more antigen peptides. Sequences may be flanked by non-mutated sequences, may be separated by linkers or may be preceded with one or more sequences targeting a subcellular compartment (See, e.g., Gros et al., Prospective identification of neoantigen- specific lymphocytes in the peripheral blood of melanoma patients, Nat Med. (2016) 22 (4):433-8, Stronen et al., Targeting of cancer neoantigens with donor-derived T cell receptor repertoires, Science.
  • a vaccine can include an epitope-encoding nucleic acid whose sequence encodes one or more tumor and/or subject- specific mutations, such as one or more of the mutations whose frequency is determined in the cfDNA.
  • a vaccine system can comprise a self- replicating alphavims-based expression system encoding an epitope-encoding nucleic acid whose sequence encodes one or more tumor and/or subject-specific mutations. Self- replicating alphavims-based expression systems for use as cancer vaccines are described in international patent application publication WO/2018/208856, which is herein incorporated by reference, in its entirety, for all purposes.
  • a vaccine system can comprise a chimpanzee adenovirus (ChAdV)-based expression system encoding an epitope-encoding nucleic acid whose sequence encodes one or more tumor and/or subject-specific mutations.
  • ChAdV-based expression system for use as cancer vaccines are described in international patent application publication WO/2018/098362, which is herein incorporated by reference, in its entirety, for all purposes.
  • a means of administering nucleic acids uses minigene constructs encoding one or multiple epitopes.
  • a human codon usage table is used to guide the codon choice for each amino acid.
  • minigene sequence examples include: helper T lymphocyte, epitopes, a leader (signal) sequence, and an endoplasmic reticulum retention signal.
  • MHC presentation of CTL epitopes can be improved by including synthetic (e.g. poly-alanine) or naturally-occurring flanking sequences adjacent to the CTL epitopes.
  • the minigene sequence is converted to DNA by assembling oligonucleotides that encode the plus and minus strands of the minigene. Overlapping oligonucleotides (30-100 bases long) are synthesized, phosphorylated, purified and annealed under appropriate conditions using well known techniques. The ends of the oligonucleotides are joined using T4 DNA ligase. This synthetic minigene, encoding the CTL epitope polypeptide, can then cloned into a desired expression vector.
  • Purified plasmid DNA can be prepared for injection using a variety of formulations. The simplest of these is reconstitution of lyophilized DNA in sterile phosphate- buffer saline (PBS). A variety of methods have been described, and new techniques can become available. As noted above, nucleic acids are conveniently formulated with cationic lipids. In addition, glycolipids, fusogenic liposomes, peptides and compounds referred to collectively as protective, interactive, non-condensing (PINC) could also be complexed to purified plasmid DNA to influence variables such as stability, intramuscular dispersion, or trafficking to specific organs or cell types.
  • PINC protective, interactive, non-condensing
  • Also disclosed is a method of manufacturing a vaccine comprising performing the steps of a method disclosed herein; and producing a vaccine comprising a plurality of antigens or a subset of the plurality of antigens.
  • Antigens disclosed herein can be manufactured using methods known in the art.
  • a method of producing an antigen or a vector (e.g., a vector including at least one sequence encoding one or more antigens) disclosed herein can include culturing a host cell under conditions suitable for expressing the antigen or vector wherein the host cell comprises at least one polynucleotide encoding the antigen or vector, and purifying the antigen or vector.
  • Standard purification methods include chromatographic techniques, electrophoretic, immunological, precipitation, dialysis, filtration, concentration, and chromatofocusing techniques.
  • Host cells can include a Chinese Hamster Ovary (CHO) cell, NS0 cell, yeast, or a HEK293 cell.
  • Host cells can be transformed with one or more polynucleotides comprising at least one nucleic acid sequence that encodes an antigen or vector disclosed herein, optionally wherein the isolated polynucleotide further comprises a promoter sequence operably linked to at least one nucleic acid sequence that encodes the antigen or vector.
  • the isolated polynucleotide can be cDNA.
  • Antigens can include nucleotides or polypeptides.
  • an antigen can be an RNA sequence that encodes for a polypeptide sequence.
  • Antigens useful in vaccines can therefore include nucleotide sequences or polypeptide sequences.
  • Antigens that can be used for cancer vaccines are described in international patent application publication WO/2019/226941, which is herein incorporated by reference, in its entirety, for all purposes.
  • Disclosed herein are isolated peptides that comprise tumor specific mutations identified by the methods disclosed herein, peptides that comprise known tumor specific mutations, and mutant polypeptides or fragments thereof identified by methods disclosed herein.
  • Neoantigen peptides can be described in the context of their coding sequence where a neoantigen includes the nucleotide sequence (e.g., DNA or RNA) that codes for the related polypeptide sequence.
  • peptides derived from any polypeptide known to or have been found to have altered expression in a tumor cell or cancerous tissue in comparison to a normal cell or tissue for example any polypeptide known to or have been found to be aberrantly expressed in a tumor cell or cancerous tissue in comparison to a normal cell or tissue.
  • Suitable polypeptides from which the antigenic peptides can be derived can be found for example in the COSMIC database. COSMIC curates comprehensive information on somatic mutations in human cancer. The peptide contains the tumor specific mutation.
  • One or more polypeptides encoded by an antigen nucleotide sequence can comprise at least one of: a binding affinity with MHC with an IC50 value of less than lOOOnM, for MHC Class I peptides a length of 8-15, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids, presence of sequence motifs within or near the peptide promoting proteasome cleavage, and presence or sequence motifs promoting TAP transport.
  • MHC Class II peptides a length 6-30, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids, presence of sequence motifs within or near the peptide promoting cleavage by extracellular or lysosomal proteases (e.g., cathepsins) or HLA-DM catalyzed HLA binding.
  • extracellular or lysosomal proteases e.g., cathepsins
  • HLA-DM catalyzed HLA binding e.g., HLA-DM catalyzed HLA binding.
  • One or more antigens can be presented on the surface of a tumor.
  • One or more antigens can be is immunogenic in a subject having a tumor, e.g., capable of eliciting a T cell response or a B cell response in the subject.
  • One or more antigens that induce an autoimmune response in a subject can be excluded from consideration in the context of vaccine generation for a subject having a tumor.
  • the size of at least one antigenic peptide molecule can comprise, but is not limited to, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, about 50, about 60, about 70, about 80, about 90, about 100, about 110, about 120 or greater amino molecule residues, and any range derivable therein.
  • the antigenic peptide molecules are equal to or less than 50 amino acids.
  • Antigenic peptides and polypeptides can be: for MHC Class I 15 residues or less in length and usually consist of between about 8 and about 11 residues, particularly 9 or 10 residues; for MHC Class II, 6-30 residues, inclusive.
  • a longer peptide can be designed in several ways.
  • a longer peptide could consist of either: (1) individual presented peptides with an extensions of 2-5 amino acids toward the N- and C-terminus of each corresponding gene product; (2) a concatenation of some or all of the presented peptides with extended sequences for each.
  • sequencing reveals a long (>10 residues) neoepitope sequence present in the tumor (e.g.
  • a longer peptide would consist of: (3) the entire stretch of novel tumor-specific amino acids— thus bypassing the need for computational or in vitro test-based selection of the strongest HLA-presented shorter peptide.
  • use of a longer peptide allows endogenous processing by patient cells and may lead to more effective antigen presentation and induction of T cell responses.
  • Antigenic peptides and polypeptides can be presented on an HLA protein. In some aspects antigenic peptides and polypeptides are presented on an HLA protein with greater affinity than a wild-type peptide. In some aspects, an antigenic peptide or polypeptide can have an IC50 of at least less than 5000 nM, at least less than 1000 nM, at least less than 500 nM, at least less than 250 nM, at least less than 200 nM, at least less than 150 nM, at least less than 100 nM, at least less than 50 nM or less.
  • antigenic peptides and polypeptides do not induce an autoimmune response and/or invoke immunological tolerance when administered to a subject.
  • compositions comprising at least two or more antigenic peptides.
  • the composition contains at least two distinct peptides.
  • At least two distinct peptides can be derived from the same polypeptide.
  • distinct polypeptides is meant that the peptide vary by length, amino acid sequence, or both.
  • the peptides are derived from any polypeptide known to or have been found to contain a tumor specific mutation or peptides derived from any polypeptide known to or have been found to have altered expression in a tumor cell or cancerous tissue in comparison to a normal cell or tissue, for example any polypeptide known to or have been found to be aberrantly expressed in a tumor cell or cancerous tissue in comparison to a normal cell or tissue.
  • Suitable polypeptides from which the antigenic peptides can be derived can be found for example in the COSMIC database or the AACR Genomics Evidence Neoplasia Information Exchange (GENIE) database.
  • COSMIC curates comprehensive information on somatic mutations in human cancer.
  • AACR GENIE aggregates and links clinical-grade cancer genomic data with clinical outcomes from tens of thousands of cancer patients.
  • the peptide contains the tumor specific mutation.
  • the tumor specific mutation is a driver mutation for a particular cancer type.
  • Antigenic peptides and polypeptides having a desired activity or property can be modified to provide certain desired attributes, e.g., improved pharmacological characteristics, while increasing or at least retaining substantially all of the biological activity of the unmodified peptide to bind the desired MHC molecule and activate the appropriate T cell.
  • antigenic peptide and polypeptides can be subject to various changes, such as substitutions, either conservative or non-conservative, where such changes might provide for certain advantages in their use, such as improved MHC binding, stability or presentation.
  • conservative substitutions is meant replacing an amino acid residue with another which is biologically and/or chemically similar, e.g., one hydrophobic residue for another, or one polar residue for another.
  • the substitutions include combinations such as Gly, Ala; Val, lie, Leu, Met; Asp, Glu; Asn, Gin; Ser, Thr; Lys, Arg; and Phe, Tyr.
  • the effect of single amino acid substitutions may also be probed using D-amino acids.
  • Modifications of peptides and polypeptides with various amino acid mimetics or unnatural amino acids can be particularly useful in increasing the stability of the peptide and polypeptide in vivo. Stability can be assayed in a number of ways. For instance, peptidases and various biological media, such as human plasma and serum, have been used to test stability. See, e.g., Verhoef et ah, Eur. J. Drug Metab Pharmacokin. 11:291-302 (1986). Half- life of the peptides can be conveniently determined using a 25% human serum (v/v) assay. The protocol is generally as follows.
  • pooled human serum (Type AB, non-heat inactivated) is delipidated by centrifugation before use. The serum is then diluted to 25% with RPMI tissue culture media and used to test peptide stability. At predetermined time intervals a small amount of reaction solution is removed and added to either 6% aqueous trichloracetic acid or ethanol. The cloudy reaction sample is cooled (4 degrees C) for 15 minutes and then spun to pellet the precipitated serum proteins. The presence of the peptides is then determined by reversed-phase HPLC using stability-specific chromatography conditions.
  • the peptides and polypeptides can be modified to provide desired attributes other than improved serum half-life. For instance, the ability of the peptides to induce CTL activity can be enhanced by linkage to a sequence which contains at least one epitope that is capable of inducing a T helper cell response.
  • Immunogenic peptides/T helper conjugates can be linked by a spacer molecule.
  • the spacer is typically comprised of relatively small, neutral molecules, such as amino acids or amino acid mimetics, which are substantially uncharged under physiological conditions.
  • the spacers are typically selected from, e.g., Ala, Gly, or other neutral spacers of nonpolar amino acids or neutral polar amino acids.
  • the optionally present spacer need not be comprised of the same residues and thus can be a hetero- or homo-oligomer.
  • the spacer will usually be at least one or two residues, more usually three to six residues.
  • the peptide can be linked to the T helper peptide without a spacer.
  • An antigenic peptide can be linked to the T helper peptide either directly or via a spacer either at the amino or carboxy terminus of the peptide.
  • the amino terminus of either the antigenic peptide or the T helper peptide can be acylated.
  • Exemplary T helper peptides include tetanus toxoid 830-843, influenza 307-319, malaria circumsporozoite 382-398 and 378-389.
  • Proteins or peptides can be made by any technique known to those of skill in the art, including the expression of proteins, polypeptides or peptides through standard molecular biological techniques, the isolation of proteins or peptides from natural sources, or the chemical synthesis of proteins or peptides.
  • the nucleotide and protein, polypeptide and peptide sequences corresponding to various genes have been previously disclosed, and can be found at computerized databases known to those of ordinary skill in the art.
  • One such database is the National Center for Biotechnology Information's Genbank and GenPept databases located at the National Institutes of Health website.
  • the coding regions for known genes can be amplified and/or expressed using the techniques disclosed herein or as would be known to those of ordinary skill in the art.
  • an antigen includes a nucleic acid (e.g. polynucleotide) that encodes an antigenic peptide or portion thereof.
  • the polynucleotide can be, e.g., DNA, cDNA, PNA, CNA, RNA (e.g., mRNA), either single- and/or double-stranded, or native or stabilized forms of polynucleotides, such as, e.g., polynucleotides with a phosphorothiate backbone, or combinations thereof and it may or may not contain introns.
  • a still further aspect provides an expression vector capable of expressing a polypeptide or portion thereof.
  • Expression vectors for different cell types are well known in the art and can be selected without undue experimentation.
  • DNA is inserted into an expression vector, such as a plasmid, in proper orientation and correct reading frame for expression. If necessary, DNA can be linked to the appropriate transcriptional and translational regulatory control nucleotide sequences recognized by the desired host, although such controls are generally available in the expression vector.
  • the vector is then introduced into the host through standard techniques. Guidance can be found e.g. in Sambrook et al. (1989) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.
  • the examples outline a cell-free DNA (cfDNA) assay used to monitor mutation frequency is provided. Additionally, data for on-treatment monitoring of mutation frequency in cfDNA from patient plasma was processed and analyzed using the provided protocol (see below) is presented. Notably, for GRANITE patients, greater than 200 mutations were monitored, representing all or a majority of high quality mutation calls associated with the tumor exome for each patient. The results demonstrate the method described provides a robust method for monitoring mutation frequency.
  • cfDNA cell-free DNA
  • RNAlater samples For genomic DNA from each sample, 50,000 PMBCs were isolated and extracted using the Qiagen Tissue AllPrep Kit. For RNAlater samples, the Qiagen DNA/RNA Mini AllPrep kit was used to isolate genomic DNA from tissue that had been preserved in RNAlater.
  • Libraries were prepared with up to 20ng cfDNA using the KAPA Hyper Prep kit per the manufacturer’s instructions (KAPA Biosystems; Wilmington, MA).
  • KAPA Biosystems KAPA Biosystems; Wilmington, MA
  • 30ng of gDNA was first fragmented using the NEBNext Ultra II FS DNA Module (NEB, Ipswich, MA) with the following conditions: 25 minutes at 37°C followed by 30 minutes at 65°C.
  • IDTT a pool of duplexed adaptors containing 5-mer non-random unique molecular identifiers
  • Fig. 1 and 2 diagram and Table 1 shows the specifications for the process used to isolate and monitor mutant alleles in an individual patient’s ctDNA.
  • Tumor-specific DNA variant alleles were identified in patients from biopsied tumor tissue and used to create baits to isolate tumor- specific DNA from all circulating cell- free DNA (cfDNA) in patient blood samples. Isolated ctDNA was duplex sequenced and analyzed for duplex consensus. Sequencing of multiple blood draws over the course of treatment allowed less-invasive monitoring of patient response.
  • GRANITE personalized neoantigen cancer vaccine
  • the GRANITE heterologous prime/boost vaccine regimen included (1) a ChAdV that is used as a prime vaccination [GRT-C901] and (2) a SAM formulated in a LNP that is used for boost vaccinations [GRT-R902] following GRT-C901.
  • the ChAdV vector is based on a modified ChAdV68 sequence.
  • the SAM vector is based on an RNA alphavims backbone. Both GRT- C901 and GRT-R902 expressed the same 20 personalized neoantigens as well as two universal CD4 T-cell epitopes (PADRE and Tetanus Toxoid).
  • Tumors were used for whole- exome and transcriptome sequencing to detect somatic mutations, and blood was used for HLA typing and detection/subtraction of germline exome variants to generate the personalized neoantigen cassette using the EDGE algorithm for 10 subjects (Patients 1-10, referred to herein as patients G1-G10).
  • a shared neoantigen cancer vaccine (“SLATE”) was administered in combination with immune checkpoint blockade in patients with advanced cancer.
  • the SLATE heterologous prime/boost vaccine regimen included (1) a ChAdV that is used as a prime vaccination [GRT-C903] and (2) a SAM formulated in a LNP that is used for boost vaccinations [GRT-R904] following GRT-C903.
  • GRT-C903 and GRT-R904 expressed the same 20 shared neoantigens derived from a specific list of oncogenic mutations as well as two universal CD4 T-cell epitopes (PADRE and Tetanus Toxoid).
  • HLA A02:01 and KRAS mutation G12C predicted to be presented by HLA A02:01 (Patients SI, S2, and S3), HLA A01:01 and KRAS mutation Q61H predicted to be presented by HLA A01:01 (Patients S4 and S7), or HLA A03:01 or All:01 and KRAS mutation G12V predicted to be presented by HLA A03:01 or All:01 (A03:01 for Patient S9; All:01 for Patients Sll and S15).
  • GRT-C901 and GRT-C903 are replication-defective, El and E3 deleted adenoviral vectors based on chimpanzee adenovirus 68.
  • the vector contained an expression cassette encoding 20 neoantigens as well as two universal CD4 T-cell epitopes (PADRE and Tetanus Toxoid).
  • GRT-C901 and GRT-C903 were formulated in solution at 5xl0 u vp/mL and 1.0 mL was injected IM at each of 2 bilateral vaccine injection sites in opposing deltoid muscles.
  • the GRT-C901 and GRT-C903 vectors differ only by the encoded neoantigens within the cassette.
  • GRT-R902 and GRT-R904 are SAM vectors derived from an alphavirus.
  • the GRT-R902 and GRT-R904 vectors encoded the viral proteins and the 5’ and 3’ RNA sequences required for RNA amplification but encoded no structural proteins.
  • the SAM vectors were formulated in LNPs that included 4 lipids: an ionizable amino lipid, a phosphatidylcholine, cholesterol, and a PEG-based coat lipid to encapsulate the SAM and form LNPs.
  • the GRT-R902 vector contained the same neoantigen expression cassette as used in GRT-C901 for each patient, respectively.
  • the GRT-R904 vector contained the same neoantigen expression cassette as used in GRT-C903.
  • GRT-R902 and GRT-R904 were formulated in solution at 1 mg/mL and was injected IM at each of 2 bilateral vaccine injection sites in opposing deltoid muscles (deltoid muscle preferred, gluteus [dorso or ventro] or rectus femoris on each side may be used).
  • the boost vaccination sites were as close to the prime vaccination site as possible.
  • the injection volume was based on the dose to be administered.
  • the dose level amount refers explicitly to the amount of the SAM vector, i.e., it does not refer to other components, such as the LNP.
  • the ratio of LNP:SAM was approximately 24:1. Accordingly, the dose of LNP was 720 pg, 2400 pg, and 7200 pg for each respective GRT-R902/GRT-R904 dose level ( see below).
  • Ipilimumab is a human monoclonal IgGl antibody that binds to the cytotoxic T- lymphocyte associated antigen 4 (CTLA-4). Ipilimumab was formulated in solution at 5 mg/mL and was injected SC proximally (within ⁇ 2 cm) to each of the bilateral vaccination sites.
  • CTL-4 cytotoxic T- lymphocyte associated antigen 4
  • Ipilimumab was administered at a dose of 30 mg of antibody in four 1.5 mL (7.5 mg) injections proximal to the vaccine draining LN at each of the bilateral vaccination sites (i.e., 1.5 mL below the vaccination site and 1.5 mL above the vaccination site on each bilateral side in each deltoid, ventrogluteal, dorsogluteal, or rectus femoris [deltoid preferred, but dependent on clinical site and patient preference])
  • Nivolumab is a human monoclonal IgG4 antibody that blocks the interaction of PD-1 and its ligands, PD-L1 and PD-L2.
  • Nivolumab was formulated in solution at 10 mg/mL and was administered as an IV infusion (480 mg) through a 0.2-micron to 1.2-micron pore size, low-protein binding in-line filter at the protocol- specified doses. It was not administered as an IV push or bolus injection. Nivolumab infusion was promptly followed by a flush of diluent to clear the line.
  • Nivolumab was administered following each vaccination (i.e., each of GRT-C901, GRT-R902, GRT-C903, or GRT-R904) with or without ipilimumab on the same day.
  • the dose and route of nivolumab was based on the Food and Drug Administration approved dose and route.
  • Fig. 3A and Fig. 3B Mean sequencing read depth (mean target duplex read coverage [x]) for targets ranged from 2817x-5017x in cfDNA samples with >87% of targets (greater than 330 variants monitored) with >2000X duplex reads and >68% of targets with > 4000X duplex read (excluding D5D1 and D6D1).
  • the sequencing profile demonstrated high target coverage at high read depth.
  • Mutation allele frequency in cfDNA was monitored over the course of treatment for GRANITE patient Gl. As shown in Fig. 3C and Table 3, 117 mutant alleles out of greater than 330 subject and tumor- specific variants were monitored in the ctDNA of Gl.
  • Fig. 4A-C also shows the frequency of mutant alleles in ctDNA isolated from Gl over the course of disease.
  • Fig. 4A shows mutant allele frequency for 11 of 20 mutations detected at baseline.
  • Fig. 4B shows average mutant allele frequency.
  • Fig. 4C shows the percent change in the average mutant allele frequency.
  • VAF tumor-specific variant allele frequency
  • MAF mutant allele frequency
  • Duplex read coverage over the course of treatment for patient G2 is shown in Fig. 3D and Fig. 3E.
  • Mean read coverage for targets ranged from 3877x-4534x after consensus in cfDNA samples with >93% of targets (greater than 240 variants monitored) with >2000X duplex reads and >76% targets with > 3000X duplex reads.
  • the sequencing profile demonstrated high target coverage at high read depth.
  • Mutation allele frequency in cfDNA was monitored over the course of treatment for GRANITE patient G2. As shown in Fig. 3F, ctDNA was not detected above the lowest call threshold over the course of the treatment regimen for patient G2, correlating with a prolonged disease free period (no evidence of disease at any timepoint on study postsurgery). Accordingly, monitoring mutation allele frequency in cfDNA served as an effective non-invasive proxy for monitoring disease, including assessing the presence of a disease and disease burden.
  • Patient G8 demonstrated a continued decrease in mutant allele frequency, including loss of some variant detection (16 of 20 variants monitored detected), correlating with stable disease. Accordingly, monitoring mutation allele frequency in cfDNA served as an effective non-invasive proxy for monitoring status of disease, including assessing disease progression.
  • Duplex read coverage over the course of treatment for patient S 1 is shown in Fig. 6A and Fig. 6B.
  • Mean read coverage for targets ranged from 2728x-3660x after consensus in cfDNA samples with >98% of targets with >1000X duplex reads and >78% targets with > 2000X duplex reads.
  • the results demonstrate mutation allele frequency in cfDNA could be monitored over the course of treatment for large numbers of tumor and subject- specific mutations.
  • the results also demonstrated monitoring mutation allele frequency in cfDNA served as an effective non-invasive proxy for monitoring status of disease, including assessing disease progression, assessing the presence of a disease and disease burden, and the efficacy of a therapeutic regimen.

Abstract

L'invention divulgue des procédés et des compositions pour surveiller une charge mutationnelle, un état cancéreux, l'efficacité d'un vaccin à l'aide d'un séquençage d'ADN acellulaire. Sous certains aspects, le procédé comprend les étapes consistant : a. à obtenir ou avoir obtenu des données de séquençage d'ADN acellulaire (ADNa) à partir d'un échantillon provenant du sujet, les données de séquençage comprenant une couverture cible d'au moins 50 % de toutes les régions polynucléotidiques d'intérêt correspondant à des mutations présentes dans un exome du cancer, et les régions polynucléotidiques séquencées d'intérêt ayant une profondeur de lecture moyenne d'au moins 1000X.
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