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WO2025213151A1 - Procédés, kits et systèmes de diagnostic et de traitement du cancer - Google Patents

Procédés, kits et systèmes de diagnostic et de traitement du cancer

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Publication number
WO2025213151A1
WO2025213151A1 PCT/US2025/023341 US2025023341W WO2025213151A1 WO 2025213151 A1 WO2025213151 A1 WO 2025213151A1 US 2025023341 W US2025023341 W US 2025023341W WO 2025213151 A1 WO2025213151 A1 WO 2025213151A1
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Prior art keywords
adc
genomic loci
genomic
gene
target antigen
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Inventor
Jonathan BEAGAN
Jamey GUESS
Anthony D’IPPOLITO
Matthew EATON
Aparna Gorthi
Rehan VERJEE
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Precede Biosciences Inc
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Precede Biosciences Inc
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Publication of WO2025213151A1 publication Critical patent/WO2025213151A1/fr
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    • 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
    • C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
    • 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
    • C12Q2600/154—Methylation markers
    • 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
    • C12Q2600/158—Expression markers

Definitions

  • ADC molecules marry the precision of antibody-mediated tumor antigen targeting with potent cytotoxic agents, thereby creating a targeted delivery vehicle for malignant tumors.
  • ADCs provide a means to reduce off-tumor toxicities by limiting payload exposure in normal tissues. While most ADC clinical candidates utilize cytotoxic chemotherapeutic payloads, recent ADC candidates have also incorporated targeted small molecules and immunomodulatory agents. Since MylotargTM’s first registration, hundreds of ADCs have been evaluated in the investigational setting and several have made it to regulatory approval including Trodelvy®.
  • Target antigen expression is currently used as the primary biomarker for patient selection and ADC efficacy has been shown to correlate with the level of target antigen expression in some studies.
  • IHC methods for quantifying target antigen expression are invasive, requiring tissue biopsies.
  • Target assessment has also been challenging as a result of known challenges with IHC assessment/interpretation.
  • the present disclosure is based, at least in part, on the demonstration that certain genomic loci associated with select genes for ADC target antigens or genes that modulate ADC response/resistance have different histone modification levels (e.g., histone methylation marks such as H3K4me3 and histone acetylation marks such as H3K27ac) in plasma samples from cancer patients as compared to plasma samples from healthy volunteers.
  • histone modification levels e.g., histone methylation marks such as H3K4me3 and histone acetylation marks such as H3K27ac
  • the present disclosure encompasses methods, kits and systems that use these epigenomic differences (alone or in combination with each other and/or with other biomarkers) to select subjects for treatment with an agent that is directed to an ADC target antigen (e.g., an ADC therapy or radioligand), to identify subpopulations of subjects that respond to treatment with an agent that is directed to an ADC target antigen (e.g., an ADC therapy or radioligand), to monitor subjects during treatment with an agent that is directed to an ADC target antigen (e.g., an ADC therapy or radioligand), etc.
  • an ADC target antigen e.g., an ADC therapy or radioligand
  • cfDNA cell-free DNA
  • a liquid biopsy sample e.g., a plasma sample obtained or derived from a subject.
  • chromatin accessibility and/or binding of one or more transcription factors are detected at the one or more genomic loci instead of (or in addition to) histone modifications.
  • the one or more genomic loci are located within +/- 200 kB of the TSS of a gene encoding an ADC target antigen or a gene that modulates ADC response/resistance, e.g., a genomic locus that is located within a region defined by a pair of genomic coordinates in Table 4, Table 5, or Table 6.
  • the subject is small cell lung cancer (SCLC) patient and the gene encoding an ADC target antigen is selected from the group consisting of TACSTD2, FOLH1, NCAM1, ERBB3, CD276, MSLN, SEZ6, DLL3, and FAP.
  • SCLC small cell lung cancer
  • the subject is a non-small cell lung cancer (NSCLC) patient and the gene encoding an ADC target antigen is selected from the group consisting of TACSTD2, F3, NECTIN4, FOLR1, ERBB3, CD276, ERBB2, AXL, CEACAM5, TFRC, SLC34A2, PTK7, GPNMB, EGFR, MET and ROR2.
  • a subject is an NEPC or PRAD patient and the gene encoding an ADC target antigen is selected from the group consisting of AR, STEAP1, CHGA, SEZ6, SSTR2, DLL3, KLK2, and KLK3.
  • the gene encoding an ADC target gene is DLL3.
  • a method comprises quantifying, in a biological sample, at (a) one or more genomic loci (i) within DLL3; and/or (ii) associated with a promoter or an enhancer of DLL3; and (b) one or more genomic loci (i) within RAB3IP, PAK5, or ITPRIPL2, or any combination thereof; and/or (ii) associated with a promoter or an enhancer of RAB3IP, PAK5, or ITPRIPL2, or any combination thereof: (A) one or more histone modifications, ( B) DNA methylation, (C) chromatin accessibility, and/or (D) binding of one or more transcription factors.
  • the one or more histone modifications are quantified using a histone modification assay that measures one or more of H3K9ac, H3K14ac, H3K18ac, H3K23ac, H3K27ac, H3K4me1, H3K4me2, H3K4me3, and pan-acetylation.
  • histone modification assay detects H3K4me3 modifications.
  • the histone modification assay detects H3K27ac modifications.
  • the histone modification assay is selected from ChIP-seq (Chromatin ImmunoPrecipitation sequencing), CUT&RUN (Cleavage Under Targets and Release Using Nuclease) sequencing, and CUT&Tag (Cleavage Under Targets and Tagmentation) sequencing.
  • chromatin accessibility is quantified using a chromatin accessibility assay selected from ATAC-seq (Assay of Transpose Accessible Chromatin sequencing), NOMe-seq (Nucleosome Occupancy and Methylome sequencing), FAIRE-seq (Formaldehyde-Assisted Isolation of Regulatory Elements sequencing), MNase-seq (Micrococcal Nuclease digestion with sequencing), a DNase hypersensitivity assay, and a fragmentomics assay.
  • ATAC-seq Assay of Transpose Accessible Chromatin sequencing
  • NOMe-seq Nucleosome Occupancy and Methylome sequencing
  • FAIRE-seq Formmaldehyde-Assisted Isolation of Regulatory Elements sequencing
  • MNase-seq Merococcal Nuclease digestion with sequencing
  • DNase hypersensitivity assay a fragmentomics assay.
  • the method comprises quantifying two or more of the following, each at one or more of the genomic loci in cell-free DNA (cfDNA) from a liquid biopsy sample obtained or derived from the subject: (i) one or more histone modifications, (ii) 12621818v1 Page 8 of 147 Attorney Docket: 2014191-0036 DNA methylation, (iii) chromatin accessibility, and (iv) transcription factor binding.
  • the method comprises quantifying two or more histone modifications for the same gene.
  • the method comprises quantifying H3K4me3 and H3K27ac modifications for the same gene.
  • the quantified H3K4me3 and H3K27ac modifications are combined to generate an activation score for the gene. In some embodiments, the quantified H3K4me3 and H3K27ac modifications are summed, optionally with weighting, to generate an activation score for the gene.
  • the gene encodes an ADC target antigen. In some embodiments, the target antigen is TROP2. In some embodiments, the gene modulates ADC response/resistance.
  • the liquid biopsy sample is a plasma sample, serum sample, or urine sample. In some embodiments, the liquid biopsy sample is a plasma sample, optionally a 1 mL plasma sample.
  • quantification of one or more histone modifications, DNA methylation, chromatin accessibility, and/or binding of one or more transcription factors, at the one or more genomic loci as compared to a reference indicates whether an ADC target is being expressed and/or provides a prediction or measurement of the expression level of the ADC target.
  • the reference is a predetermined threshold, a measurement from a liquid biopsy sample, and/or a normalized value, optionally wherein the reference is a measurement from a liquid biopsy sample obtained from a cohort of subjects who have previously been determined to have undetectable or low expression of the ADC target based on IHC testing, or to be cancer free.
  • the reference is a predetermined threshold, a measurement from a liquid biopsy sample, and/or a normalized value, optionally wherein the reference is a measurement from a liquid biopsy sample obtained from a cohort of subjects who have previously been determined to have high expression of the ADC target based on IHC testing.
  • quantification of one or more histone modifications, DNA methylation, chromatin accessibility, and/or binding of one or more transcription factors, at the one or more genomic loci as compared to a reference indicates whether a gene that modulates ADC response/resistance is active.
  • the reference is a predetermined threshold, a measurement from a liquid biopsy sample, and/or a normalized value, optionally 12621818v1 Page 9 of 147 Attorney Docket: 2014191-0036 wherein the reference is a measurement from a liquid biopsy sample obtained from a cohort of subjects who have previously been determined to have low or undetectable activity of the gene that modulates ADC response/resistance.
  • the reference is a predetermined threshold, a measurement from a liquid biopsy sample, and/or a normalized value, optionally wherein the reference is a measurement from a liquid biopsy sample obtained from a cohort of subjects who have previously been determined to have high activity of a gene that modulates ADC response/resistance.
  • quantification of one or more histone modifications, DNA methylation, chromatin accessibility, and/or binding of one or more transcription factors, at the one or more genomic loci as compared to a reference indicates whether the subject is likely to respond to treatment with an ADC for the ADC target.
  • the reference is a predetermined threshold, a measurement from a liquid biopsy sample, and/or a normalized value, optionally wherein the reference is a measurement from a liquid biopsy sample obtained from a cohort of subjects who have previously been determined to not respond to treatment with the ADC for the ADC target.
  • the reference is a predetermined threshold, a measurement from a liquid biopsy sample, and/or a normalized value, optionally wherein the reference is a measurement from a liquid biopsy sample obtained from a cohort of subjects who have previously been determined to respond to treatment with the ADC for the ADC target.
  • the method comprises quantifying one or more histone modifications, DNA methylation, chromatin accessibility, and/or binding of one or more transcription factors at one or more genomic loci that are located within one or more regions defined by pairs of genomic coordinates in Table 1, Table 2, Table 3, Table 4, Table 5, or Table 6.
  • the method comprises quantifying H3K4me3 modifications for at least 1, 2, 3, 4, 5, 10, 15, 20, or 25 genomic loci that are located within one or more regions defined by pairs of genomic coordinates in Table 1. In some embodiments, the method comprises quantifying H3K4me3 modifications for at least 1, 2, 3, 4, 5, 10, 15, 20, or 25 genomic loci that are located within one or more regions defined by pairs of genomic coordinates in Table 2. In some embodiments, the method comprises quantifying H3K27ac modifications for at least 1, 2, 3, 4, 5, 10, 15, 20, or 25 genomic loci that are located within one or more regions defined by pairs of genomic coordinates in Table 3.
  • the method comprises quantifying H3K27ac or H3K4me3 modifications and/or DNA methylation for at least 12621818v1 Page 10 of 147 Attorney Docket: 2014191-0036 1, 2, 3, 4, 5, 10, 15, 20, or 25 genomic loci that are located within one or more regions defined by pairs of genomic coordinates in Table 4. In some embodiments, the method comprises quantifying H3K4me3 modifications for at least 1, 2, 3, 4, 5, 10, 15, 20, or 25 genomic loci that are located within one or more regions defined by pairs of genomic coordinates in Table 5.
  • the method comprises quantifying H3K27ac or H3K4me3 modifications for at least 1, 2, 3, 4, 5, 10, 15, 20, or 25 genomic loci that are located within one or more regions defined by pairs of genomic coordinates in Table 6.
  • the subject has previously been determined to have cancer.
  • the subject has previously been determined to have breast cancer, prostate cancer, or lung cancer.
  • the lung cancer is SCLC
  • the prostate cancer is PRAD or NEPC
  • the breast cancer is metastatic breast cancer (e.g., HR+/HER2- metastatic breast cancer).
  • the method further comprises administering an agent that is directed to an ADC target antigen (e.g., an ADC therapy or radioligand) to the subject.
  • an ADC target antigen e.g., an ADC therapy or radioligand
  • the agent that is directed to an ADC target antigen is selected at least in part based on a quantified level of (i) one or more histone modifications, (ii) DNA methylation, (iii) chromatin accessibility, and/or (iv) transcription factor binding at the one or more of the genomic loci in cell-free DNA (cfDNA) from a liquid biopsy sample obtained or derived from the subject.
  • the quantified level is indicative of activity at one or more genomic loci associated with a promoter or an enhancer of a gene encoding a first ADC target antigen and the selected agent that is directed to an ADC target antigen (e.g., ADC therapy or radioligand) targets the first ADC target antigen.
  • the quantified level is indicative of activity at one or more genomic loci associated with a promoter or an enhancer of a gene that modulates response/resistance to a first ADC and the selected agent that is directed to an ADC target antigen (e.g., ADC therapy or radioligand) does not include the first ADC.
  • the present disclosure provides a method of treating a subject having cancer with an agent that is directed to an ADC target antigen (e.g., an ADC therapy or radioligand), the method comprising administering an agent that is directed to an ADC target antigen (e.g., an ADC therapy or radioligand) to the subject that has been selected at least in part based on a quantified level, at one or more genomic loci, of: (i) one or more histone modifications, (ii) DNA methylation, (iii) chromatin accessibility, and/or (iv) transcription factor binding at one or more genomic loci in cell-free DNA (cfDNA) from a liquid biopsy sample obtained or derived from the subject, wherein the one or more genomic loci are (a) within a gene encoding an ADC target antigen or a gene that modulates ADC response/resistance and/or (b) are associated with a promoter or an enhancer of a gene encoding an ADC target antigen or
  • the one or more genomic loci are: (a) within a gene encoding an ADC target antigen or within a gene that modulates ADC response/resistance; and/or (b) are associated with a promoter or an enhancer of:(I) a gene that modulates ADC response/resistance; and/or (II) a gene encoding an ADC target antigen.
  • the present disclosure provides, a method of determining whether a subject is likely to respond to treatment with an agent that is directed to an ADC target antigen (e.g., an ADC therapy or radioligand), comprising quantifying, at one or more genomic loci in cell-free DNA (cfDNA) from a liquid biopsy sample obtained or derived from the subject:(i) one or more histone modifications, (ii) DNA methylation, (iii) chromatin accessibility, and/or (iv) binding of one or more transcription factors, wherein the one or more genomic loci are: (a) within a gene encoding an ADC target antigen or within a gene that modulates ADC response/resistance; and/or (b) are associated with a promoter or an enhancer of: (I) a gene that modulates ADC response/resistance; and/or (II) a gene encoding an ADC target antigen.
  • an ADC target antigen e.g., an ADC therapy or radioligand
  • the gene that modulates ADC response/resistance is selected from the group consisting of SH3GL1, HSP90AA1, SLC46A3, ABCB1, ABCG2, SLFN11, CCNB1, and TUBB3. 12621818v1 Page 12 of 147 Attorney Docket: 2014191-0036 [0045]
  • the quantification of (i) one or more histone modifications, (ii) DNA methylation, (iii) chromatin accessibility, and/or (iv) transcription factor binding at one or more of the genomic loci in cell-free DNA (cfDNA) from a liquid biopsy sample obtained or derived from the subject was performed in accordance with a method described herein.
  • the method further comprises quantifying (i) one or more histone modifications, (ii) DNA methylation, (iii) chromatin accessibility, and/or (iv) transcription factor binding at one or more of the genomic loci in cell-free DNA (cfDNA) from a liquid biopsy sample obtained or derived from the subject in accordance with a method described herein.
  • the quantified level is indicative of activity at one or more genomic loci associated with a promoter or an enhancer of a gene encoding a first ADC target antigen and the selected agent that is directed to an ADC target antigen (e.g., ADC therapy or radioligand) targets the first ADC target antigen.
  • an ADC target antigen e.g., ADC therapy or radioligand
  • the quantified level is indicative of activity at one or more genomic loci associated with a promoter or an enhancer of a gene that modulates response/resistance to a first ADC and the selected ADC therapy does not include the first ADC.
  • an ADC target antigen e.g., an ADC therapy or radioligand
  • the method comprising quantifying (i) one or more histone modifications, (ii) DNA methylation, (iii) chromatin accessibility, and/or (iv) transcription factor binding at one or more of the genomic loci in cell-free DNA (cfDNA) from a liquid biopsy sample obtained or derived from the subject in accordance with a method described herein at first and second time points during the treatment period.
  • the present disclosure provides a kit comprising reagents for quantifying one or more histone modifications, DNA methylation, chromatin accessibility, and/or binding of one or more transcription factors at one or more genomic loci, wherein the one or more genomic loci are located within one or more regions defined by pairs of genomic coordinates in Table 1, Table 2, Table 3, Table 4, Table 5, or Table 6.
  • the kit comprises reagents for quantifying H3K4me3 modifications for at least 1, 2, 3, 4, 5, 10, 15, 20, or 25 genomic loci that are located within one or more regions defined by pairs of genomic coordinates in Table 1.
  • the kit comprises reagents for quantifying H3K4me3 modifications for at least 1, 2, 3, 4, 5, 10, 15, 20, or 25 genomic loci that are located within one or more regions defined by pairs of genomic coordinates in Table 5. In some embodiments, the kit comprises reagents for quantifying H3K4me3 or H3K27ac modifications for at least 1, 2, 3, 4, 5, 10, 15, 20, or 25 genomic loci that are located within one or more regions defined by pairs of genomic coordinates in Table 6. In some embodiments, the kit comprises one or more antibodies for use in ChIP-seq, optionally wherein the one or more antibodies specifically bind H3K4me3- or H3K27ac-modified histones.
  • the kit comprises reagents for isolation of cell-free DNA (cfDNA) from a liquid biopsy sample. In some embodiments, the kit comprises reagents for library preparation for sequencing. In some embodiments, kit comprises reagents for sequencing. [0048] In one aspect the present disclosure provides a non-transitory computer readable storage medium encoded with a computer program, wherein the program comprises instructions that when executed by one or more processors cause the one or more processors to perform operations to perform a method described herein.
  • the present disclosure provides a computer system comprising a memory and one or more processors coupled to the memory, wherein the one or more processors are configured to perform operations to perform a method described herein.
  • the present disclosure provides a system comprising a sequencer configured to generate a sequencing dataset from a sample; and the non-transitory computer readable storage medium and/or the computer system.
  • the sequencer is configured to generate a Whole Genome Sequencing (WGS) dataset from the sample.
  • the system further comprises a sample preparation device configured to prepare the sample for sequencing from a biological sample, optionally a liquid biopsy sample.
  • the sample preparation device comprises reagents for quantifying one or more histone modifications, DNA methylation, chromatin accessibility, and/or binding of one or more transcription factors at one or more genomic loci in cell-free DNA (cfDNA) from the biological sample, optionally the liquid biopsy sample.
  • the device comprises reagents for quantifying one or more histone modifications, DNA methylation, chromatin accessibility, and/or binding of one or more transcription factors at one or more genomic loci that are located within one or more regions defined by pairs of genomic coordinates in Tables 1-6 .
  • the device comprises reagents for quantifying H3K27ac or H3K4me3 modifications or DNA methylation for at least 1, 2, 3, 4, 5, 10, 15, 20, or 25 genomic loci that are located within one or more regions defined by pairs of genomic coordinates in Table 4. In some embodiments, the device comprises reagents for quantifying H3K4me3 modifications for at least 1, 2, 3, 4, 5, 10, 15, 20, or 25 genomic loci that are located within one or more regions defined by pairs of genomic coordinates in Table 5.
  • the device comprises reagents for quantifying H3K27ac or H3K4me3 modifications for at least 1, 2, 3, 4, 5, 10, 15, 20, or 25 12621818v1 Page 15 of 147 Attorney Docket: 2014191-0036 genomic loci that are located within one or more regions defined by pairs of genomic coordinates in Table 6.
  • the reagents comprise one or more antibodies for use in ChIP- seq, optionally wherein the one or more antibodies specifically bind H3K4me3- or H3K27ac- modified histones.
  • the device comprises reagents for isolation of cell-free DNA (cfDNA) from the biological sample, optionally the liquid biopsy sample.
  • the device comprises reagents for library preparation for sequencing.
  • the sequencer comprises reagents for sequencing.
  • BRIEF DESCRIPTION OF THE DRAWING [0051]
  • Fig. 1 (A) shows a qualitative track view displaying promoter (H3K4me3) signals across breast cancer-relevant ADC target antigens (plus housekeeping genes GAPDH and ACTB). Rows represent select breast cancer patient plasma samples sorted by ichorCNA ctDNA fraction estimate or healthy volunteer plasma samples.
  • Fig. 1 (B) shows a quantitative heatmap of normalized and ctDNA-corrected epigenomic activation score (combining enhancer and promoter signals) for each ADC target antigen.
  • FIG. 9 shows a quantitative heatmap of normalized and ctDNA-corrected epigenomic activation score (combining enhancer and promoter signals) for each ADC target antigen. Patient-specific epigenomic activation scores for each gene are represented on a white (low) to dark (high) gradient.
  • Figs. 10-18 show graphs showing trend lines and confidence intervals for promoter signals (H3K4me3; (A) in each of Figs. 10-18) and enhancer signals (H3K27ac; (B) in each of Figs.
  • Fig. 19 (A) shows a qualitative track view displaying promoter (H3K4me3) signals across NSCLC-relevant ADC target antigens (plus housekeeping genes GAPDH and 12621818v1 Page 16 of 147 Attorney Docket: 2014191-0036 ACTB). Rows represent select NSCLC patient plasma samples sorted by ichorCNA ctDNA fraction estimate or healthy volunteer plasma samples.
  • Fig. 19 (B) shows a quantitative heatmap of normalized and ctDNA-corrected epigenomic activation score (combining enhancer and promoter signals) for each ADC target antigen.
  • Figs. 20-35 show graphs showing trend lines and confidence intervals for promoter signals (H3K4me3; (A) in each of Figs.20-35) and enhancer signals (H3K27ac; (B) in each of Figs.20-35(B)) based on ctDNA% for individual NSCLC-relevant ADC target antigens.
  • Figs. 36-51 show data obtained for breast cancer-relevant ADC target antigens using breast cancer patient plasma samples and healthy volunteer plasma samples.
  • (B) shows a quantitative heatmap of normalized and ctDNA-corrected epigenomic activation score (combining enhancer and promoter signals) for the indicated ADC target antigen compared to the other transcripts of interest in breast cancer (HER2, FOXA1 and MYC).
  • Patient-specific epigenomic activation scores for each gene are represented on a gradient (regressed out and scaled to the min and the max of the ADC target antigen in these samples).
  • (C) and (D) in each figure show graphs showing trend lines and confidence intervals for the promoter signal (H3K4me3) and enhancer signal (H3K27ac), respectively, relative to ctDNA% for the indicated ADC target antigen.
  • FIG. 56 shows graphs predicting expression of clinically relevant drug targets in breast cancer.
  • RNA expression (as measured by RNA-seq) and plotted 12621818v1 Page 18 of 147 Attorney Docket: 2014191-0036 on the y-axis is predicted RNA expression (predicted using cfDNA epigenomic features).
  • C Shows graphs showing the stability of predictions across varying ctDNA fractions for certain genes. Plotted on the x-axis of each graph is tumor (ctDNA) fraction. Plotted on the y-axis of each graph is the Spearman correlation coefficient between RNA expression (as measured by RNA-seq) and predicted expression (as measured using epigenomic features).
  • Fig. 58 provides a graph depicting correlation between certain loci and DLL3 expression at varying ctDNA%.
  • “Pearson’s r” refers to the Pearson correlation coefficient determined by comparing predicted expression (as determined using epigenetic modifications) in (a) plasma samples having a high ctDNA fraction (30% ctDNA), and (b) the same samples serially diluted in silico with healthy plasma sequencing data.
  • “Single Gene Model” (purple data points, connected by the bottom purple line) refers to prediction values generated using a model that incorporates only loci that are proximal to the DLL3 locus.
  • “Multigene Model” black points connected by the top black line) refers to prediction values determined using a model that incorporates loci that are proximal to DLL3 and several additional genes whose expression is correlated with that of DLL3.
  • FIG. 1 Shows a combined DLL3, SEZ6, CHGA score, calculated by taking the mean of the promoter (H3K4me3) signal measured in a promoter region associated with each gene.
  • Circled is a group of patients 12621818v1 Page 19 of 147 Attorney Docket: 2014191-0036 having a high DLL3/SEZ6/CHGA score despite having been diagnosed with PRAD, indicating that these patients may have been misdiagnosed.
  • left box-and-whiskers bar indicates signal measured in NEPC subjects
  • right box-and-whiskers bar indicates signal measured in PRAD subjects, and each point represents a measurement from an individual patient.
  • the present disclosure is based, at least in part, on the demonstration that certain genomic loci associated with select genes for ADC target antigens or genes that modulate ADC response/resistance have different histone modification levels (e.g., histone methylation marks such as H3K4me3 and histone acetylation marks such as H3K27ac) in plasma samples from cancer patients as compared to plasma samples from healthy volunteers.
  • histone modification levels e.g., histone methylation marks such as H3K4me3 and histone acetylation marks such as H3K27ac
  • the present disclosure encompasses methods, kits and systems that use these epigenomic differences (alone or in combination with each other and/or with other biomarkers) to select subjects for treatment with an agent that is directed to an ADC target antigen (e.g., an ADC therapy or radioligand), to identify subpopulations of subjects that respond to ADCs, to monitor subjects during treatment with an agent that is directed to an ADC target antigen (e.g., an ADC therapy or radioligand), etc. by detecting and quantifying the presence of histone modifications at these one or more genomic loci in cell-free DNA (cfDNA) from a liquid biopsy sample, e.g., a plasma sample obtained or derived from a subject.
  • cfDNA cell-free DNA
  • Sacituzumab govitecan is an ADC that includes the TROP2-targeted antibody sacituzumab conjugated to the chemotherapeutic agent SN-38, an active metabolite of irinotecan, a TOP1 inhibitor (DNA targeting).
  • Enfortumab vedotin is an ADC that includes the NECTIN4-targeted antibody enfortumab conjugated to the chemotherapeutic agent Monomethyl auristatin E (MMAE) (microtubule targeting).
  • MMAE Monomethyl auristatin E
  • Trastuzumab emtansine is an ADC that includes the HER2-targeted antibody trastuzumab conjugated to the chemotherapeutic agent DM1, a maytansinoid derivative (microtubule targeting). It is used by itself to treat early-stage breast cancer after surgery (when 12621818v1 Page 21 of 147 Attorney Docket: 2014191-0036 chemotherapy and trastuzumab were given before surgery, and there was cancer still present at the time of surgery), or to treat advanced breast cancer in women who have already been treated with trastuzumab and chemotherapy. This therapeutic agent is administered intravenously.
  • Trastuzumab deruxtecan is an ADC that includes the HER2-targeted antibody trastuzumab conjugated to the chemotherapeutic agent deruxtecan, which is a Topoisomerase I (TOP1) inhibitor (DNA targeting) similar to irinotecan. It can be used by itself to treat breast cancer that cannot be removed with surgery or that has spread (metastasized) to another part of the body, typically after at least one other HER2-targeted agent has been tried. This therapeutic agent is administered intravenously.
  • TOP1 Topoisomerase I
  • Trastuzumab deruxtecan can also be used to treat HER2-low breast cancers that cannot be removed with surgery or that have spread to another part of the body, typically after chemotherapy has been tried or if the cancer recurs within 6 months of finishing adjuvant chemotherapy.
  • Tisotumab vedotin is an ADC that includes the Tissue factor-targeted antibody tisotumab conjugated to the chemotherapeutic agent MMAE (microtubule targeting).
  • Mirvetuximab soravtansine-gynx is an ADC that includes the FOLR1-targeted antibody mirvetuximab conjugated to the chemotherapeutic agent DM4, a maytansinoid derivative (microtubule targeting).
  • ADCs currently in development include ADCs targeting ROR1 (e.g., MK-2140, NBE-002, and CS5001), CRC2 (e.g., TAK-500), CD44 (e.g., bivatuzumab mertansine (BIWI 1), which targets CD44v6), CLDN6 (e.g., TOLR-1-32, and DS-9606), TNFRSF10B (e.g., Oba01), FN-1 (e.g., PYX-201), FGFR2 (e.g., BAY 1187982), GD2 (e.g., M3554), GPC1, GRPR, ITGN6 (e.g., SGN-B6A), IGF1R (e.g., W0101), MUC1 (e.g., MUC1-C-ADC and DS-3939), CDH3 (e.g., PCA062), PD-L1 (e.g.,
  • TAK-500 is a CCR2-binding ADC that comprises a STING agonist dazostinag, and is designed to target myeloid cells that express CCR2, thereby inducing activation of IFN 12621818v1 Page 22 of 147 Attorney Docket: 2014191-0036 response, reprogramming of suppressive intratumoral CCR2+ myeloid cells, and blockade of suppressive tumor-associated macrophage recruitment.
  • Bivatuzumab mertansine (BIWI 1) comprises a monoclonal antibody targeting isoform 6 of CD44 (CD44v6) conjugated to a thiol-containing maytansinoid (a highly potent anti-microtubule agent).
  • TORL-1-32 comprises an MMAE moiety conjugated to an anti-CLDN6 fully humanized monoclonal antibody.
  • TORL-1-32 ADC has a favorable safety/tolerability profile and pharmacokinetic (PK) characteristics and has demonstrated preliminary antitumor activity in patients with heavily-pretreated CLDN6-expressing ovarian and testicular cancers (see, e.g., Konecny et al., ESMO Annals of Oncol. 2024. 54(S2, S551)).
  • DS-9606 comprises a humanized anti-CLDN6 antibody conjugated to modified pyrrolobenzodiazepine.
  • DS-9606 has also shown favorable safety and preliminary efficacy results in ovarian, gastric, NSCLC, giant cell tumor, breast, and endometrial cancer patients.
  • PYX-201 comprises a fully human anti-extra-domain B splice variant of fibronectin antibody, a cleavable mcValCitPABC linker, and four Auristatin 0101 (Aur0101, PF- 06380101) payload molecules.
  • PYX-201 has been granted a Food and Drug Administration (FDA) fast track designation for the treatment of adult patients with recurrent or metastatic head and neck squamous cell carcinoma (R/M HNSCC) whose disease has progressed following treatment with platinum-based chemotherapy and an anti-PD-(L)1 antibody.
  • FDA Food and Drug Administration
  • PYX-201 was shown to inhibit tumor growth in patient-derived xenografts in mice by 90% or more (Severe et al., Cancer Res.2024.84(6_Supplement)). 12621818v1 Page 23 of 147 Attorney Docket: 2014191-0036 [0078]
  • BAY 1187982 (Aprutumab ixadotin) comprises a fully human anti-FGFR2 monoclonal antibody linked to an auristatin W derivative, toxophore. Aprutumab ixadotin was found to be poorly tolerated in a phase I, first-in-human trial in patients with advanced solid tumors from cancer indications known to be FGFR2-positive.
  • 212 Pb-GRPR is a radioligand being developed for therapeutic and diagnostic (e.g., theragnostic) purposes.
  • 212 Pb-GRPR comprises a lead-212 radioisotope conjugated to a GRPR- targeting peptide, GRPR1 (see Saidi et al., J Nucl Med. 2024. 165(11)).
  • SGN-B6A is an ITGB6-directed vedotin ADC that comprises a monomethyl auristatin E (MMAE) payload.
  • MMAE monomethyl auristatin E
  • SGN-B6A demonstrated a manageable safety profile, and preliminary antitumor activity and response durability in dose escalation in a heavily pretreated patient population (see Hollebecque et al., J Clin Oncol.2023.41(16_Supplement)).
  • MUC1-targeting ADCs constitute an area of high interest for targeting stromal and difficult to treat tumors, and are likely to be expanded to various metastatic cancer 12621818v1 Page 24 of 147 Attorney Docket: 2014191-0036 indications in future clinical trials.
  • MUC1-C-ADC comprises an anti-glycosylated Mucin 1 (MUC1) monoclonal antibody (targeting the extracellular domain of MUC1), conjugated to MMAE via vc-PAB linkers.
  • DS-3939 comprises a humanized anti- TA-MUC1 antibody attached to a number of topoisomerase I inhibitor payloads (an exatecan derivative, DXd) via tetrapeptide-based cleavable linkers.
  • PCA062 is a P-cadherin-targeting ADC, and has been evaluated in HNSCC, EC, and triple-negative, Cadherin-3 positive breast cancer patients for safety and preliminary efficacy. High incidence of adverse events and limited antitumor activity led to termination of the clinical trial evaluating PCA062.
  • PF-08046054 / SGN-PDL1V is an investigational ADC comprising a monoclonal anti-PD-L1 antibody conjugated to MMAE via a protease-cleavable mc-vc (maleimidocaproyl- valine-citrulline) linker.
  • PEN-221 is an emtansine-conjugated, SSTR2-targeting ADC, which has been investigated in a Phase 2 clinical trial in a patient population with advanced GI mid-gut neuroendocrine tumors.
  • DXC008 is another STEAP1-targeting ADC, comprising an antibody having high binding affinity to PSMA conjugated to a tubulysin B analogue.
  • H1H7814N is an early-development ADC comprising an anti-STEAP2 monoclonal antibody that is conjugated to either auristatin or a maytansinoid.
  • H1H7814N has shown preliminary anti-tumor activity in murine models of prostate cancer, when compared to an unconjugated anti-STEAP2 antibody.
  • AGX101 is a first-in-class anti-TM4SF1 ADC comprising a conjugated tubulin inhibitor, and targets nuclear membranes of cancer cells to deliver payloads directly to the nucleus.
  • AGX101 has shown potent anti-tumor activity in colon and pancreatic cancer murine xenografts and a favorable safety profile in animal studies (see Jaminet et al. J Clin Oncol. 2024. 43 (4_Supplement)).
  • a phase 1 clinical trial is currently ongoing to test the safety and efficacy of AGX101 in patients with unresectable, locally advanced, or metastatic solid tumors.
  • Exemplary agents targeting DLL3 include ADCs (e.g., Rova-T, an ADC comprising a DLL3-specific humanized monoclonal antibody (SC16) conjugated to a membrane-permeable pyrrolobenzodiazepine (PBD) dimer toxin (warhead) via a lysosomal, protease-sensitive dipeptide linker; and SC-002, a bioengineered, DLL3-directed antibody-drug conjugate (ADC) that delivers the cytotoxic pyrrolobenzodiazepine (PBD) dimer warhead, SC-DR002, to cells expressing DLL3).
  • a method of the present disclosure predicts high levels of ADC target expression (relative to a reference) and/or detects an increase in ADC target expression (e.g., relative to a prior measurement during treatment) this may be used to select an ADC or radioligand for that ADC target as an ADC therapy radioligand for a subject.
  • ADC selection is based on an assessment of ADC target status using a method of the present disclosure in combination with IHC testing.
  • a “gene that modulates ADC response/resistance,” refers to a gene encoding a protein that has an activity that can modulate ADC efficacy.
  • proteins having an activity that can modulate ADC response/resistance include proteins that can interfere with ADC internalization, proteins that can interfere with pathway signaling, proteins that increase stability of an ADC target antigen, proteins involved in lysosomal transport, proteins involved in a DNA damage repair pathway, and proteins that function as a drug efflux pump.
  • genes that modulate ADC response/resistance include SH3GL1 (Endophilin A2), HSP90AA1 (HSP90), SLC46A3, CCNB1 (Cyclin B1), SLFN11, ABCB1 (MDR1), ABCG2 (BCRP), and TUBB3 (Class III beta-tubulin).
  • epigenomic differences (alone or in combination with each other and/or with other biomarkers) associated with these genes may be used in combination with epigenomic differences (or other biomarkers, e.g., HER2 IHC test results) associated with a gene for an ADC target antigen (e.g., ERRB2 gene encoding HER2).
  • Endophilin A2 is a scaffolding protein involved in clathrin-independent endocytosis. Reduced EndophilinA2 expression has been shown to decrease HER2 internalization, reduce downstream signaling, and decrease the migratory potential of the BRCA cells.
  • a decrease in Endophilin A2 results in reduced cytotoxicity of HER2-targeted ADCs (e.g., trastuzumab emtansine) against HER2-positive cells. See Baldassarre et al., Breast Cancer Res (2017) 19:110 which is incorporated herein by reference in its entirety.
  • HER2-targeted ADCs e.g., trastuzumab emtansine
  • HER2-targeted ADCs e.g., trastuzumab emtansine
  • a method of the present disclosure predicts high levels of Endophilin A2 expression (relative to a reference) and/or detects an increase in Endophilin A2 expression (e.g., relative to a prior measurement during treatment) this may be used to select a HER2-targeted ADC (e.g., trastuzumab emtansine) as an ADC therapy for a subject.
  • the selection is based in part on detecting or quantifying a level of activity (relative to a reference) and/or detecting an increase or decrease in the level of activity (e.g., relative to a prior measurement during treatment) at one or more genomic loci associated with a promoter or an enhancer of SH3GL1.
  • HER2-targeted ADC selection is also based on an assessment of HER2 status using a method of the present disclosure and/or based on IHC testing.
  • HSP90 is a chaperone protein that is important for HER2 stability (by preventing ubiquitination). Increase in HSP90 results in more stable HER2, reduced internalization and reduced ADC efficacy. See Li et al., Cancer Discov (2020) 10(5):674-687 which is incorporated herein by reference in its entirety.
  • a method of the present disclosure predicts low levels of HSP90 expression (relative to a reference) and/or detects a decrease in HSP90 expression (e.g., relative to a prior measurement during treatment) this may be used to select a HER2-targeted ADC (e.g., trastuzumab emtansine) as an ADC therapy for a subject.
  • a HER2-targeted ADC e.g., trastuzumab emtansine
  • a method of the present disclosure predicts high levels of 12621818v1 Page 28 of 147 Attorney Docket: 2014191-0036 HSP90 expression (relative to a reference) and/or detects an increase in HSP90 expression (e.g., relative to a prior measurement during treatment) this may be used to select a therapy other than a HER2-targeted ADC for a subject.
  • the selection is based in part on detecting or quantifying a level of activity (relative to a reference) and/or detecting an increase or decrease in the level of activity (e.g., relative to a prior measurement during treatment) at one or more genomic loci associated with a promoter or an enhancer of HSP90AA1.
  • HER2-targeted ADC selection is also based on an assessment of HER2 status using a method of the present disclosure and/or based on IHC testing.
  • SLC46A3 is a lysosomal membrane transporter protein relevant for efflux of maytansine ⁇ based catabolites from the lysosome. Loss/decrease in SLC46A3 has been shown to lead to accumulation of maytansine ⁇ based catabolites in lysosomes resulting in resistance to the HER2-targeted ADC trastuzumab emtansine. See Li et al., Mol Cancer Ther (2016) 17(7):1441- 1453 which is incorporated herein by reference in its entirety.
  • HER2-targeted ADC selection is also based on an assessment of HER2 status using a method of the present disclosure and/or based on IHC testing.
  • Cyclin B1 is essential for mitosis/ mitotic catastrophe.
  • Reduced CCNB1 has been shown to result in resistance to the HER2-targeted ADC trastuzumab emtansine. See Sabbaghi et al., Clin Cancer Res (2017) 23(22):7006-7019 which is incorporated herein by reference in its entirety.
  • a method of the present disclosure predicts low levels of Cyclin B1 expression (relative to a reference) and/or detects a decrease in Cyclin B1 expression 12621818v1 Page 29 of 147 Attorney Docket: 2014191-0036 (e.g., relative to a prior measurement during treatment) this may be used to select a therapy other than a HER2-targeted ADC for a subject.
  • a method of the present disclosure predicts high levels of Cyclin B1 expression (relative to a reference) and/or detects an increase in Cyclin B1 expression (e.g., relative to a prior measurement during treatment) this may be used to select a HER2-targeted ADC (e.g., trastuzumab emtansine) as an ADC therapy for a subject.
  • the selection is based in part on detecting or quantifying a level of activity (relative to a reference) and/or detecting an increase or decrease in the level of activity (e.g., relative to a prior measurement during treatment) at one or more genomic loci associated with a promoter or an enhancer of CCNB1.
  • HER2-targeted ADC selection is also based on an assessment of HER2 status using a method of the present disclosure and/or based on IHC testing.
  • SLFN11 is a DNA/RNA helicase important in DNA damage response and replication stress.
  • SLFN11 sensitizes cells to a broad range of anti-cancer drugs including platinum derivatives (cisplatin and carboplatin), inhibitors of topoisomerases (camptothecin, irinotecan, topotecan, doxorubicin, daunorubicin, mitoxantrone and etoposide), DNA synthesis inhibitors (gemcitabine, cytarabine, hydroxyurea and nucleoside analogues), and poly(ADPribose) polymerase (PARP) inhibitors (olaparib, rucaparib, niraparib and talazoparib).
  • platinum derivatives cisplatin and carboplatin
  • inhibitors of topoisomerases camptothecin, irinotecan, topotecan, doxorubicin, daunorubicin, mitoxantrone and etoposide
  • DNA synthesis inhibitors gemcitabine, cytarabine
  • a method of the present disclosure predicts high levels of SLFN11 expression (relative to a reference) and/or detects an increase in SLFN11 expression (e.g., relative to a prior measurement during treatment) this may be used to select an ADC with one of the aforementioned anti-cancer drugs, e.g., an ADC with a TOP1 inhibitor payload (e.g., an ADC with an camptothecin, topotecan, or irinotecan payload) for a subject.
  • an ADC with a TOP1 inhibitor payload e.g., an ADC with an camptothecin, topotecan, or irinotecan payload
  • the selection is based in 12621818v1 Page 30 of 147 Attorney Docket: 2014191-0036 part on detecting or quantifying a level of activity (relative to a reference) and/or detecting an increase or decrease in the level of activity (e.g., relative to a prior measurement during treatment) at one or more genomic loci associated with a promoter or an enhancer of SLFN11.
  • Multi-Drug Resistance Protein 1 is a drug efflux pump with MMAE as a substrate. Increased expression of MDR1 results in reduced sensitivity to ADCs with MMAE payloads.
  • a method of the present disclosure predicts high levels of BCRP expression (relative to a reference) and/or detects an increase in BCRP expression (e.g., relative to a prior measurement during treatment) this may be used to combine ADC therapy with a BCRP inhibitor, e.g., Ko143 (see Allen et al., Mol Cancer Ther (2002) 1:417-425), YHO-13351 (see Yamazaki al., Mol Cancer Ther (2011) 10:1252-1263), FTC (see Rabindran et al., Cancer Res (2000) 60:47-50), cyclosporine A (see Qadir et al., Clin Cancer Res (2005) 11:2320-2326, or GF120918 (see de Bruin et al., Cancer Lett (1999) 146:117-12
  • the selection is based in part on detecting or quantifying a level of activity (relative to a reference) and/or detecting an increase or decrease in the level of activity (e.g., relative to a prior measurement during treatment) at one or more genomic loci associated with a promoter or an enhancer of ABCG2. 12621818v1 Page 31 of 147 Attorney Docket: 2014191-0036 [0104]
  • Class III beta-tubulin is a microtubule protein. Increased expression is associated with resistance to microtubule-targeting agents (e.g., MMAE). See Kanakkanthara and Miller, Biochim Biophys Rev Cancer (2021) 1876(2):188607 which is incorporated herein by reference in its entirety.
  • a method of the present disclosure predicts low levels of Class III beta-tubulin expression (relative to a reference) and/or detects a decrease in Class III beta-tubulin expression (e.g., relative to a prior measurement during treatment) this may be used to select an ADC with microtubule-targeting agent (e.g., MMAE) payload for a subject.
  • microtubule-targeting agent e.g., MMAE
  • a method of the present disclosure predicts high levels of Class III beta-tubulin expression (relative to a reference) and/or detects an increase in Class III beta- tubulin expression (e.g., relative to a prior measurement during treatment) this may be used to select an ADC with a payload other than a microtubule-targeting agent (e.g., MMAE) for a subject.
  • the selection is based in part on detecting or quantifying a level of activity (relative to a reference) and/or detecting an increase or decrease in the level of activity (e.g., relative to a prior measurement during treatment) at one or more genomic loci associated with a promoter or an enhancer of TUBB3.
  • a sample analyzed using methods, kits and systems provided herein can be any biological sample including any processed sample that includes circulating tumor DNA (ctDNA) derived from a biological sample.
  • a sample analyzed using methods, kits and systems provided herein can be a sample obtained from a mammalian subject.
  • a sample analyzed using methods, kits and systems provided herein can be a sample obtained from a human subject.
  • a human subject is a subject diagnosed or seeking diagnosis as having, diagnosed as, or seeking diagnosis as at risk of having, and/or diagnosed as or seeking diagnosis as at immediate risk of having cancer, e.g., breast cancer, small cell lung cancer (SCLC) or non-small cell lung cancer (NSCLC), neuroendocrine prostate cancer (NEPC), prostate adenocarcinoma (PRAD), etc.
  • a human subject is a subject identified as needing treatment with an agent that is directed to an ADC target antigen (e.g., an ADC therapy or radioligand).
  • an ADC target antigen e.g., an ADC therapy or radioligand
  • a human subject is a subject identified as needing ADC therapy or radioligand screening by a medical practitioner.
  • a subject may not have undergone previous treatments for cancer, such as the treatments recited in this disclosure. In other embodiments, the subject has undergone previous treatments for cancer, such as the treatments recited in this disclosure. [0108] In various embodiments a subject has one or more biomarkers and/or risk factors for cancer, e.g., breast cancer, small cell lung cancer (SCLC) or non-small cell lung cancer (NSCLC), neuroendocrine prostate cancer (NEPC), prostate adenocarcinoma (PRAD), etc.
  • biomarkers and/or risk factors for cancer e.g., breast cancer, small cell lung cancer (SCLC) or non-small cell lung cancer (NSCLC), neuroendocrine prostate cancer (NEPC), prostate adenocarcinoma (PRAD), etc.
  • a human subject is identified as in need of ADC therapy or radioligand screening based on an initial cancer diagnosis, e.g., a breast cancer, small cell lung cancer (SCLC) or non-small cell lung cancer (NSCLC), neuroendocrine prostate cancer (NEPC), prostate adenocarcinoma (PRAD), etc. diagnosis.
  • a human subject is a subject not yet diagnosed as having, not at risk of having, not at immediate risk of having, not diagnosed as having, and/or not seeking diagnosis for a cancer. Genetic factors may also contribute to cancer risk, as evidenced by individuals with a family history of cancer.
  • a sample from a subject can be obtained from a liquid biopsy.
  • a sample and/or reference is obtained from serum, plasma, or urine.
  • the sample is serum.
  • a sample comprises circulating tumor DNA (ctDNA).
  • ctDNA circulating tumor DNA
  • a sample is derived from about 1 mL of blood obtained from the subject.
  • a sample is derived from about 0.5-2 mL of blood obtained from the subject, e.g., about 0.5 to 1.75 mL, about 0.5 to 1.5 mL, about 0.75 to 1.25 mL or about 0.9 to 1.1 mL of blood.
  • a sample is a sample of cell-free DNA (cfDNA).
  • cfDNA is typically found in human biofluids (e.g., plasma, serum, or urine) in short, double- stranded fragments.
  • the concentration of cfDNA is typically low, but can significantly increase under particular conditions, including without limitation pregnancy, autoimmune disorders, myocardial infarction, and cancer.
  • Circulating tumor DNA (ctDNA) is the component of cell- free DNA specifically derived from cancer cells.
  • ctDNA can be present in human biofluids bound to leukocytes and erythrocytes or not bound to leukocytes and erythrocytes.
  • Various tests for detection of tumor-derived ctDNA are based on detection of genetic or epigenetic modifications that are characteristic of cancer (e.g., of a relevant cancer).
  • Genetic or epigenetic factors characteristic of cancer can include, without limitation, oncogenic or cancer-associated mutations in tumor-suppressor genes, activated oncogenes, chromosomal disorders, histone 12621818v1 Page 33 of 147 Attorney Docket: 2014191-0036 modifications (e.g., histone methylation and/or histone acetylation), chromatin accessibility, binding of one or more transcription factors and/or DNA methylation.
  • ctDNA comprises less than 30%, less than 20%, or less than 10% of the cfDNA in the liquid biopsy sample obtained from the subject, e.g., less than 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or less than 1% of the cfDNA in the sample.
  • the percentage of ctDNA in the liquid biopsy sample is assessed using ichorCNA which estimates the percentage of ctDNA in a sample probabilistically (see Adalsteinsson et al., Nat Commun (2017) 8(1):1324 the entire contents of which are incorporated herein by reference).
  • Nucleic acids can be isolated using, without limitation, standard DNA purification techniques, by direct gene capture (e.g., by clarification of a sample to remove assay-inhibiting agents and capturing a target nucleic acid, if present, from the clarified sample with a capture agent to produce a capture complex and isolating the capture complex to recover the target nucleic acid).
  • direct gene capture e.g., by clarification of a sample to remove assay-inhibiting agents and capturing a target nucleic acid, if present, from the clarified sample with a capture agent to produce a capture complex and isolating the capture complex to recover the target nucleic acid.
  • samples can be collected from individuals repeatedly over a period of time (e.g., once daily, weekly, monthly, annually, biannually, etc.). In various embodiments, such samples can be used to verify results from earlier detections and/or to identify an alteration in biological pattern because of, for example, disease progression, resistance to therapy, treatment, remission, and the like.
  • subject samples can be 12621818v1 Page 34 of 147 Attorney Docket: 2014191-0036 taken and monitored every month, every two months, or combinations of one, two, or three- month intervals according to the present disclosure.
  • samples can be collected for monitoring over time beginning at or at certain clinically determined stages, such as at resistance to a therapy, before radiographic progression, after radiographic progression, and/or at tissue biopsy.
  • results from samples obtained at different points in time can be conveniently compared with each other, as well as with those of normal controls during the monitoring period, thereby providing the subject’s own values, as an internal, or personal, control for long-term monitoring.
  • Samples include materials prepared by processes including, without limitation, steps such as concentration, dilution, adjustment of pH, removal of high abundance polypeptides (e.g., albumin, gamma globulin, and transferrin, etc.), addition of preservatives, addition of calibrants, addition of protease inhibitors, addition of denaturants, desalting, concentration and/or extraction of sample nucleic acids, and/or amplification of sample nucleic acids (e.g., by PCR or other nucleic acid amplification techniques). Samples also include materials prepared by techniques that isolate, e.g., nucleosomes or transcription factors and/or nucleic acids associated with nucleosomes or transcription factors.
  • steps such as concentration, dilution, adjustment of pH, removal of high abundance polypeptides (e.g., albumin, gamma globulin, and transferrin, etc.), addition of preservatives, addition of calibrants, addition of protease inhibitors, addition of denaturants
  • Removal from a sample of proteins that are not desirable for a relevant purpose or context can be achieved using high affinity reagents, high molecular weight filters, ultracentrifugation and/or electrodialysis.
  • High affinity reagents include antibodies or other reagents (e.g., aptamers) that selectively bind to high abundance proteins.
  • Sample preparation can also include ion exchange chromatography, metal ion affinity chromatography, gel filtration, hydrophobic chromatography, chromatofocusing, adsorption chromatography, isoelectric focusing and related techniques.
  • Molecular weight filters include membranes that separate molecules based on size and molecular weight.
  • Such filters may further employ reverse osmosis, nanofiltration, ultrafiltration and microfiltration.
  • Ultracentrifugation is the centrifugation of a sample at about 15,000-60,000 rpm while monitoring with an optical system the sedimentation (or lack thereof) of particles.
  • Electrodialysis is a procedure which uses an electromembrane or semipermeable membrane in a process in which ions are transported through semi-permeable membranes from one solution to another under the influence of a potential gradient.
  • Separation and purification in the present disclosure may include any procedure known in the art, such as capillary electrophoresis (e.g., in capillary or on-chip) or chromatography (e.g., in capillary, column or on a chip).
  • Electrophoresis is a method that can be used to separate ionic molecules under the influence of an electric field. Electrophoresis can be conducted in a gel, capillary, or in a microchannel on a chip.
  • gels used for electrophoresis include starch, acrylamide, polyethylene oxides, agarose, or combinations thereof.
  • a gel can be modified by its cross-linking, addition of detergents, or denaturants, immobilization of enzymes or antibodies (affinity electrophoresis) or substrates (zymography) and incorporation of a pH gradient.
  • capillaries used for electrophoresis include capillaries that interface with an electrospray.
  • CE Capillary electrophoresis
  • CZE capillary zone electrophoresis
  • CIEF capillary isoelectric focusing
  • CITP capillary isotachophoresis
  • CEC capillary electrochromatography
  • An embodiment to couple CE techniques to electrospray ionization involves the use of volatile solutions, for example, aqueous mixtures containing a volatile acid and/or base and an organic such as an alcohol or acetonitrile.
  • Capillary isotachophoresis is a technique in which the analytes move through the capillary at a constant speed but are nevertheless separated by their respective mobilities.
  • Capillary zone electrophoresis also known as free-solution CE (FSCE)
  • FSCE free-solution CE
  • Capillary isoelectric focusing allows weakly-ionizable amphoteric molecules, to be separated by electrophoresis in a pH gradient.
  • Histone methylation is understood to increase or decrease expression of associated coding sequences, depending on which histone residue is methylated. Histone methylation is an essential modification that can cause monomethylation (me1), dimethylation (me2), and trimethylation (me3) of several amino acids, thus directly affecting heterochromatin formation, gene imprinting, X chromosome inactivation, and gene transcriptional regulation. Histone methyltransferases promote monomethylation, dimethylation, or trimethylation of histones while histone demethylases promote demethylation of histones.
  • Histone methylation In general, lysine (Lys or K), arginine (Arg or R), and rarely histidine (His or H) are the most common histone methyl acceptors. Histone methylation only occurs at specific lysine and arginine sites of histone H3 and H4. In histone H3, lysine 4, 9, 26, 27, 36, 56, and 79 and arginine 2, 8, and 17 can be methylated. By comparison, histone H4 has fewer methylation sites, in which only lysine 5, 12, and 20 and arginine 3 can be methylated. Histone methylation is often associated with transcriptional activation or inhibition of downstream genes.
  • HATs can acetylate histones and recruit HAT-containing complexes to activate the transcriptional process.
  • H3K9ac and H3K27ac levels can be associated with promoter and enhancer activities.
  • H3K27ac enhances not only the kinetics of transcriptional activation, but also accelerates the transition of RNA polymerase II from the initiation state to the elongation state.
  • Modified histones can regulate chromatin accessibility through a variety of mechanisms, such as altering transcription factor (TF) binding through steric hindrance and modulating nucleosome affinity for active chromatin remodelers.
  • TF transcription factor
  • the topological organization of nucleosomes across the genome is non-uniform: while histones can be densely arranged within facultative and constitutive heterochromatin, histones can be depleted at regulatory loci, including within enhancers, insulators and transcribed gene bodies. Active regulatory elements of the genome are generally accessible.
  • a reference value can be a predetermined threshold value, a value that varies in accordance with circumstances (e.g., according to patient subpopulation, age, weight, or other variables), or a ratio. Reference ratios can be ratios relating to the modification and/or accessibility of multiple loci within individual samples and/or references, or across or between samples and/or references.
  • a reference can have or represent a normal, non-diseased state.
  • a reference can have or represent a diseased state, e.g., a cancer, stage of cancer, or subtype of cancer.
  • a reference can represent a particular level of ADC target expression based on IHC testing, e.g., ADC target-positive or ADC target-negative cancer.
  • a reference is a non-contemporaneous sample from the same source, e.g., a prior sample from the same source, e.g., from the same subject.
  • a reference for the modification status of one or more genomic loci can be the modification status of the one or more genomic loci (e.g., one or more differentially modified genomic loci) in a sample (e.g., a sample from a subject), or a plurality of samples, known to represent a particular state (e.g., ADC target- positive or ADC target-negative cancer).
  • a reference for the accessibility status of one or more genomic loci can be the accessibility status of the one or more genomic loci (e.g., one or more differentially accessible genomic loci) in a sample (e.g., a sample from a subject), or a plurality of samples, known to represent a particular state (e.g., ADC target-positive or ADC target-negative cancer).
  • differential modification or differential accessibility can refer to a differential (e.g., between a sample and a reference) with an absolute log2(fold-change) that is greater than or equal to 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0 or more, or any range in between, inclusive, e.g., as measured according to an assay provided herein.
  • Enhancers are genomic loci that can be differentially modified or differentially accessible in and/or between conditions, diseases, and other states. Enhancers are cis-acting DNA regulatory regions that are thought to bind trans-acting proteins that contribute to expression patterns of associated genes.
  • Chromatin ImmunoPrecipitation sequencing 12621818v1 Page 39 of 147 Attorney Docket: 2014191-0036 of histone modifications (e.g., acetylation) have identified millions of enhancers in mammalian genomes. The number of active enhancers in any given cell type is estimated to be in the tens of thousands. Certain transcription factors (TFs), sometimes referred to as “master” transcription factors, associate with active enhancers with important impacts on gene expression and cell function. Certain such transcription factors preferentially associate with enhancers that regulate genes required for establishing cell identity and function, including enhancer domains known as “super-enhancers”.
  • master TFs can participate in inter-connected auto-regulatory circuitries or “cliques” that are self-reinforcing, show marked cell selectivity, and function to maintain cell state and/or cell survival.
  • Techniques for Detecting and Quantifying Histone Modifications and Transcription Factor Binding [0132] Various techniques of molecular biology are well known in the art and/or disclosed in the present application for detecting and quantifying histone modifications and/or transcription factor binding. In some embodiments, the methods, kits and systems of present disclosure involve the detection and quantification of histone modifications and/or transcription factor binding in samples, e.g., in liquid biopsy samples including cfDNA such as plasma samples including cfDNA.
  • Chromatin ImmunoPrecipitation is one technique of molecular biology useful in detecting and quantifying histone modifications and transcription factor binding in samples.
  • CUT&RUN or CUT&Tag are other more recent techniques that can also be used to detect and quantify histone modifications and transcription factor binding sites.
  • ChIP-chip, ChIP-exo, ChIP Re-ChIP, and ChIPmentation are other alternative techniques that could be used.
  • ChIP can involve various steps including one or more of fixation, sonication, immunoprecipitation, and analysis of the immunoprecipitated DNA. ChIP has become a very widely used tissue-based technique for determining the in vivo location of binding sites of various transcription factors and histones.
  • an antibody used to bind a target epitope can be a “pan” antibody (e.g., a pan- acetylation antibody, a pan-methylation antibody, an antibody that binds a group of histone modifications associated with increased transcription activation, and/or an antibody that binds a group of histone modifications associated with increased transcription repression).
  • the antibody 12621818v1 Page 41 of 147 Attorney Docket: 2014191-0036 against the protein of interest is allowed to bind to the protein-DNA complex, and the complex can be then precipitated.
  • ChIP-seq can be used to map DNA-binding proteins, e.g., transcription factor binding sites and histone modifications in a genome-wide manner.
  • Cell-free Chromatin ImmunoPrecipitation sequencing involves applying ChIP-seq to samples that include cell-free DNA, e.g., liquid biopsy samples including cfDNA such as plasma samples including cfDNA (e.g., see Sadeh et al., Nat Biotechnol (2021) 39: 586–598 and Jang et al., Life Sci Alliance (2023) 6(12):e202302003 the entire contents of each of which are incorporated herein by reference).
  • cfChIP-seq uses antibodies or antibody fragments that bind specific histone modifications (e.g., H3K4me3 and/or H3K27ac) and/or transcription factors that are coupled (covalently or non-covalently) to beads, e.g., magnetic beads such as Dynabeads® magnetic beads and incubated with a volume, e.g., about 1 mL of thawed plasma obtained from a subject.
  • specific histone modifications e.g., H3K4me3 and/or H3K27ac
  • transcription factors that are coupled (covalently or non-covalently) to beads, e.g., magnetic beads such as Dynabeads® magnetic beads and incubated with a volume, e.g., about 1 mL of thawed plasma obtained from a subject.
  • exemplary antibodies that bind H3K4me3 include PA5-27029 (available from Thermo Fisher Scientific in Waltham, MA) and C15410003 (available from Diagenode in Denville, NJ) and exemplary antibodies that bind H3K27ac include ab21623 or ab4729 (both available from Abcam in Cambridge, UK) and C15210016 (available from Diagenode in Denville, NJ).
  • the antibodies or antibody fragments can be covalently coupled to beads, e.g., epoxy beads.
  • the antibodies or antibody fragments can be non-covalently coupled to beads, e.g., Protein A or Protein G beads such as Dynabeads® Protein A or Dynabeads® Protein G beads.
  • a cfDNA library is then typically prepared from the captured cfDNA.
  • Library preparation can be done on-bead or after releasing the captured cfDNA by digestion of bound histones, e.g., using proteinase K.
  • the cfDNA library is then sequenced to generate reads of captured cfDNA sequences, e.g., by next-generation 12621818v1 Page 42 of 147 Attorney Docket: 2014191-0036 sequencing (NGS) as is known in the art.
  • the reads are then analyzed, e.g., aligned and counted using standard bioinformatic techniques as is known in the art.
  • a cfChIP-seq bioinformatic pipeline can include, e.g., alignment of sequence reads to a reference genome with BWA or Bowtie2. Aligned reads can be used to call and quantify peaks as compared to a reference.
  • CUT&Tag involves antibody-based binding of a target protein, e.g., transcription factor or histone modification of interest, where antibody incubation is directly followed by the shearing of the chromatin and library preparation (see Kaya-Okur et al., Nat Comm (2019) 10:1930).
  • CUT&Tag assays take advantage of a Tn5 transposase that is fused with Protein A to direct the enzyme to the antibody bound to its target on chromatin.
  • Tn5 transposase is pre- loaded with sequencing adapters (generating the assembled pA-Tn5 adapter transposome) to carry out antibody-targeted tagmentation.
  • sequencing adapters generating the assembled pA-Tn5 adapter transposome
  • samples are incubated with an antibody immobilized on Concanavalin A-coated magnetic beads to facilitate subsequent washing steps.
  • Cells can be incubated with a primary antibody specific for the target protein of interest followed by incubation with a secondary antibody.
  • Samples can then be incubated with assembled transposomes, which consist of Protein A fused to the Tn5 transposase enzyme that is conjugated to NGS adapters. After incubation, unbound transposome can be washed away using stringent conditions.
  • CUT&RUN has low background levels.
  • a sample is incubated with an antibody or antibody fragment that binds the target protein, e.g., transcription factor or histone modification of interest.
  • the sample is then incubated with Protein-A-MNase after which CaCl 2 can be added to initiate the calcium dependent nuclease activity of MNase to cleave the DNA around the target protein.
  • the protein- A-MNase reaction can be quenched by adding chelating agents (EDTA and EGTA). Cleaved DNA fragments are then liberated, extracted, and used to construct a sequencing library.
  • FAIRE-seq is a method in which nucleosome-depleted regions of DNA (NDRs) are isolated from chromatin.
  • a typical FAIRE-seq assay can include a first step in which cells are fixed using formaldehyde so that histones are crosslinked to interacting DNA. Crosslinked chromatin can then be sheared by sonication that generates protein-free DNA and protein- crosslinked DNA fragments. Protein-free DNA can be isolated using a phenol–chloroform extraction: DNA crosslinked with protein stays in organic phase, while protein-free DNA stays in aqueous phase. Highly crosslinked DNA remains in the organic phase and the non- crosslinked DNA is pulled to the aqueous phase.
  • a typical NOMe-seq protocol can include a step in which samples are treated with M.CviPI and S-adenosylhomocysteine (SAM) to methylate accessible GpC sites.
  • M.CviPI treated DNA can be sheared using a sonicator, so that 12621818v1 Page 45 of 147 Attorney Docket: 2014191-0036 DNA fragments can be sequenced.
  • DNA is treated with bisulfite, which converts unmethylated cytosine to uracil using sodium bisulfite, while methylated cytosine is unaffected.
  • a library is generated using adapters and sequenced. Accessible chromatin is expected to have high levels of GpCm but low levels of CmpG.
  • NOMe-seq identifies NDRs using the two separate methylation analyses that serve as independent (but opposite) measures, providing matched chromatin designations for each regulatory element.
  • ATAC-seq uses hyperactive Tn5 transposase that preferentially cuts accessible chromatin regions and simultaneously inserts adapters to the fragmented region (Buenrostro et al., Nat Methods (2013) 10(12):1213-1218 the entirety of which is incorporated herein by reference).
  • a typical ATAC-seq assay can include a first step in which samples are incubated with Tn5 transposase. DNA can then be isolated and purified.
  • DNA fragmented and tagged by Tn5 transposase can be purified and then amplified to generate a library and sequenced for analysis.
  • Framentomics or a “fragmentomics assay” refers to methods that use certain size and sequence characteristics of cfDNA to gain insight into the epigenetic state of cells at the time their genomic DNA was released into the extracellular environment. Without wishing to be bound by theory, upon release of genomic DNA from a cell into the extracellular environment, nucleases rapidly cleave the genomic DNA into short fragments.
  • the cleavage pattern and sequences of the fragments reflect the positioning of nucleosomes genome-wide at the point of cell death, and by finding nucleosomes that are consistently genomically positioned across cancer cells (i.e. many of the circulating tumor DNA fragments that map to that small region of the genome have the same start and end positions or similar fragment length characteristics) fragmentomics attempts to infer the location of stably positioned nucleosomes at regulatory sites, and thus to infer where the active regulatory sites are in a given cell type. Accordingly, analysis of cfDNA fragmentation patterns can be used to infer characteristics of the cells at the time they released genomic DNA.
  • fragment size examples include fragment size, preferred ends, end motifs, single-stranded jagged ends, and nucleosomal footprints.
  • Approaches for measuring fragmentomics metrics include, e.g., qPCR, electron microscopy, single molecule sequencing, and next-generation sequencing.
  • a relationship between fragmentomic metrics and histone modifications has been established. See Bai, Jinyue, et al. "Histone modifications of circulating nucleosomes are 12621818v1 Page 46 of 147 Attorney Docket: 2014191-0036 associated with changes in cell-free DNA fragmentation patterns.” Proceedings of the National Academy of Sciences 121.42 (2024): e2404058121.
  • the present disclosure includes the identification of exemplary genomic loci that are differentially modified and/or differentially accessible in samples from different cancer patients (e.g., different breast cancer patients, different SCLC patients or different NSCLC patients) and/or samples from cancer patients (e.g., breast cancer patients, SCLC patients or NSCLC patients) and samples from healthy volunteers.
  • the present disclosure also includes the identification of exemplary genomic loci that are differentially modified and/or differentially accessible depending on ADC target status.
  • Table 1 which shows exemplary genomic coordinates of H3K4me3 regions associated with genes that encode ADC target antigens
  • Table 2 which shows exemplary genomic coordinates of H3K4me3 regions associated with genes that modulate ADC response/resistance
  • Table 3 which shows exemplary genomic coordinates of H3K27ac regions associated with genes that encode ADC target antigens
  • Table 4 which shows exemplary genomic coordinates of H3K4me3, H3K27ac, or DNA methylation regions associated with genes that encode ADC target antigens
  • Table 5 which shows exemplary genomic coordinates of H3K4me3 methylation regions associated with DLL3
  • Table 6 which shows exemplary genomic coordinates of H3K4me3, H3K27ac, or DNA methylation regions associated with DLL3 or genes whose expression levels are correlated with expression level of DLL3.
  • Table 1 references chr1:59,039,915-59,043,420 as a genomic locus for detecting and/or quantifying H3K4me3 modification
  • this encompasses methods that detect and/or quantify H3K4me3 modification at any position or sub-region of chr1:59,039,915-59,043,420, e.g., 12621818v1 Page 47 of 147 Attorney Docket: 2014191-0036 methods that detect and/or quantify H3K4me3 modification within chr1:59,039,915-59,043,420, etc.
  • a subregion may have a different central coordinate as a genomic locus recited in Tables 1-6. It is also to be understood that the lower/upper coordinates of the genomic loci in Tables 1-6 are approximate and that the present disclosure encompasses methods where any one or more of the genomic loci are expanded by increasing the size of the genomic locus by 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40% or up to 50% in one or both directions.
  • a classifier is generated using a set of differentially modified and/or differentially accessible genomic loci that are correlated with ADC target- positive status and a set of differentially modified and/or differentially accessible loci that are correlated with ADC target-negative status (e.g., based on IHC testing). Sequence reads that fall into each selected genomic locus are analyzed and counted, e.g., as described herein including the Examples. In some embodiments, counts from genomic loci that are correlated with ADC target-positive status are aggregated and counts from genomic loci that are correlated with ADC target-negative status are aggregated.
  • exemplary genomic loci from Table 1, 2, 3, 4, 5, 12621818v1 Page 48 of 147 Attorney Docket: 2014191-0036 and/or 6 are used in combination in a multimodal classifier, e.g., a classifier that uses more than one histone modification (e.g., H3K4me3 and H3K27ac) or more than one histone modification (e.g., H3K4me3 and H3K27ac) and DNA methylation at one or more genomic loci for purposes of determining ADC target status.
  • a multimodal classifier e.g., a classifier that uses more than one histone modification (e.g., H3K4me3 and H3K27ac) or more than one histone modification (e.g., H3K4me3 and H3K27ac) and DNA methylation at one or more genomic loci for purposes of determining ADC target status.
  • exemplary genomic loci from Table 6 associated with DLL3, RAB3IP, PAK5, ITPRIPL2, or any combination thereof are used in a monomodal or multimodal classifier for purposes of determining DLL3 status.
  • exemplary genomic loci from Table 6 associated with DLL3 are used in a monomodal or multimodal classifier for purposes of determining DLL3 status.
  • exemplary genomic loci from Table 6 associated with RAB3IP are used in a monomodal or multimodal classifier for purposes of determining DLL3 status.
  • exemplary genomic loci from Table 6 associated with PAK5 are used in a monomodal or multimodal classifier for purposes of determining DLL3 status.
  • exemplary genomic loci from Table 6 associated with ITPRIPL2 are used in a monomodal or multimodal classifier for purposes of determining DLL3 status.
  • exemplary genomic loci from Table 1, Table 3, Table 5, or Table 6 associated with DLL3 are used in a monomodal or multimodal classifier that also uses one or more genomic loci associated with RAB3IP, PAK5, or ITPRIPL2, or any combination thereof for purposes of determining DLL3 status.
  • exemplary genomic loci associated with RAB3IP, PAK5, or ITPRIPL2, or any combination thereof are provided in Table 6.
  • exemplary genomic loci from Table 1 associated with DLL3 are used in a monomodal or multimodal classifier that also uses one or more genomic loci from Table 6 associated with RAB3IP, PAK5, or ITPRIPL2, or any combination thereof for purposes of determining DLL3 status.
  • exemplary genomic loci from Table 3 associated with DLL3 are used in a monomodal or multimodal classifier that also uses one or more genomic loci from Table 6 associated with RAB3IP, PAK5, or ITPRIPL2, or any combination thereof for purposes of determining DLL3 status.
  • exemplary genomic loci from Table 5 associated with DLL3 are used in a monomodal or multimodal classifier that also uses one or more genomic loci from Table 6 associated with RAB3IP, PAK5, or ITPRIPL2, or any combination thereof for purposes of determining DLL3 status.
  • exemplary genomic loci from Table 6 associated with DLL3 are used in a monomodal or multimodal classifier that also uses one or more genomic loci from Table 6 associated with RAB3IP, PAK5, or ITPRIPL2, or any combination thereof for purposes of determining DLL3 status.
  • Genomic loci demonstrating differential H3K4 methylation in particular H3K4 trimethylation, H3K4me3 in samples from different cancer patients (e.g., different breast cancer patients, different SCLC patients, different NSCLC patients, different NEPC patients, or different PRAD patients) and/or samples from cancer patients (e.g., breast cancer patients, SCLC patients, NSCLC patients, NEPC patients, or PRAD patients) and samples from healthy volunteers are provided in Tables 1-2 and 4-6 which show the chromosomal coordinates of each genomic locus. The chromosomal coordinates are based on human genome build hg19.
  • Subsets of the genomic loci of Tables 1-2 and 4-6 can be selected (e.g., for use in determining ADC target status) based on the type of ADC screening required (e.g., the ADC target or ADC targets of interest), based on various performance criteria, e.g., to select genomic loci that demonstrate differential modification with a particular level of statistical significance, progression free survival (PFS) of subjects, from whom the sample is obtained or derived, and/or a particular threshold of differential between relevant states (e.g., a measured log2(fold-change)).
  • Subsets of the genomic loci may also be selected based on an algorithm, e.g., during the process of obtaining a classifier.
  • Such subsets of loci of Tables 1-2 and 4-6and loci included in such subsets are together, individually, and/or in randomly selected subsets, at least as informative (e.g., as statistically significant and/or reliable) for uses disclosed herein, e.g., for determining ADC target status.
  • an increase or decrease in a value measuring methylation can be, or is expressed as, a log2(fold-change), e.g., a log2(fold-change) of at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 75%, 100%, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, or greater, or any range in between, inclusive, such as an increase or decrease of 0.1-fold to 10- fold, 0.2-fold to 5-fold, 0.2-fold to 4.0-fold, 0.4-4.0-fold, 0.4-fold to 4.0-fold, 0.6-fold to 4.0- fold, 0.8-fold to 4.0-fold, 1.0-fold to 4.0-fold.1.2-fold to 4.0-fold.1.4-fold to 4.0-fold, 1.6-fold
  • Genomic loci demonstrating differential H3K27 acetylation (H3K27ac) in samples from different cancer patients e.g., different breast cancer patients, different SCLC 12621818v1 Page 51 of 147 Attorney Docket: 2014191-0036 patients, different NSCLC patients, different NEPC patients, or different PRAD patients
  • samples from cancer patients e.g., breast cancer patients, SCLC patients, NSCLC patients, NEPC patients, or PRAD patients
  • Tables 3, 4 and 6 show the chromosomal coordinates of each genomic locus. The chromosomal coordinates are based on human genome build hg19.
  • Subsets of the genomic loci of Tables 3, 4 and 6 can be selected (e.g., for use in determining ADC target status or the status of one or more genes that modulate ADC response/resistance) based on the type of ADC screening required (e.g., the ADC target or ADC targets of interest), based on various performance criteria, e.g., to select genomic loci that demonstrate differential modification with a particular level of statistical significance and/or a particular threshold of differential between relevant states (e.g., a measured log2(fold-change)). Subsets of the genomic loci may also be selected based on an algorithm, e.g., during the process of obtaining a classifier.
  • Such subsets of loci of Tables 3, 4 and 6, and loci included in such subsets are together, individually, and/or in randomly selected subsets, at least as informative (e.g., as statistically significant and/or reliable) for uses disclosed herein, e.g., for determining ADC target status or the status of one or more genes that modulate ADC response/resistance.
  • a sample or subject from whom the sample is obtained or derived is determined to have a particular ADC target status (e.g., ADC target-positive) if at least 1, 2, 3, 4, 5 or more loci identified in Table 4 or 6 (or any subset thereof) are differentially 12621818v1 Page 52 of 147 Attorney Docket: 2014191-0036 H3K27ac modified as compared to a reference (e.g., a sample from an ADC target-negative subject or healthy volunteer).
  • ADC target status e.g., ADC target-positive
  • differentially H3K27ac modified refers to an acetylation status characterized by an increase or decrease in a value measuring acetylation (e.g., of read counts and/or normalized read counts for a given genomic locus), and/or a mean, median and/or mode thereof, and/or a log thereof (e.g., log base 2 (log2)), of at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 75%, 100%, 2-fold, 3-fold, 4- fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40- fold, 45-fold, 50-fold, or greater, or any range in between, inclusive, such as 1% to 50%, 50% to 2-fold, 25% to 50-fold, 25% to 30-fold, 25% to
  • an increase or decrease in a value measuring acetylation can be, or is expressed as, a log2(fold-change), e.g., a log2(fold-change) of at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 75%, 100%, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, or greater, or any range in between, inclusive, such as an increase or decrease of 0.1-fold to 10- fold, 0.2-fold to 5-fold, 0.2-fold to 4.0-fold, 0.4-4.0-fold, 0.4-fold to 4.0-fold, 0.6-fold to 4.0- fold, 0.8-fold to 4.0-fold, 1.0-fold to 4.0-fold, 1.2-fold to 4.0-fold, 1.4-fold to 4.0-fold,
  • an agent directed to an ADC target antigen is an ADC.
  • an agent directed to an ADC target antigen is a radioligand, e.g., an antibody or antibody fragment, peptide or small molecule ligand that (i) associates with the ADC target antigen and (ii) is linked to a chelator that is bonded to a radioisotope.
  • Responsiveness can refer to the ability or likelihood of a therapy to cause a reduction in tumor size or inhibit tumor growth or metastasis.
  • Responsiveness can refer to 12621818v1 Page 60 of 147 Attorney Docket: 2014191-0036 improvement in prognosis (e.g., increased time to cancer recurrence or increased life expectancy, e.g., overall survival, recurrence-free survival, metastasis-free survival, progression-free survival, or disease-free survival).
  • Responsiveness can refer to achievement of a treatment benefit, including e.g., improvement in one or more symptoms of cancer, e.g., breast cancer, gastric/gastroesophageal cancer, colorectal cancer, prostate cancer, or lung cancer.
  • Responsiveness can be measured quantitatively (e.g., as in the case of tumor size; as in the case of measurement of histone modification, chromatin accessibility, transcription factor binding, or DNA methylation at one or more genomic loci; or as in the calculation of clinical benefit (CBR)), or qualitatively (e.g., by measures such as “pathological complete response” (pCR), “clinical complete remission” (cCR), “clinical partial remission” (cPR), “clinical stable disease” (cSD), “clinical progressive disease” (cPD), or other qualitative criteria).
  • CBR clinical benefit
  • Resistance can refer to the inability or unlikelihood of a therapy to achieve a desired therapeutic effect (e.g., a reduction in tumor size, improvement in prognosis, or other treatment benefit such as, e.g., improvement in one or more symptoms of cancer) in a subject and/or cancer. Resistance includes both acquired and natural resistance. In certain embodiments, resistance includes the extent to which one or more desired therapeutic benefits results from administration of a therapy to a subject and/or cancer is less than that expected and/or achieved in a reference (e.g., less than 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, or 10% of benefit achieved in a reference).
  • methods, kits and systems can be used to detect the clinical efficacy of a course of therapy for cancer, e.g., breast cancer, gastric/gastroesophageal cancer, colorectal cancer, prostate cancer, or lung cancer.
  • a course of therapy for cancer e.g., breast cancer, gastric/gastroesophageal cancer, colorectal cancer, prostate cancer, or lung cancer.
  • methods and/or compositions of the present disclosure could be used to determine the presence, absence, or ADC target status of a cancer in a subject over the course of treatment.
  • compositions of the present disclosure could be used in conjunction with, or confirmed by, other means of determining the presence, absence, or ADC target status of a cancer including, for example measurements of tumor size or character by techniques such as CT, PET, mammogram, ultrasound, palpation, histology, caliper measurement after biopsy or surgical resection, or by various qualitative, quantitative, or semi quantitative scoring systems including without limitation based on IHC testing, residual cancer burden (Symmans et al., J Clin Oncol (2007) 25:4414-4422, incorporated by reference herein in its entirety) or Miller-Payne score (Ogston et al., Breast (2003) 12:320-327, incorporated by reference herein in its entirety) in a qualitative 12621818v1 Page 61 of 147 Attorney Docket: 2014191-0036 fashion like “pathological complete response” (pCR), “clinical complete remission” (cCR), “clinical partial remission” (cPR), “clinical stable disease” (cSD), “clin
  • methods, kits and systems for ADC target status determination provided herein can inform treatment and/or payment (e.g., reimbursement for or reduction of cost of medical care, such as detecting or treatment) decisions and/or actions, e.g., by individuals, healthcare facilities, healthcare practitioners, health insurance providers, governmental bodies, or other parties interested in healthcare cost.
  • treatment and/or payment e.g., reimbursement for or reduction of cost of medical care, such as detecting or treatment
  • decisions and/or actions e.g., by individuals, healthcare facilities, healthcare practitioners, health insurance providers, governmental bodies, or other parties interested in healthcare cost.
  • methods, kits and systems for ADC target status determination provided herein are used as the basis for, to contribute to, or support a determination as to whether a reimbursement or cost reduction will be provided to a healthcare cost payer or recipient.
  • a party seeking reimbursement or cost reduction can provide results of ADC target status determination conducted in accordance with the present disclosure together with a request for such reimbursement or reduction of a healthcare cost.
  • a party making a determination as to whether or not to provide a reimbursement or reduction of a healthcare cost will reach a determination based in whole or in part upon receipt and/or review of results of ADC target status determination conducted in accordance with the present disclosure.
  • a sample obtained from a subject e.g., a liquid biopsy sample including cfDNA, e.g., a plasma sample including cfDNA
  • ChIP-seq for a histone modification e.g., H3K4me3 and/or H3K27ac
  • ChIP-seq sequence reads are aligned to human genome build hg19, e.g., using the Burrows-Wheeler Aligner (BWA).
  • BWA Burrows-Wheeler Aligner
  • the normalized data is then used as input into a classifier that was trained using the same histone modification and selected genomic loci.
  • the classifier uses the inputted data to determine ADC target status of the subject’s cancer. It will be appreciated that this or similar approaches can be applied to assays of the present disclosure that quantify chromatin accessibility and/or transcription factor binding. [0200] For the avoidance of any doubt, those of skill in the art will appreciate from the present disclosure that methods, kits and systems for ADC target status determination of the present disclosure are at least for in vitro use. Accordingly, all aspects and embodiments of the present disclosure can be performed and/or used at least in vitro.
  • methods of the present disclosure can be implemented on and/or in conjunction with a computer program and computer system.
  • methods of the present disclosure can be implemented on and/or in conjunction with a non-transitory computer readable storage medium encoded with the computer program, wherein the program comprises instructions that when executed by one or more processors cause the one or more processors to perform operations to perform the method.
  • a computer system can also store and manipulate data generated by methods of the present disclosure that comprise a plurality of genomic locus modification status and/or accessibility status changes/profiles, which data can be used by a computer system in implementing methods disclosed herein.
  • a computer system receives modification status and/or accessibility status data; (ii) stores the data; and (iii) compares the data in any number of ways described herein (e.g., analysis relative to appropriate references), e.g., to determine ADC target status.
  • a computer system (i) compares the genomic locus modification and/or accessibility status to a reference; and (ii) outputs an indication of whether the modification status and/or accessibility status of the genomic locus is significantly different from the reference and/or provides a determination regarding ADC target status.
  • Numerous types of computer systems can be used to implement methods of the present disclosure according to knowledge possessed by a skilled artisan in the bioinformatics and/or computer arts.
  • the software components can comprise both software components that are standard in the art and components that are special to the present disclosure (e.g., dCHIP software described in Lin et al., Bioinformatics (2004) 20:1233-1240, incorporated herein by reference in its entirety; radial basis machine learning algorithms (RBM) known in the art).
  • Methods of the present disclosure can also be programmed or modeled in mathematical software packages that allow symbolic entry of equations and high-level specification of processing, including specific algorithms to be used, thereby freeing a user of the need to procedurally program individual equations and algorithms.
  • a computer system comprises a database for storage of genomic locus modification status and/or accessibility status data. Such stored profiles can be accessed and used to perform comparisons of interest at a later point in time.
  • a single learning statistical classifier system such as a classification tree (e.g., random forest) is used.
  • a combination of 2, 3, 4, 5, 6, 7, 8, 9, 10, or more learning statistical classifier systems are used, preferably in tandem.
  • Examples of learning statistical classifier systems include, but are not limited to, those described in the Examples and also those using inductive learning (e.g., decision/classification trees such as random forests, classification and regression trees (C&RT), boosted trees, etc.), Probably Approximately Correct (PAC) learning, connectionist learning (e.g., neural networks (NN), artificial neural networks (ANN), neuro fuzzy networks (NFN), network structures, perceptrons such as multi-layer perceptrons, multi-layer feed-forward networks, applications of neural networks, Bayesian learning in belief networks, etc.), reinforcement learning (e.g., passive learning in a known environment such as naive learning, adaptive dynamic learning, and temporal difference learning, passive learning in an unknown environment, active learning in an unknown environment, learning action-value functions, applications of reinforcement learning, etc.), and genetic algorithms and evolutionary programming.
  • inductive learning e.g., decision/classification trees such as random forests, classification and regression trees (C&RT), boosted trees, etc.
  • PAC Probably Approximately Correct
  • connectionist learning
  • an ADC target-negative cancer is greater than 0.5 (e.g., greater than 0.55, greater than 0.6, greater than 0.65, greater than 0.7, greater than 0.75, greater than 0.8, greater than 0.85, greater than 0.9, or greater than 0.95).
  • an agent that is directed to an ADC target antigen e.g., an ADC therapy or radioligand
  • a subject e.g., breast cancer, SCLC, NSCLC, etc.
  • the agent that is directed to an ADC target antigen provided herein will be available, appropriate, and/or preferred for the determined ADC target status.
  • compositions for delivery of one or more ADCs to a subject include pharmaceutical compositions for delivery of one or more ADCs to a subject.
  • a pharmaceutical composition may be in any form known in the art, including formulations for administration according to any route known in the art.
  • a suitable means of administration can be selected based on the age and condition of a subject.
  • Pharmaceutical composition forms of the present disclosure can include, e.g., liquid, semi-solid and solid dosage forms.
  • the compositions provided herein are present in unit dosage form, which unit dosage form can be suitable for self-administration.
  • unit dosage 12621818v1 Page 66 of 147 Attorney Docket: 2014191-0036 form may be provided within a container, e.g., a pill, vial, cartridge, prefilled syringe, or disposable pen.
  • a pharmaceutical composition of the present disclosure can be in an injectable or infusible form.
  • the present disclosure includes sterile formulations for injection or infusion, which can be formulated in accordance with conventional pharmaceutical practices.
  • Sterile solutions can be prepared by incorporating a composition described herein in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filter sterilization.
  • Solutions can be formulated, e.g., using distilled water, physiological saline, or an isotonic solution containing glucose and other supplements such as D- sorbitol, D-mannose, D-mannitol, or sodium chloride as an aqueous solution for injection, optionally in combination with a suitable solubilizing agent, for example, an alcohol such as ethanol and/or a polyalcohol such as propylene glycol or polyethylene glycol, and/or a nonionic surfactant such as polysorbate 80TM or HCO-50, and the like.
  • a suitable solubilizing agent for example, an alcohol such as ethanol and/or a polyalcohol such as propylene glycol or polyethylene glycol, and/or a nonionic surfactant such as polysorbate 80TM or H
  • sterile powders for the preparation of sterile injectable solutions methods for preparation include vacuum drying and freeze-drying that yield a powder of a composition described herein plus any additional desired ingredient (see below) from a previously sterile-filtered solution thereof.
  • the proper fluidity of a solution can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • Prolonged absorption of injectable compositions can be brought about by including in the composition a reagent that delays absorption, for example, monostearate salts, and gelatin.
  • compositions can be formulated with a carrier that will protect the ADC against rapid release, such as a controlled release formulation, including implants and microencapsulated delivery systems.
  • a carrier that will protect the ADC against rapid release, such as a controlled release formulation, including implants and microencapsulated delivery systems.
  • Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Many methods for the preparation of such formulations are known in the art.
  • a pharmaceutical composition can be formulated by suitably combining the therapeutic molecule with pharmaceutically acceptable vehicles or media, such as sterile water and physiological saline, vegetable oil, emulsifier, suspension agent, surfactant, stabilizer, flavoring excipient, diluent, vehicle, preservative, binder, followed by mixing in a unit dose form required for generally accepted pharmaceutical practices.
  • pharmaceutically acceptable vehicles or media such as sterile water and physiological saline, vegetable oil, emulsifier, suspension agent, surfactant, stabilizer, flavoring excipient, diluent, vehicle, preservative, binder.
  • examples of oily liquid include sesame oil and soybean oil, and it may be combined with benzyl benzoate or benzyl alcohol as a solubilizing agent.
  • a buffer such as a phosphate buffer, or sodium acetate buffer
  • a soothing agent such as procaine 12621818v1 Page 68 of 147 Attorney Docket: 2014191-0036 hydrochloride
  • a stabilizer such as benzyl alcohol or phenol
  • an antioxidant such as an antioxidant.
  • Such devices can include at least one injection needle, are typically pre-filled with one or more therapeutic unit doses of a solution that includes the ADC and are useful for rapidly delivering solution to a subject with as little pain as possible.
  • One medication delivery pen includes a vial holder into which a vial of a therapeutic or other medication may be received.
  • the pen may be an entirely mechanical device or it may be combined with electronic circuitry to accurately set and/or indicate the dosage of medication that is injected into the user. See, e.g., U.S. Pat. No.6,192,891.
  • the needle of the pen device is disposable and the kits include one or more disposable replacement needles.
  • Pen devices suitable for delivery of any one of the presently featured compositions are also described in, e.g., U.S.
  • a therapeutically effective amount can be an amount at which any toxic or detrimental effects of the composition are outweighed by therapeutically beneficial effects.
  • a dose can also be chosen to reduce or avoid production of antibodies or other host immune responses against an ADC.
  • the amount of active ingredient included in a pharmaceutical composition is such that a suitable dose within the designated range can be administered to subjects.
  • the dose and method of administration can vary depending on weight, age, condition, and other characteristics of a patient, and can be suitably selected as needed by those skilled in the art.
  • Exemplary dosages of a composition described herein include, without limitation, 0.1 mg/kg, 0.5 mg/kg, 1 mg/kg, 2 mg/kg, 4 mg/kg, 8 mg/kg, or 20 mg/kg. The present disclosure is not limited to such ranges or dosages.
  • the present disclosure further includes methods of preparing pharmaceutical compositions of the present disclosure and kits including pharmaceutical compositions of the present disclosure.
  • ADCs of the present disclosure can be administered to a subject in a course of treatment that further includes administration of one or more additional therapeutic agents or therapies that are not ADCs (e.g., surgery or radiation).
  • Combination therapies of the present disclosure can include simultaneous exposure of a subject to therapeutic agents of two or more therapeutic regimens.
  • the kit comprises reagents for quantifying H3K27ac modifications for at least 1, 2, 3, 4, 5, 10, 15, 20, or 25 genomic loci that are located within one or more regions defined by pairs of genomic coordinates in Table 3.
  • the kit comprises reagents for quantifying H3K27ac or H3K4me3 modifications or DNA methylation for at least 1, 2, 3, 4, 5, 10, 15, 20, or 25 genomic loci that are located within one or more regions defined by pairs of genomic coordinates in Table 4.
  • the kit comprises reagents for quantifying H3K4me3 modifications for 1 or 2 genomic loci that are located within one or more regions defined by pairs of genomic coordinates in Table 5.
  • the kit comprises instructions for determining if a subject has an ADC target-positive cancer.
  • the present disclosure includes systems for detecting modification and/or accessibility of one or more genomic loci.
  • the present disclosure provides systems for quantifying one or more histone modifications, DNA methylation, chromatin accessibility, and/or binding of one or more transcription factors at one or more genomic loci.
  • Systems of the present disclosure can include a sequencer configured to generate a sequencing dataset from a sample; and a non-transitory computer readable storage medium and/or a computer system.
  • the non-transitory computer readable storage medium is encoded with a computer program, wherein the program comprises instructions that when executed by one or more processors cause the one or more processors to perform operations to perform a method of the present disclosure.
  • the computer system comprises a memory and one or more processors coupled to the memory, wherein the one or more processors are configured to perform a method of the present disclosure. 12621818v1 Page 73 of 147 Attorney Docket: 2014191-0036 [0232]
  • the sequencer is configured to generate a Whole Genome Sequencing (WGS) dataset from the sample.
  • WGS Whole Genome Sequencing
  • the system comprises reagents for quantifying H3K27ac or H3K4me3 modifications or DNA methylation for at least 1, 2, 3, 4, 5, 10, 15, 20, or 25 genomic loci that are 12621818v1 Page 74 of 147 Attorney Docket: 2014191-0036 located within one or more regions defined by pairs of genomic coordinates in Table 4.
  • the system comprises reagents for quantifying H3K4me3 modifications for 1 or 2 genomic loci that are located within one or more regions defined by pairs of genomic coordinates in Table 5.
  • the system comprises reagents for quantifying H3K4me3 or H3K27ac modifications for 1, 2, 3, 4, 5, 10, 15, or 16 genomic loci that are located within one or more regions defined by pairs of genomic coordinates in Table 6.
  • the system comprises one or more antibodies for use in ChIP-seq, optionally wherein the one or more antibodies specifically bind H3K4me3- or H3K27ac-modified histones or methylated DNA.
  • the system comprises one or more methyl binding domains (MBDs) for use in MBD-seq.
  • the system comprises reagents for isolation of cell-free DNA (cfDNA) from a liquid biopsy sample.
  • the sequencer comprises reagents for library preparation for sequencing. In some embodiments, the sequencer comprises reagents for sequencing. In some embodiments, the system comprises instructions for determining if a subject has an ADC target-positive cancer. Definitions [0237] “A” or “An”: The articles “a” and “an” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” refers to one element or more than one element. [0238] About: The term “about”, when used herein in reference to a value, refers to a value that is similar, in context, to the referenced value.
  • a sample that has an accessibility status that differs in accessibility status from a standard or reference can be referred to as differentially modified.
  • Suitable assays for determining chromatin accessibility are known in the art. Exemplary assays include ATAC-seq (Assay of Transpose Accessible Chromatin sequencing), NOMe-seq (Nucleosome Occupancy and Methylome sequencing), FAIRE-seq (Formaldehyde-Assisted Isolation of Regulatory Elements sequencing), MNase-seq (Micrococcal Nuclease digestion with sequencing), a DNase hypersensitivity assay, and/or a fragmentomics assay.
  • agent may refer to any chemical or physical entity, including without limitation any of one or more of an atom, e.g., a radioactive atom, molecule, compound, conjugate, polypeptide, polynucleotide, polysaccharide, lipid, cell, or combination or complex thereof.
  • Antibody refers to a polypeptide that includes one or more canonical immunoglobulin sequence elements sufficient to confer specific binding to a particular antigen (e.g., a heavy chain variable domain, a light chain variable domain, and/or one or more CDRs).
  • antibody includes, without limitation, human antibodies, non-human antibodies, synthetic and/or engineered antibodies, fragments thereof, and agents including the same.
  • Antibodies can be naturally occurring immunoglobulins (e.g., generated by an organism reacting to an antigen). Synthetic, non-naturally occurri or engineered antibodies can be produced by recombinant engineering, chemical synthesis, or other artificial systems or methodologies known to those of skill in the art.
  • each heavy chain includes a heavy chain variable domain (VH) and a heavy chain constant domain (CH).
  • VH heavy chain variable domain
  • CH heavy chain constant domain
  • a short region known as the “switch”, connects the heavy chain variable and constant regions.
  • Each light chain includes a light chain variable domain (VL) and a light chain constant domain (CL), separated from one another by another “switch.”
  • VL light chain variable domain
  • CL light chain constant domain
  • Each variable domain contains three hypervariable loops known as “complement determining regions” (CDR1, CDR2, and CDR3) and four somewhat invariant “framework” regions (FR1, FR2, FR3, and FR4).
  • CDR1, CDR2, and CDR3 three hypervariable loops known as “complement determining regions” (CDR1, CDR2, and CDR3) and four somewhat invariant “framework” regions (FR1, FR2, FR3, and FR4).
  • CDR1, CDR2, and CDR3 three hypervariable loops known as “complement determining regions”
  • FR1, FR2, FR3, and FR4 four somewhat invariant “framework” regions
  • variable regions of a heavy and/or a light chain are typically understood to provide a binding moiety that can interact with an antigen. Constant domains can mediate binding of an antibody to various immune system cells (e.g., effector cells and/or cells that mediate cytotoxicity), receptors, and elements of the complement system. Heavy and light chains are linked to one another by a single disulfide bond, and two other disulfide bonds connect the heavy chain hinge regions to one another, so that the dimers are connected to one another and the tetramer is formed.
  • an antibody is a polyclonal, monoclonal, monospecific, or multispecific antibody (e.g., a bispecific antibody).
  • an antibody includes at least one light chain monomer or dimer, at least one heavy chain monomer or dimer, at least one heavy chain-light chain dimer, or a tetramer that includes two heavy chain monomers and two light chain monomers.
  • antibody can include (unless otherwise stated or clear from context) any art-known constructs or formats utilizing antibody structural and/or functional features including without limitation intrabodies, domain antibodies, antibody 12621818v1 Page 77 of 147 Attorney Docket: 2014191-0036 mimetics, Zybodies®, Fab fragments, Fab’ fragments, F(ab’)2 fragments, Fd’ fragments, Fd fragments, isolated CDRs or sets thereof, single chain antibodies, single-chain Fvs (scFvs), disulfide-linked Fvs (sdFv), polypeptide-Fc fusions, single domain antibodies (e.g., shark single domain antibodies such as IgNAR or fragments thereof), cameloid antibodies, camelized antibodies, masked antibodies (e.g., Probodies®), affybodies, anti-idiotypic (anti-Id) antibodies (including, e.g., anti-anti-Id antibodies), Small Modular ImmunoPharmaceuticals
  • An antibody including a heavy chain constant domain can be, without limitation, an antibody of any known class, including but not limited to, IgA, secretory IgA, IgG, IgE and IgM, based on heavy chain constant domain amino acid sequence (e.g., alpha ( ⁇ ), delta ( ⁇ ), epsilon ( ⁇ ), gamma ( ⁇ ) and mu ( ⁇ )).
  • IgG subclasses are also well known to those in the art and include but are not limited to human IgG1, IgG2, IgG3 and IgG4.
  • “Isotype” refers to the Ab class or subclass (e.g., IgM or IgG1) that is encoded by the heavy chain constant region genes.
  • a “light chain” can be of a distinct type, e.g., kappa ( ⁇ ) or lambda ( ⁇ ), based on the amino acid sequence of the light chain constant domain.
  • an antibody has constant region sequences that are characteristic of mouse, rabbit, primate, or human immunoglobulins.
  • Naturally produced immunoglobulins are glycosylated, typically on the CH2 domain.
  • affinity and/or other binding attributes of Fc regions for Fc 12621818v1 Page 78 of 147 Attorney Docket: 2014191-0036 receptors can be modulated through glycosylation or other modification.
  • an antibody may lack a covalent modification (e.g., attachment of a glycan) that it would have if produced naturally.
  • antibodies produced and/or utilized in accordance with the present invention include glycosylated Fc domains, including Fc domains with modified or engineered glycosylation.
  • an antibody can be specific for a particular histone modification (e.g., an antibody can bind one histone modification, e.g., H3K27ac with a higher affinity than other histone modifications, under conditions that are commonly used in ChIP-seq experiments).
  • a pan antibody is a pan-methylation antibody (e.g., an antibody that can bind a histone, e.g., H3 that comprises at least one methylated lysine, wherein the at least one methylated lysine can be at any one of a plurality of amino acid positions, e.g., in some embodiments, a pan-methylation antibody can bind an H3 protein comprising a methylated lysine at any position).
  • a pan-methylation antibody e.g., an antibody that can bind a histone, e.g., H3 that comprises at least one methylated lysine, wherein the at least one methylated lysine can be at any one of a plurality of amino acid positions, e.g., in some embodiments, a pan-methylation antibody can bind an H3 protein comprising a methylated lysine at any position).
  • an “antibody fragment” refers to a portion of an antibody or antibody agent as described herein, and typically refers to a portion that includes an antigen-binding portion or variable region thereof.
  • An antibody fragment can be produced by any means. For example, in some embodiments, an antibody fragment can be enzymatically or chemically produced by fragmentation of an intact antibody or antibody agent. Alternatively, in 12621818v1 Page 79 of 147 Attorney Docket: 2014191-0036 some embodiments, an antibody fragment can be recombinantly produced, i.e., by expression of an engineered nucleic acid sequence. In some embodiments, an antibody fragment can be wholly or partially synthetically produced.
  • two or more entities are physically “associated” with one another if they interact, directly or indirectly, so that they are and/or remain in physical proximity with one another.
  • two or more entities that are physically associated with one another are covalently linked to one another; in some embodiments, two or more entities that are physically associated with one another are not covalently linked to one another but are non-covalently associated, for example by means of hydrogen bonds, van der Waals interaction, hydrophobic interactions, magnetism, or a combination thereof.
  • Bodily fluids refer to fluids that are excreted or secreted from the body as well as fluids that are normally not (e.g., blood, serum, plasma, Cowper’s fluid or pre- ejaculate fluid, chyle, chyme, stool, interstitial fluid, intracellular fluid, lymph, menses, saliva, sebum, semen, serum, sweat, synovial fluid, tears, urine, vitreous humor, vomit).
  • a biological sample is a liquid biopsy sample obtained from a bodily fluid.
  • a biological sample is or includes DNA obtained from a single subject or from a plurality of subjects.
  • a biological sample can be a “primary sample” obtained directly from a biological source or can be a “processed sample”, i.e., a sample that was derived from a primary sample, e.g., via dilution, purification, mixing with one or more reagents, or any other processing step(s) as described herein.
  • Blood component refers to any component of whole blood, including red blood cells, white blood cells, plasma, platelets, endothelial cells, mesothelial cells, epithelial cells, cell-free DNA (cfDNA), and circulating- tumor DNA (ctDNA). Blood components also include the components of plasma, including proteins, metabolites, lipids, nucleic acids, and carbohydrates, and any other cells that can be present in blood, e.g., due to pregnancy, organ transplant, infection, injury, or disease.
  • cancer As used herein, the terms “cancer,” “malignancy,” “tumor,” and “carcinoma,” are used interchangeably to refer to a disease, disorder, or condition in which cells exhibit or exhibited relatively abnormal, uncontrolled, and/or autonomous growth, so that they display or displayed an abnormally elevated proliferation rate and/or aberrant growth phenotype.
  • a cancer can include one or more tumors.
  • a cancer 12621818v1 Page 81 of 147 Attorney Docket: 2014191-0036 can be or include cells that are precancerous (e.g., benign), malignant, pre-metastatic, metastatic, and/or non-metastatic.
  • a cancer can be or include a solid tumor.
  • Combination therapy refers to administration to a subject of two or more therapeutic agents or therapeutic regimens such that the two or more therapeutic agents or therapeutic regimens together treat a disease, condition, or disorder of the subject.
  • the two or more therapeutic agents or therapeutic regimens can be administered simultaneously, sequentially, or in overlapping dosing regimens.
  • combination therapy includes but does not require that the two therapeutic agents or therapeutic regimens be administered together in a single composition, nor at the same time.
  • corresponding to may be used to designate the position/identity of a structural element in a compound or composition through comparison with an appropriate reference compound or composition.
  • a monomeric residue in a polymer e.g., an amino acid residue in a polypeptide or a nucleic acid residue in a polynucleotide
  • corresponding to a residue in an appropriate reference polymer.
  • corresponding positions can be readily identified, e.g., by alignment of sequences, and that such alignment is commonly accomplished by any of a variety of known tools, strategies, and/or algorithms, including without limitation software programs such as, for example, BLAST, CS-BLAST, CUDASW++, DIAMOND, FASTA, GGSEARCH/GLSEARCH, Genoogle, HMMER, HHpred/HHsearch, IDF, Infernal, KLAST, USEARCH, parasail, PSI-BLAST, PSI-Search, ScalaBLAST, Sequilab, SAM, SSEARCH, SWAPHI, SWAPHI-LS, SWIMM, or SWIPE.
  • software programs such as, for example, BLAST, CS-BLAST, CUDASW++, DIAMOND, FASTA, GGSEARCH/GLSEARCH, Genoogle, HMMER, HHpred/HHsearch, IDF, Infernal, KLAST, USEARCH, parasail, PSI
  • Two sequences can be identified as corresponding if they are identical or if they share substantial identity, e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, e.g., over a length of at 12621818v1 Page 82 of 147 Attorney Docket: 2014191-0036 least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500 or more residues.
  • a nucleic acid sequence can correspond to a sequence that is identical or substantially identical (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical) to the complement of the nucleic acid sequence, e.g., over a length of at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500 or more nucleic acid residues.
  • diagnosis includes the act, process, and/or outcome of determining whether, and/or the qualitative of quantitative probability that, a subject has or will develop the condition, disease, or related state.
  • diagnosing can include a determination relating to prognosis and/or likely response to one or more general or particular therapeutic agents or regimens.
  • Differentially accessible describes a genomic locus for which chromatin accessibility status differs between a first condition or sample and a second condition or sample (e.g., a standard or reference).
  • a differentially accessible genomic locus can include a greater or smaller measured accessibility under a selected condition of interest, such as disease state, as compared to a reference state, such as healthy state.
  • Differentially modified describes a genomic locus for which histone modification status differs between a first condition or sample and a second condition or sample (e.g., a standard or reference).
  • a differentially modified genomic locus can include a greater or smaller number or frequency of histone modification under a selected condition of interest, such as a disease state, as compared to a reference state, such as healthy state.
  • Gene refers to a DNA sequence in a chromosome encoding a gene product (e.g., an RNA product and/or a polypeptide product).
  • a gene includes a coding sequence (e.g., a sequence encoding a particular gene product); in some embodiments, a gene includes a non-coding sequence.
  • a gene may include both coding (e.g., exonic) and non-coding (e.g., intronic) sequences.
  • a gene may include one or more regulatory elements (e.g. 12621818v1 Page 83 of 147 Attorney Docket: 2014191-0036 promoters, silencers, termination signals) that, for example, may control or impact one or more aspects of gene expression (e.g., cell-type-specific expression).
  • identity refers to the overall relatedness between polymeric molecules, e.g., between nucleic acid molecules (e.g., DNA molecules) and/or between polypeptide molecules.
  • % sequence identity refers to a relationship between two or more sequences, as determined by comparing the sequences.
  • identity also means the degree of sequence relatedness between protein and nucleic acid sequences as determined by the match between strings of such sequences.
  • Identity (often referred to as “similarity”) can be readily calculated by known methods, including those described in: Computational Molecular Biology (Lesk, A. M. ed.) Oxford University Press, NY (1988); Biocomputing: Informatics and Genome Projects (Smith, D. W. ed.) Academic Press, NY (1994); Computer Analysis of Sequence Data, Part I (Griffin, A.
  • the percent identity between the two sequences is a function of the number of identical positions shared by the sequences, optionally accounting for the number of gaps, and the length of each gap, which may need to be introduced for optimal alignment of the two sequences.
  • the comparison of sequences and determination of percent identity between two sequences can be accomplished using a computational algorithm, such as BLAST (basic local alignment search tool).
  • Sequence 12621818v1 Page 84 of 147 Attorney Docket: 2014191-0036 alignments and percent identity calculations may be performed using the Megalign program of the LASERGENE bioinformatics computing suite (DNASTAR, Inc., Madison, Wisconsin).
  • Modification status or “histone modification status” of a genomic locus refers to the frequency with which DNA sequences corresponding to the genomic locus are identified in an assay for detection of DNA sequences associated with histones bearing one or more histone modifications (e.g., one or more particular histone modifications) and/or the density (e.g., the measured density) of histone modifications (e.g., one or more particular histone modifications) corresponding to the genomic locus. Modification status can be determined by various assays known in the art, including without limitation ChIP-seq as one example.
  • a regulatory sequence is a nucleic acid sequence that controls expression of a coding sequence, e.g., a promoter sequence or an enhancer sequence. In some embodiments, a regulatory sequence can control or impact one or more aspects of gene expression (e.g., cell-type-specific expression, inducible expression, etc.).
  • Subject As used herein, the term “subject” refers to an organism, typically a mammal (e.g., a human).
  • a subject is suffering from a disease, disorder or condition (e.g., breast cancer, small cell lung cancer or SCLC, non-small cell lung cancer or NSCLC, neuroendocrine prostate cancer or NEPC, prostate adenocarcinoma or PRAD, etc.).
  • a subject is susceptible to a disease, disorder, or condition.
  • a subject displays one or more symptoms or characteristics of a disease, disorder or condition.
  • a subject is not suffering from a disease, disorder or condition.
  • a subject does not display any symptom or characteristic of a disease, disorder, or condition.
  • a subject has one or more features characteristic of susceptibility to or risk of a disease, disorder, or condition.
  • a subject is a subject that has been tested for a disease, disorder, or condition, and/or to whom therapy has been administered.
  • a human subject can be interchangeably referred to as a “patient” or “individual”. References herein to a subject that is a “cancer patient” or a “breast cancer patient”, etc. are not intended to limit the subject to a subject that has been diagnosed with cancer, breast cancer, etc. and is intended to encompass any of the aforementioned subjects.
  • therapeutic agent refers to any agent that elicits a desired pharmacological effect when administered to a subject.
  • an agent is considered to be a therapeutic agent if it demonstrates a statistically significant effect across an appropriate population.
  • the appropriate population can be a population of model organisms or a human population.
  • an appropriate population can be defined by various criteria, such as a certain age 12621818v1 Page 86 of 147 Attorney Docket: 2014191-0036 group, gender, genetic background, preexisting clinical conditions, etc.
  • a therapeutic agent is a substance that can be used for treatment of a disease, disorder, or condition (e.g., breast cancer, small cell lung cancer or SCLC, non-small cell lung cancer or NSCLC, neuroendocrine prostate cancer or NEPC, prostate adenocarcinoma or PRAD, etc.).
  • a therapeutic agent is an agent that has been or is required to be approved by a government agency before it can be marketed for administration to humans.
  • a therapeutic agent is an agent for which a medical prescription is required for administration to humans.
  • a therapeutic agent is an ADC.
  • a therapeutically effective amount is one that reduces the incidence and/or severity of, and/or delays onset of, one or more symptoms of the disease, disorder, and/or condition.
  • a therapeutically effective amount does not in fact require successful treatment be achieved in a particular individual. Rather, a therapeutically effective amount may be that amount that provides a particular desired pharmacological response in a significant number of subjects when administered to patients in need of such treatment.
  • reference to a therapeutically effective amount may be a reference to an amount as measured in one or more specific tissues (e.g., a tissue affected by the disease, disorder or condition) or fluids (e.g., blood, saliva, serum, sweat, tears, urine, etc.).
  • tissue e.g., a tissue affected by the disease, disorder or condition
  • fluids e.g., blood, saliva, serum, sweat, tears, urine, etc.
  • a therapeutically effective amount of a particular agent or therapy may be formulated and/or administered in a single dose.
  • a therapeutically effective amount of a particular agent or therapy may be formulated and/or administered in a plurality of doses, for example, as part of a dosing regimen.
  • such treatment can be of a subject who does not exhibit signs of the relevant disease, disorder, or condition and/or of a subject who exhibits only early signs of the disease, disorder, or condition (e.g., breast cancer, small cell lung cancer or SCLC, non-small cell lung cancer or NSCLC, neuroendocrine prostate cancer or NEPC, prostate adenocarcinoma or PRAD, etc.).
  • a subject who exhibits one or more established signs of the relevant disease, disorder and/or condition can be of a subject who has been diagnosed as suffering from the relevant disease, disorder, and/or condition.
  • treatment can be of a subject known to have one or more susceptibility factors that are statistically correlated with increased risk of development of the relevant disease, disorder, or condition.
  • a “prophylactic treatment” includes a treatment administered to a subject who does not display signs or symptoms of a condition to be treated or displays only early signs or symptoms of the condition to be treated such that treatment is administered for the purpose of diminishing, preventing, or decreasing the risk of developing the condition. Thus, a prophylactic treatment functions as a preventative treatment against a condition.
  • a “therapeutic treatment” includes a treatment administered to a subject who displays symptoms or signs of a condition and is administered to the subject for the purpose of reducing the severity or progression of the condition.
  • Chromatin immunoprecipitation for histone marks (H3K4me3 and H3K27ac) in plasma samples was performed using methods similar to those previously described in Sadeh et al., Nat Biotechnol (2021) 39:586–598; Jang et al., Life Sci Alliance (2023) 6(12):e202302003; and Baca et al., Nat Medicine (2023) 29:2737-2741. Briefly, about 1 mL frozen plasma was thawed and then prepared for ChIP.
  • ChIP-seq data analysis [0272] ChIP-sequencing reads were aligned to the human genome build hg19 using the Burrows-Wheeler Aligner (BWA) version 0.7.15. Non-uniquely mapping and redundant reads were discarded. MACS v2.2.7.1 was used for peak calling with a q-value (FDR) threshold of 0.01.
  • I ndication Gene (Target) Genomic coordinates 12621818v1 Page 90 of 147 Attorney Docket: 2014191-0036 S CLC TACSTD2 (TROP2) chr1:59,040,748-59,043,452 SCLC FOLH1 (PSMA) chr11:49,228,902-49,230,855 genes that modulate ADC response/resistance.
  • I ndication Gene Protein Genomic coordinates 12621818v1 Page 91 of 147 Attorney Docket: 2014191-0036 S CLC (Fig. 54) ABCB1 (MDR1) chr7:87,220,000-87,260,500 SCLC (Fig.
  • ABCG2 (BCRP) chr4:89,077,500-89,081,500 ct genes for ADC target antigens we defined a peak within +/- 10kb of the transcription site (TSS) for each gene. If there was no peak, the entire region within +/- 10kb of the TSS was used. If there were multiple peaks or multiple TSSs the peak with the largest differential between samples from cancer patients and healthy volunteers was selected. For simplicity, these peaks or regions are called “regions of interest” below. Exemplary genomic coordinates of the H3K27ac regions of interest for select genes for ADC target antigens are provided in Table 3. As a control, H3K27ac epigenomic activation signals were also measured at housekeeping genes using a similar approach.
  • Table 3 Exemplary genomic coordinates of H3K27ac regions of interest for select genes for ADC target antigens.
  • I ndication Gene (Target) Genomic coordinates 12621818v1 Page 92 of 147 Attorney Docket: 2014191-0036 B reast cancer F3 (Tissue factor) chr1:95,005,901-95,008,635 Breast cancer FOLR1 chr11:71,910,584-71,911,554 12621818v1 Page 93 of 147 Attorney Docket: 2014191-0036 N EPC/PRAD DLL3 Chr19:39,985,409-39,994,931 NEPC/PRAD SEZ6 Chr17:27,305,415-27,356,950 on of all base pairs covered by any peak in any of the cancer patient or healthy volunteer plasma samples.
  • This set of regions was then combined with the set of “regions of interest” defined above to produce a set of “enriched regions”.
  • the number of sequencing fragments (reads) overlapping each enriched region were quantified for each analyte.
  • Counts of reads in all enriched regions between experiments were quantile normalized together.
  • Quantile normalized counts of reads in the regions of interest were corrected for local ChIP-seq background to improve signal-to-noise. Promoter and enhancer epigenomic activation signals were ctDNA corrected independently.
  • Fig. 1 To correct for ctDNA% we used the ichorCNA estimated values for each sample and regressed the log of the normalized, corrected counts against logit-transformed estimated ctDNA% with standard linear regression, and then subtracted the estimated percent of each count due to ctDNA% based on its regression weight. Corrected enhancer and promoter counts were summed for each gene to produce an integrated activation score. The mean and standard-deviation of the summed enhancer and promoter counts within the healthy volunteers were used to calculate a z-score for each patient sample, which was then logged and 0-1 scaled for the final activation score. [0276] Fig.
  • Fig. 9 shows a qualitative track view displaying promoter (H3K4me3) signals across SCLC-relevant ADC target antigens (plus housekeeping genes GAPDH and ACTB). Rows represent select SCLC patient plasma samples sorted by ichorCNA ctDNA fraction estimate or healthy volunteer plasma samples.
  • FIG. 9 shows a quantitative heatmap of normalized and ctDNA-corrected epigenomic activation score (combining enhancer and promoter signals) for each ADC target antigen.
  • Patient-specific epigenomic activation scores for each gene are represented on a white (low) to dark (high) gradient.
  • Figs. 10-18 show trend lines and confidence intervals for promoter signals (H3K4me3; left graph) and enhancer signals (H3K27ac; right graph) based on ctDNA% for individual SCLC-relevant ADC target antigens.
  • Fig. 10-18 show trend lines and confidence intervals for promoter signals (H3K4me3; left graph) and enhancer signals (H3K27ac; right graph) based on ctDNA% for individual SCLC-relevant ADC target antigens.
  • Fig. 10-18 show trend lines and confidence intervals for promoter signals (H3K4me3; left graph) and enhancer signals (H3K27ac; right graph) based on
  • FIG. 19 shows a qualitative track view displaying promoter (H3K4me3) signals across NSCLC-relevant ADC target antigens (plus housekeeping genes GAPDH and ACTB). Rows represent select NSCLC patient plasma samples sorted by ichorCNA ctDNA fraction estimate or healthy volunteer plasma samples.
  • Fig. 19 shows a quantitative heatmap of normalized and ctDNA-corrected epigenomic activation score (combining enhancer and promoter signals) for each ADC target antigen. Patient-specific epigenomic activation scores for each gene are represented on a white (low) to dark (high) gradient. [0281] Figs.
  • FIGs. 20-35 show trend lines and confidence intervals for promoter signals (H3K4me3; left graph) and enhancer signals (H3K27ac; right graph) based on ctDNA% for individual NSCLC-relevant ADC target antigens.
  • Figs. 36-51 show additional data obtained for breast cancer-relevant ADC target antigens using breast cancer patient plasma samples and healthy volunteer plasma samples. Each figure is for a different ADC target antigen identified in the top left-hand corner of the figure (e.g., TROP2 in Figure 35, ROR2 in Figure 36, etc.).
  • the left-hand image shows a qualitative track view displaying promoter (H3K4me3) signals for the gene encoding the relevant ADC target antigen and other transcripts of interest in breast cancer for context (HER2, FOXA1 and MYC) plus housekeeping genes (GAPDH and ACTB) as controls.
  • Rows represent select breast cancer patient plasma samples sorted by ichorCNA ctDNA fraction estimate or healthy volunteer plasma samples.
  • the ER and HER2 status (based on IHC) is also shown for each 12621818v1 Page 95 of 147 Attorney Docket: 2014191-0036 sample. Blue boxes identify samples for which ER status was unknown.
  • the central image shows a quantitative heatmap of normalized and ctDNA-corrected epigenomic activation score (combining enhancer and promoter signals) for the ADC target antigen compared to the other transcripts of interest in breast cancer (HER2, FOXA1 and MYC).
  • Patient-specific epigenomic activation scores for each gene are represented on a gradient (regressed out and scaled to the min and the max of the ADC target antigen in these samples).
  • the right-hand images show trend lines and confidence intervals for the promoter signal (H3K4me3; top graph) and enhancer signal (H3K27ac; bottom graph) based on ctDNA% for the relevant ADC target antigen.
  • Fig. 54 shows promoter epigenomic activation signals at select genes known to modulate ADC response/resistance in SCLC patient plasma samples. Altered expression of these genes could impart payload resistance (SLFN11: topoisomerase inhibitor resistance, TUBB3: microtubule inhibitor resistance, CCNB1) or induce payload efflux (increase in drug efflux pumps ABCG2, ABCB1).
  • SLFN11 topoisomerase inhibitor resistance
  • TUBB3 microtubule inhibitor resistance
  • CCNB1 microtubule inhibitor resistance
  • CCNB1 microtubule inhibitor resistance
  • Fig. 55 shows a qualitative track view displaying histone modification signal across NEPC and PRAD-relevant ADC target antigens (plus housekeeping genes GAPDH and ACTB) in plasma samples obtained from patients with NEPC or PRAD or healthy subjects.
  • Rows represent select NEPC or PRAD patient plasma samples sorted by 12621818v1 Page 96 of 147 Attorney Docket: 2014191-0036 ichorCNA ctDNA fraction estimate or healthy volunteer plasma samples.
  • Right-hand top plot shows H3K27ac signal at an enhancer region associated with AR in plasma samples obtained from AR+/NE- PRAD subjects. Also shown is a measure of H3K27ac signal at the locus, which demonstrates an ability to detect increased H3K27ac signal at the AR locus in prostate cancer patients as compared to healthy subjects.
  • Right-hand bottom plot shows H3K27ac signal at an enhancer region associated with DLL3 in NE+ tumors.
  • a method described herein comprises quantifying, at one or more genomic loci in a plasma sample obtained from a subject with prostate cancer, optionally in cell-free DNA (cfDNA), one or more histone modifications, wherein the one or more genomic loci are associated with a promoter or an enhancer of a gene encoding an ADC target antigen.
  • cfDNA cell-free DNA
  • the one or more genomic loci are associated with an enhancer and are located within +/- 10kb of the transcription site (TSS) of a gene encoding an ADC target antigen.
  • TSS transcription site
  • the present disclosure described a method of treating prostate cancer, where the method comprises determining the expression status of one or more ADC targets by quantifying one or more H3K27ac modifications at one or more enhancers of a gene encoding an ADC target antigen (e.g., one or more PRAD/NEPC associated target loci listed in Table 3).
  • Example 3 Correlation Between Predicted Expression and Measured Expression in Cell Lines
  • RNA-seq, ChIP-Seq, and MBD-seq assays were performed for a number of relevant cancer cell lines to measure RNA expression and determine H3K4me3-, H3K27ac-, and DNAme- modification patterns.
  • Table 4 provides genomic loci, each one of which was shown to have the indicated epigenetic modifications correlate with expression of the indicated target of interest. These loci were determined using breast cancer cell lines and were obtained by analyzing loci within a gene of interest and within 200 kB of the gene of interest.
  • Fig. 56(A) provides spearman correlations coefficients (determined by comparing predicted expression vs.
  • Fig. 56(B) shows predicted and measured expression levels of B7-H4 (VTCN1) in 35 different breast cancer cell lines.
  • Fig. 56(C) shows Spearman correlation coefficient values (determined by comparing predicted and measured expression) measured at different tumor fractions (ctDNA) in simulated plasma samples for 9 different targets. Simulated plasma samples were generated by diluting breast cancer cell line data in silico with sequencing data from healthy plasma samples. This data demonstrates that technologies described herein can predict ADC target expression with high accuracy even at lower ctDNA levels. [0294] Lastly, the loci described in Table 4 were used to predict target expression in plasma samples obtained from breast cancer patients.
  • Example 4 Prediction of DLL3 Expression using a Single Gene Model and a Multigene Model [0295] The present Example provides data demonstrating that loci described herein can be used to accurately predict DLL3 expression.
  • the present Example provides results from two models – a “Single Gene Model,” which incorporated loci that are proximal to the DLL3 gene, and a “Multigene Model,” which incorporated loci that are proximal to the DLL3 gene and loci proximal to other genes whose expression correlated with that of DLL3.
  • proximal refers to loci within a gene or within 200 kB of a gene.
  • Table 5 Loci Proximal to DLL3, in Which Epigenetic Modifications Correlate with DLL3 Expression C hr Start End Gene Analyte chr19 39990062 39991061 DLL3 H3K4me3 3 [0297]
  • the AB3IP RAB3A Interacting Protein
  • PAK5 p21 (RAC1) activated kinase 5
  • ITPRIPL2 ITPRIPL2
  • Table 6 Loci Proximal to DLL3, RAB3IP, PAK5 and ITPRIPL2, in Which Epigenetic Modifications Correlate with DLL3 Expression C hr Start End Gene Analyte 3 3 3 3 3 3 3 12621818v1 Page 99 of 147 Attorney Docket: 2014191-0036 c hr16 19119335 19120334 ITPRIPL2 H3K27ac chr16 19123335 19124334 ITPRIPL2 H3K27ac [0298] In the genomic position (positions shown relative to the hg19 genome), Gene refers to the gene that the indicated loci is proximal to, and “analyte” refers to the epigenetic modification at the loci that correlates with DLL3 expression.
  • Example 5 Predicting Expression of NEPC and PRAD Markers in Patient Plasma Samples
  • the present Example provides further data demonstrating that technologies provided herein can be used to measure expression of ADC targets.
  • the present Example demonstrates that technologies described herein can be used to measure expression of DLL3, SEZ6, and CHGA in subjects having NEPC or PRAD.
  • Plasma samples were obtained from subjects with NEPC or PRAD.
  • DLL3, SEZ6, and CHGA are markers of NEPC and are not highly expressed in PRAD. Promoter regions having H3K4me3 signal that correlated with expression of each of these markers was determined. For each sample, promoter (H3K4me3) signal at each of these promoter regions was measured and corrected for ctDNA%.
  • Results are provided in Fig. 59(A).
  • promoter signal for each of DLL3, SEZ6, and CHGA was increased in NEPC subjects as 12621818v1 Page 100 of 147 Attorney Docket: 2014191-0036 compared to PRAD subjects, demonstrating that technologies provided herein can be used to measure expression of each of these targets in subjects with prostate cancer.
  • Expression of KLK3 and KLK2 was also measured as control, as these genes are expressed in PRAD but not NEPC. Both KLK2 and KLK3 expression was found to be elevated in PRAD as compared to NEPC, again demonstrating that technologies provided herein can be used to measure ADC target expression.
  • DLL3, SEZ6, and CHGA are each markers of NEPC, they can also be used to classify subjects as having NEPC or PRAD, which can be useful, e.g., for diagnosing a subject, informing therapy selection, and/or monitoring disease progression.
  • a score for distinguishing PRAD and NEPC was developed by combining DLL3, SEZ6, and CHGA promoter signal. Results are provided in Fig.59(B). As shown, a clear difference in NEPC/PRAD score was observed in the two patient populations, demonstrating that technologies provided herein can be used to classify NEPC and PRAD subjects. Also, as shown, several subjects were found to have elevated DLL3/SEZ6/CHGA promoter signal, despite having been diagnosed with PRAD.
  • epigenetic markers may provide an alternative and/or complementary method for diagnosing subjects that, in some embodiments, may provide more accurate results as compared to histology-based methods for diagnosing subjects.
  • Table 4 describes loci with epigenetic modification levels that correlate with expression of certain ADC targets. “Target Name” indicates an ADC target, “Gene” indicates a gene in which the indicated epigenetic modification is correlated with expression, “Direct of Effect” refers to whether the association is positive or negative (meaning whether epigenetic modifications increase or decrease as expression increases), “Chromosome No.,” “Start,” and “End” provide the genomic location of the loci, and “Width” refers to the length of the genomic loci.
  • sequence location is shown relative to the positive strand of the hg19 genome. Assays described herein, however, can use sequence reads from the positive and/or negative strands. 12621818v1 Page 101 of 147 Attorney Docket: 2014191-0036 Table 4: Exemplary Genomic Loci With Epigenetic Modifications That Correlate With Expression of ADC Targets of Interest. Gene Analyte Direction of Effect Chromosome No.

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Abstract

La présente divulgation concerne, entre autres, des procédés, des kits et des systèmes qui peuvent être utilisés pour sélectionner des sujets pour un traitement avec un agent qui est dirigé vers un antigène cible ADC (par exemple, une thérapie ADC ou un radioligand), pour identifier des sous-populations de sujets qui répondent à un traitement avec un agent qui est dirigé vers un antigène cible ADC (par exemple, une thérapie ADC ou un radioligand), pour surveiller des sujets pendant un traitement avec un agent qui est dirigé vers un antigène cible ADC (par exemple, une thérapie ADC ou un radioligand), etc. Dans divers modes de réalisation, la présente divulgation concerne l'utilisation d'une ou de plusieurs de modifications d'histone, de méthylation de l'ADN, d'accessibilité de la chromatine et/ou de liaison d'un ou de plusieurs facteurs de transcription qui sont caractéristiques de l'activité promotrice et/ou activatrice qui est associée à des gènes sélectionnés pour des antigènes ou des gènes cibles ADC qui modulent la réponse/résistance ADC.
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