Methods, kits and systems for cancer diagnosis and treatment

Abstract

The present disclosure includes, among other things, methods, kits, and systems that can be used 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. In various embodiments, the present disclosure relates to the use of one or more histone modifications, DNA methylation, chromatin accessibility, and / or binding of one or more transcription factors that that are characteristic of promoter and / or enhancer activity that is associated with select genes for ADC target antigens or genes that modulate ADC response / resistance.

Description

Attorney Docket: 2014191-0036 METHODS, KITS AND SYSTEMS FOR CANCER DIAGNOSIS AND TREATMENT CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No.63 / 575,697, filed on 6 April 2024 which is incorporated herein in its entirety by this reference.BACKGROUND

[0002] Cancer ranks as the primary or secondary cause of premature death in manycountries. Antibody-drug conjugates (ADCs) were ushered into oncology clinical practice in 2000 with the FDA’s approval of Mylotarg™for the treatment of acute myeloid leukemia (AML). ADC molecules marry the precision of antibody-mediated tumor antigen targeting with potent cytotoxic agents, thereby creating a targeted delivery vehicle for malignant tumors. In this manner, 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 andimmunomodulatory agents. Since Mylotarg™’s first registration, hundreds of ADCs have beenevaluated in the investigational setting and several have made it to regulatory approval including Trodelvy®. Adcetris™, Kadcyla™, Besponsa™, Enhertu™, Padcev™, Polivy™, Blenrep™, Todelvy™, Tivdak™, Zylonta™and Elahere™.

[0003] Target antigen expression is currently used as the primary biomarker for patientselection and ADC efficacy has been shown to correlate with the level of target antigenexpression in some studies. However, IHC methods for quantifying target antigen expression areinvasive, requiring tissue biopsies. In addition, for many targets, there is limited information about their expression across a variety of tumor types, as well as intratumoral heterogeneity and evolution of expression with tumor progression and metastasis. The effects of differentintervening therapies on expression are also largely unknown. Data also suggests that there canbe clinical efficacy in patients who also have low target antigen expression, shifting toward a threshold expression that is still unknown for many target antigens over which ADCs have therapeutic efficacy.

[0004] Target assessment has also been challenging as a result of known challenges withIHC assessment / interpretation. There therefore remains a need for new biomarkers and12621818v1 Page 1 of 147Attorney Docket: 2014191-0036improved methods of selecting patients for treatment with an agent that is directed to an ADCtarget antigen (e.g., an ADC therapy or radioligand). Improved diagnostic methods would alsobetter support future clinical trials that seek to identify subpopulations of patients that respond to ADCs. They would also expand our understanding of the underlying biology of cancer and help identify new treatments. SUMMARY

[0005] The present disclosure is based, at least in part, on the demonstration that certaingenomic loci associated with select genes for ADC target antigens or genes that modulate ADCresponse / resistance have different histone modification levels (e.g., histone methylation markssuch as H3K4me3 and histone acetylation marks such as H3K27ac) in plasma samples fromcancer patients as compared to plasma samples from healthy volunteers.

[0006] The present disclosure encompasses methods, kits and systems that use theseepigenomic 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., anADC therapy or radioligand), to identify subpopulations of subjects that respond to treatmentwith an agent that is directed to an ADC target antigen (e.g., an ADC therapy or radioligand), tomonitor 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 biopsysample, e.g., a plasma sample obtained or derived from a subject. The present disclosure alsoencompasses methods where 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 present disclosure also encompasses methods, kits and systems where the genomic loci that are differentially modified based on different types of histone modifications (e.g., histone methylation marks such as H3K4me3 and histone acetylation marks such asH3K27ac) are combined into multimodal classifiers to select subjects for treatment with an agentthat is directed to an ADC target antigen (e.g., an ADC therapy or radioligand), etc. These monomodal and multimodal classifiers can provide minimally invasive ways of selecting subjects for treatment with an agent that is directed to an ADC target antigen (e.g., an ADC12621818v1 Page 2 of 147Attorney Docket: 2014191-0036therapy or radioligand), etc. that are more accurate, objective, and comprehensive than thecurrent tissue-based approaches.

[0007] The present disclosure includes, among other things, technologies for thedetermination of the activation status of genes for ADC target antigens (“ADC target status”) or genes that modulate ADC response / resistance and for the detection, monitoring, and / or treatmentof cancer (including, e.g., breast, small cell lung cancer (SCLC), non-small cell lung cancerNSCLC, neuroendocrine prostate cancer (NEPC), prostate adenocarcinoma (PRAD), etc.) basedon the activation status of these genes. In various embodiments, the present disclosure relates tothe measurement of histone modifications in a sample obtained or derived from a subject todetect and / or treat cancer (including, e.g., breast, SCLC, NSCLC, NEPC, PRAD, etc.) based onthe activation status of these genes. The present disclosure includes, among other things, histonemodification measurements in cell-free DNA (cfDNA) that are characteristic of the activationstatus of genes for ADC target antigens (“ADC target status”) or genes that modulate ADCresponse / resistance, and which in various embodiments are useful, e.g., for detecting,monitoring, selecting treatment for, and / or treating cancer (including, e.g., breast, SCLC,NSCLC, NEPC, PRAD, etc.) based on the activation status of these genes. In someembodiments, histone modification measurements in cfDNA can be used to detect or determineresistance of a cancer (e.g., breast, SCLC, NSCLC, NEPC, PRAD, etc.) to treatment with anagent that is directed to an ADC target antigen (e.g., an ADC therapy or radioligand) ortransformation of a cancer from one subtype to another. In various embodiments, the presentdisclosure includes exemplary genomic loci that are differentially modified in different cancer patients (including, e.g., breast cancer, SCLC, NSCLC, NEPC, PRAD, etc. patients) and / or between cancer patients and healthy volunteers. In various embodiments, genomic locidifferentially modified in cfDNA are or include one or more enhancers. In variousembodiments, genomic loci differentially modified in cfDNA are or include one or more promoters.

[0008] In various embodiments, a genomic locus is differentially modified if it ischaracterized by increased or decreased histone modification as compared to a reference (e.g., asample from an ADC target-negative or healthy subject). Increased or decreased histonemodification can be or include, e.g., increased or decreased histone methylation(hypermethylation or hypomethylation, respectively) of one or more particular methylation12621818v1 Page 3 of 147Attorney Docket: 2014191-0036 marks, or a combination thereof; increased or decreased pan-methylation; increased or decreased histone acetylation (hyperacetylation or hypoacetylation, respectively) of one or more particular acetylation marks, or a combination thereof; and / or increased or decreased pan-acetylation (e.g., pan-H3 acetylation). In various embodiments, histone methylation can be or include histone methylation marks selected from H3K4me1, H3K4me2, H3K4me3, or a combination thereof. In various embodiments, histone methylation can be or include H3K4me3. In various embodiments, histone acetylation can be or include histone acetylation marks selected from H3K9ac, H3K14ac, H3K18ac, H3K23ac, H3K27ac, or a combination thereof. In various embodiments, histone acetylation can be or include H3K27ac.

[0009] The present disclosure further relates, in various embodiments, to themeasurement of chromatin accessibility in cell-free DNA (cfDNA) to determine the activationstatus of genes for ADC target antigens (“ADC target status”) or genes that modulate ADC response / resistance. The present disclosure includes, among other things, chromatinaccessibility measurements in cfDNA that are characteristic of ADC target-positive cancers,which in various embodiments are useful, e.g., in detecting, monitoring, selecting treatment for,and / or treating an ADC target-positive cancer. In some embodiments, chromatin accessibilitymeasurements in cfDNA can be used to detect or determine resistance of a cancer (e.g., breast,SCLC, NSCLC, NEPC, PRAD, etc.) to treatment with an agent that is directed to an ADC targetantigen (e.g., an ADC therapy or radioligand) or transformation of a cancer from one subtype toanother. In various embodiments, the present disclosure includes genomic loci that aredifferentially accessible in ADC target-positive vs. ADC target-negative cancers. In variousembodiments, genomic loci differentially accessible in cfDNA are or include one or more enhancers. In various embodiments, genomic loci differentially accessible in cfDNA are or include one or more promoters.

[0010] In various embodiments, without wishing to be bound by any particular scientifictheory, histone acetylation (e.g., H3K27ac) corresponds and / or is correlated with chromatin accessibility. In various embodiments, without wishing to be bound by any particular scientific theory, histone methylation (e.g., H3K4me3) corresponds and / or is correlated with chromatin accessibility.

[0011] In various embodiments, a genomic locus is differentially accessible if it ischaracterized by increased or decreased chromatin accessibility as compared to a reference (e.g.,12621818v1 Page 4 of 147Attorney Docket: 2014191-0036 a sample from an ADC target-negative or healthy subject). Increased or decreased histonemodification can be or include, e.g., increased or decreased accessibility as determined byvarious chromatin accessibility assays known in the art.

[0012] The present disclosure further relates, in various embodiments, to themeasurement of transcription factor binding in cell-free DNA (cfDNA) to determine the activation status of genes for ADC target antigens (“ADC target status”) or genes that modulateADC response / resistance. The present disclosure includes, among other things, transcriptionfactor binding measurements in cfDNA that are characteristic of ADC target-positive cancers, which in various embodiments are useful, e.g., in detecting, monitoring, selecting treatment for,and / or treating an ADC target-positive cancer. In some embodiments, transcription factorbinding measurements in cfDNA can be used to detect or determine resistance of a cancer (e.g.,breast, SCLC, NSCLC, NEPC, PRAD, etc.) to a therapy or transformation of a cancer from onesubtype to another. In various embodiments, the present disclosure includes genomic loci thatare differentially bound by transcription factors in ADC target-positive vs. ADC target-negativecancers. In various embodiments, genomic loci that are differentially bound by transcriptionfactors in cfDNA are or include one or more enhancers. In various embodiments, genomic loci that are differentially bound by transcription factors in cfDNA are or include one or more promoters.

[0013] In various embodiments, without wishing to be bound by any particular scientifictheory, histone acetylation (e.g., H3K27ac) corresponds and / or is correlated with transcription factor binding. In various embodiments, without wishing to be bound by any particular scientific theory, histone methylation (e.g., H3K4me3) corresponds and / or is correlated with transcription factor binding.

[0014] In various embodiments, a genomic locus is differentially bound by transcriptionfactors if it is characterized by increased or decreased transcription factor binding as compared toa reference (e.g., a sample from an ADC target-negative or healthy subject). Increased ordecreased transcription factor binding can be or include, e.g., increased or decreasedtranscription factor binding as determined by various transcription factor binding assays known in the art.

[0015] In one aspect the present disclosure provides a method comprising quantifying, atone or more genomic loci in a biological sample, optionally in cell-free DNA (cfDNA) from a12621818v1 Page 5 of 147Attorney Docket: 2014191-0036 liquid biopsy sample, obtained or derived from a 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)associated with a promoter or an enhancer of: (I) a gene that modulates ADCresponse / resistance; and / or (II) a gene encoding an ADC target antigen.

[0016] In some embodiments, a method comprises quantifying one or more histonemodifications, 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 regionsdefined by pairs of genomic coordinates in Table 1, Table 2, Table 3, Table 4, Table 5, orTable 6.

[0017] In some embodiments, the one or more genomic loci are associated with apromoter and are located within + / - 200 kB (e.g., + / - 10 kB or + / - 1kb) of the transcription site(TSS) of a gene encoding an ADC target antigen or a gene that modulates ADCresponse / resistance, e.g., a genomic locus that is located within a region defined by a pair ofgenomic coordinates in Table 1, Table 2, Table 4, Table 5, or Table 6.

[0018] In some embodiments, the one or more genomic loci are associated with anenhancer and are located within + / - 200 kB (e.g., + / - 10kb) of the transcription site (TSS) of agene encoding an ADC target antigen or a gene that modulates ADC response / resistance, e.g., agenomic locus that is located within a region defined by a pair of genomic coordinates in Table3, Table 4, Table 5, or Table 6.

[0019] In some embodiments, the one or more genomic loci are located within + / - 200kB of the TSS of a gene encoding an ADC target antigen or a gene that modulates ADCresponse / resistance, e.g., a genomic locus that is located within a region defined by a pair ofgenomic coordinates in Table 4, Table 5, or Table 6.

[0020] In some embodiments, the subject is a breast cancer patient and the gene encodingan ADC target antigen is selected from the group consisting of AXL, CCR2, CEACAM5, CD44,CLDN6, DLL3, EGFR, FAP, FOLH1, TNFRSF10B, FN-1 , ESR1, FGFR2, B4GALNT1, GPC1,GRPR, ITGB6, IGF1R, MUC1, CDH3, CD274, PTK7, SSTR2, STEAP1, STEAP2, TM4SF1,ROR1, SEZ6, SLC34A2, TACSTD2, VTCN1, NECTIN4, ERBB3, ERBB2, EPHA5, ROR2, F3,FOLR1, CD276, MSLN, SLC39A6, LY75, LY75-CD302, CDH6, KAAG1, TPBG and MET. In12621818v1 Page 6 of 147Attorney Docket: 2014191-0036some embodiments, the subject is a breast cancer patient and the gene encoding an ADC targetantigen is selected from the group consisting of VTCN1, NECTIN4, ERBB2, EPHA5, ROR2,CDH6 and MET. In some embodiments, the subject is a breast cancer patient and the geneencoding an ADC target antigen is selected from the group consisting of TACSTD2, ERBB3, F3,CD276, LY75 and KAAG1.

[0021] In some embodiments, the subject is small cell lung cancer (SCLC) patient andthe gene encoding an ADC target antigen is selected from the group consisting of TACSTD2,FOLH1, NCAM1, ERBB3, CD276, MSLN, SEZ6, DLL3, and FAP.

[0022] In some embodiments, 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 ofTACSTD2, F3, NECTIN4, FOLR1, ERBB3, CD276, ERBB2, AXL, CEACAM5, TFRC, SLC34A2,PTK7, GPNMB, EGFR, MET and ROR2.

[0023] In some embodiments, a subject is an NEPC or PRAD patient and the geneencoding an ADC target antigen is selected from the group consisting of AR, STEAP1, CHGA,SEZ6, SSTR2, DLL3, KLK2, and KLK3.

[0024] In some embodiments, the gene encoding an ADC target gene is DLL3.

[0025] In some embodiments, 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 anenhancer of DLL3; and (b) one or more genomic loci (i) within RAB3IP, PAK5, or ITPRIPL2, orany 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.

[0026] In some embodiments, one or more genomic loci within RAB3IP, PAK5, orITPRIPL2, or any combination thereof, and / or associated with a promoter or an enhancer ofRAB3IP, PAK5, or ITPRIPL2, or any combination thereof, are located within + / - 200 kB of theTSS of RAB3IP, PAK5, or ITPRIPL2, or any combination thereof, e.g., a genomic locus that isdefined by a pair of genomic coordinates in Table 6.12621818v1 Page 7 of 147Attorney Docket: 2014191-0036

[0027] In some embodiments, the subject is a breast cancer patient. In someembodiments, the subject is a metastatic breast cancer patient.

[0028] In some embodiments, the gene that modulates ADC response / resistance isselected from the group consisting of SH3GL1, HSP90AA1, SLC46A3, ABCB1, ABCG2,SLFN11, CCNB1, and TUBB3. In some embodiments, the subject is a breast cancer patient, asmall cell lung cancer (SCLC) patient, a non-small cell lung cancer (NSCLC) patient, aneuroendocrine prostate cancer (NEPC) patient, or prostate adenocarcinoma patient (PRAD).

[0029] In some embodiments, the one or more histone modifications are quantified usinga histone modification assay that measures one or more of H3K9ac, H3K14ac, H3K18ac, H3K23ac, H3K27ac, H3K4me1, H3K4me2, H3K4me3, and pan-acetylation. In some embodiments, histone modification assay detects H3K4me3 modifications. In some embodiments, the histone modification assay detects H3K27ac modifications. In some embodiments, 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.

[0030] In some embodiments, chromatin accessibility is quantified using a chromatinaccessibility 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.

[0031] In some embodiments, the binding of one or more transcription factors isquantified using a transcription factor binding assay that detects binding of one or more of p300,mediator complex, cohesin complex, RNA pol II, FOXA1, ESR1, PR, MYC, EN1, FOXM1,KLF4, AP-2, RARa, or RUNX1. In some embodiments, the transcription factor binding assay isselected 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.

[0032] In some embodiments, the method comprises quantifying two or more of thefollowing, each at one or more of the genomic loci in cell-free DNA (cfDNA) from a liquidbiopsy sample obtained or derived from the subject: (i) one or more histone modifications, (ii)12621818v1 Page 8 of 147Attorney Docket: 2014191-0036 DNA methylation, (iii) chromatin accessibility, and (iv) transcription factor binding. In someembodiments, the method comprises quantifying two or more histone modifications for the samegene. In some embodiments, the method comprises quantifying H3K4me3 and H3K27acmodifications for the same gene. In some embodiments, the quantified H3K4me3 and H3K27acmodifications are combined to generate an activation score for the gene. In some embodiments,the quantified H3K4me3 and H3K27ac modifications are summed, optionally with weighting, togenerate an activation score for the gene. In some embodiments, the gene encodes an ADC targetantigen. In some embodiments, the target antigen is TROP2. In some embodiments, the genemodulates ADC response / resistance.

[0033] In some embodiments, the liquid biopsy sample is a plasma sample, serumsample, or urine sample. In some embodiments, the liquid biopsy sample is a plasma sample,optionally a 1 mL plasma sample.

[0034] In some embodiments, quantification of one or more histone modifications, DNAmethylation, chromatin accessibility, and / or binding of one or more transcription factors, at theone or more genomic loci as compared to a reference indicates whether an ADC target is beingexpressed and / or provides a prediction or measurement of the expression level of the ADCtarget.

[0035] In some embodiments, the reference is a predetermined threshold, a measurementfrom 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 onIHC testing, or to be cancer free. In some embodiments, the reference is a predeterminedthreshold, 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.

[0036] In some embodiments, quantification of one or more histone modifications, DNAmethylation, chromatin accessibility, and / or binding of one or more transcription factors, at theone or more genomic loci as compared to a reference indicates whether a gene that modulatesADC response / resistance is active. In some embodiments, the reference is a predeterminedthreshold, a measurement from a liquid biopsy sample, and / or a normalized value, optionally12621818v1 Page 9 of 147Attorney 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 genethat modulates ADC response / resistance. In some embodiments, the reference is apredetermined 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 acohort of subjects who have previously been determined to have high activity of a gene thatmodulates ADC response / resistance.

[0037] In some embodiments, quantification of one or more histone modifications, DNAmethylation, chromatin accessibility, and / or binding of one or more transcription factors, at theone or more genomic loci as compared to a reference indicates whether the subject is likely torespond to treatment with an ADC for the ADC target. In some embodiments, the reference is apredetermined 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 theADC for the ADC target. In some embodiments, the reference is a predetermined threshold, ameasurement 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.

[0038] In some embodiments, the method comprises quantifying one or more histonemodifications, 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 regionsdefined by pairs of genomic coordinates in Table 1, Table 2, Table 3, Table 4, Table 5, orTable 6. In some embodiments, the method comprises quantifying H3K4me3 modifications forat least 1, 2, 3, 4, 5, 10, 15, 20, or 25 genomic loci that are located within one or more regionsdefined by pairs of genomic coordinates in Table 1. In some embodiments, the methodcomprises quantifying H3K4me3 modifications for at least 1, 2, 3, 4, 5, 10, 15, 20, or 25genomic loci that are located within one or more regions defined by pairs of genomic coordinatesin Table 2. In some embodiments, the method comprises quantifying H3K27ac modificationsfor at least 1, 2, 3, 4, 5, 10, 15, 20, or 25 genomic loci that are located within one or more regionsdefined by pairs of genomic coordinates in Table 3. In some embodiments, the methodcomprises quantifying H3K27ac or H3K4me3 modifications and / or DNA methylation for at least12621818v1 Page 10 of 147Attorney Docket: 2014191-0036 1, 2, 3, 4, 5, 10, 15, 20, or 25 genomic loci that are located within one or more regions defined bypairs of genomic coordinates in Table 4. In some embodiments, the method comprisesquantifying H3K4me3 modifications for at least 1, 2, 3, 4, 5, 10, 15, 20, or 25 genomic loci thatare located within one or more regions defined by pairs of genomic coordinates in Table 5. Insome embodiments, 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.

[0039] In some embodiments, the subject has previously been determined to have cancer. Insome embodiments, the subject has previously been determined to have breast cancer, prostatecancer, or lung cancer. In some embodiments, the lung cancer is SCLC, the prostate cancer isPRAD or NEPC, or the breast cancer is metastatic breast cancer (e.g., HR+ / HER2- metastaticbreast cancer).

[0040] In some embodiments, the method further comprises administering an agent thatis directed to an ADC target antigen (e.g., an ADC therapy or radioligand) to the subject. Insome embodiments, the agent that is directed to an ADC target antigen (e.g., ADC therapy orradioligand) is selected at least in part based on a quantified level of (i) one or more histonemodifications, (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 biopsysample obtained or derived from the subject. In some embodiments, the quantified level isindicative of activity at one or more genomic loci associated with a promoter or an enhancer of agene encoding a first ADC target antigen and the selected agent that is directed to an ADC targetantigen (e.g., ADC therapy or radioligand) targets the first ADC target antigen. In someembodiments, 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. In some embodiments, the quantified level is indicative ofactivity 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: (i) includes the firstADC if increased activity of a gene associated with resistance to the first ADC is not detected;and (ii) does not include the first ADC if increased activity of a gene associated with resistanceto the first ADC is detected.12621818v1 Page 11 of 147Attorney Docket: 2014191-0036

[0041] In some embodiments, the agent that is directed to an ADC target antigen is ananti-TROP2 ADC and / or comprises a Topoisomerase I (TOPI) inhibitor moiety, optionallywherein the ADC is sacituzumab govitecan (SG). In some embodiments, the TOPI inhibitormoiety comprises SN-38.

[0042] In one aspect the present disclosure provides a method of treating a subject havingcancer 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 targetantigen (e.g., an ADC therapy or radioligand) to the subject that has been selected at least in partbased on a quantified level, at one or more genomic loci, of: (i) one or more histonemodifications, (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 sampleobtained or derived from the subject, wherein the one or more genomic loci are (a) within a geneencoding an ADC target antigen or a gene that modulates ADC response / resistance and / or (b) areassociated with a promoter or an enhancer of a gene encoding an ADC target antigen or a genethat modulates ADC response / resistance. In some embodiments, the one or more genomic lociare: (a) within a gene encoding an ADC target antigen or within a gene that modulates ADCresponse / resistance; and / or (b) are associated with a promoter or an enhancer of:(I) a gene thatmodulates ADC response / resistance; and / or (II) a gene encoding an ADC target antigen.

[0043] In one aspect the present disclosure provides, a method of determining whether asubject 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.

[0044] In some embodiments, the gene that modulates ADC response / resistance isselected from the group consisting of SH3GL1, HSP90AA1, SLC46A3, ABCB1, ABCG2,SLFN11, CCNB1, and TUBB3.12621818v1 Page 12 of 147Attorney Docket: 2014191-0036

[0045] In some embodiments, the quantification of (i) one or more histone modifications,(ii) DNA methylation, (iii) chromatin accessibility, and / or (iv) transcription factor binding at oneor more of the genomic loci in cell-free DNA (cfDNA) from a liquid biopsy sample obtained orderived from the subject was performed in accordance with a method described herein. In someembodiments, the method further comprises quantifying (i) one or more histone modifications,(ii) DNA methylation, (iii) chromatin accessibility, and / or (iv) transcription factor binding at oneor more of the genomic loci in cell-free DNA (cfDNA) from a liquid biopsy sample obtained orderived from the subject in accordance with a method described herein. In some embodiments,the quantified level is indicative of activity at one or more genomic loci associated with apromoter or an enhancer of a gene encoding a first ADC target antigen and the selected agentthat is directed to an ADC target antigen (e.g., ADC therapy or radioligand) targets the first ADCtarget antigen. In some embodiments, the quantified level is indicative of activity at one or moregenomic loci associated with a promoter or an enhancer of a gene that modulatesresponse / resistance to a first ADC and the selected ADC therapy does not include the first ADC.

[0046] In one aspect the present disclosure provides a method of monitoring a subjectreceiving an agent that is directed to an ADC target antigen (e.g., an ADC therapy orradioligand) during a treatment period, 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 describedherein at first and second time points during the treatment period. In some embodiments, theagent that is directed to an ADC target antigen (e.g., ADC therapy or radioligand) is modified (e.g., the ADC therapy or radioligand is discontinued, the ADC used in the ADC therapy orradioligand is changed to a different ADC or radioligand, the dose of the ADC used in the ADCtherapy or the radioligand is increased or decreased, the frequency of administration of the ADC used in the ADC therapy or the radioligand is increased or decreased) after the second time pointat least in part based on a quantified level of (i) one or more histone modifications, (ii) DNAmethylation, (iii) chromatin accessibility, and / or (iv) transcription factor binding at the one ormore of the genomic loci in cell-free DNA (cfDNA) from a liquid biopsy sample obtained or derived from the subject at the second time point.12621818v1 Page 13 of 147Attorney Docket: 2014191-0036

[0047] In one aspect the present disclosure provides a kit comprising reagents forquantifying 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 genomiccoordinates in Table 1, Table 2, Table 3, Table 4, Table 5, or Table 6. In some embodiments,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 ofgenomic coordinates in Table 1. In some embodiments, the kit comprises reagents forquantifying H3K4me3 modifications for at least 1, 2, 3, 4, 5, 10, 15, 20, or 25 genomic loci thatare located within one or more regions defined by pairs of genomic coordinates in Table 2. Insome embodiments, 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 regionsdefined by pairs of genomic coordinates in Table 3. In some embodiments, the kit comprisesreagents for quantifying H3K4me3 or H3K27ac 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 bypairs of genomic coordinates in Table 4. In some embodiments, the kit comprises reagents forquantifying H3K4me3 modifications for at least 1, 2, 3, 4, 5, 10, 15, 20, or 25 genomic loci thatare located within one or more regions defined by pairs of genomic coordinates in Table 5. Insome 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 oneor more regions defined by pairs of genomic coordinates in Table 6. In some embodiments, thekit comprises one or more antibodies for use in ChIP-seq, optionally wherein the one or moreantibodies specifically bind H3K4me3- or H3K27ac-modified histones. In some embodiments,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 someembodiments, kit comprises reagents for sequencing.

[0048] In one aspect the present disclosure provides a non-transitory computer readablestorage medium encoded with a computer program, wherein the program comprises instructionsthat when executed by one or more processors cause the one or more processors to performoperations to perform a method described herein.12621818v1 Page 14 of 147Attorney Docket: 2014191-0036

[0049] In one aspect the present disclosure provides a computer system comprising amemory 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.

[0050] In one aspect the present disclosure provides a system comprising a sequencerconfigured to generate a sequencing dataset from a sample; and the non-transitory computerreadable storage medium and / or the computer system. In some embodiments, the sequencer isconfigured to generate a Whole Genome Sequencing (WGS) dataset from the sample. In someembodiments, the system further comprises a sample preparation device configured to preparethe sample for sequencing from a biological sample, optionally a liquid biopsy sample. In someembodiments, the sample preparation device comprises reagents for quantifying one or morehistone modifications, DNA methylation, chromatin accessibility, and / or binding of one or moretranscription factors at one or more genomic loci in cell-free DNA (cfDNA) from the biologicalsample, optionally the liquid biopsy sample. In some embodiments, the device comprisesreagents 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 thatare located within one or more regions defined by pairs of genomic coordinates in Tables 1-6 .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 regionsdefined by pairs of genomic coordinates in Table 1. In some embodiments, the devicecomprises 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 genomiccoordinates in Table 2. In some embodiments, the device comprises reagents for quantifyingH3K27ac modifications for at least 1, 2, 3, 4, 5, 10, 15, 20, or 25 genomic loci that are locatedwithin one or more regions defined by pairs of genomic coordinates in Table 3. In someembodiments, 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 least1, 2, 3, 4, 5, 10, 15, 20, or 25 genomic loci that are located within one or more regions defined bypairs of genomic coordinates in Table 5. In some embodiments, the device comprises reagents for quantifying H3K27ac or H3K4me3 modifications for at least 1, 2, 3, 4, 5, 10, 15, 20, or 2512621818v1 Page 15 of 147Attorney Docket: 2014191-0036 genomic loci that are located within one or more regions defined by pairs of genomic coordinatesin Table 6. In some embodiments, 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. In some embodiments, the device comprises reagents for isolation of cell-freeDNA (cfDNA) from the biological sample, optionally the liquid biopsy sample. In someembodiments, the device comprises reagents for library preparation for sequencing. In someembodiments, the sequencer comprises reagents for sequencing. BRIEF DESCRIPTION OF THE DRAWING

[0051] Fig. 1 (A) shows a qualitative track view displaying promoter (H3K4me3) signalsacross breast cancer-relevant ADC target antigens (plus housekeeping genes GAPDH andACTB). Rows represent select breast cancer patient plasma samples sorted by ichorCNA ctDNAfraction estimate or healthy volunteer plasma samples. Fig. 1 (B) shows a quantitative heatmapof normalized and ctDNA-corrected epigenomic activation score (combining enhancer andpromoter signals) for each ADC target antigen. Patient-specific epigenomic activation scores foreach gene are represented on a white (low) to dark (high) gradient.

[0052] Figs. 2-8 show graphs showing trend lines and confidence intervals for promotersignals (H3K4me3; (A) in each of Figs. 2-8) and enhancer signals (H3K27ac; (B) in each ofFigs. 2-8) relative to ctDNA% for individual breast cancer-relevant ADC target antigens.

[0053] Fig. 9 (A) shows a qualitative track view displaying promoter (H3K4me3) signalsacross SCLC-relevant ADC target antigens (plus housekeeping genes GAPDH and ACTB).Rows represent select SCLC patient plasma samples sorted by ichorCNA ctDNA fractionestimate or healthy volunteer plasma samples. Fig. 9 (B) shows a quantitative heatmap ofnormalized and ctDNA-corrected epigenomic activation score (combining enhancer andpromoter signals) for each ADC target antigen. Patient-specific epigenomic activation scores foreach gene are represented on a white (low) to dark (high) gradient.

[0054] Figs. 10-18 show graphs showing trend lines and confidence intervals forpromoter signals (H3K4me3; (A) in each of Figs. 10-18) and enhancer signals (H3K27ac; (B) ineach of Figs. 10-18) relative to ctDNA% for individual SCLC-relevant ADC target antigens.

[0055] Fig. 19 (A) shows a qualitative track view displaying promoter (H3K4me3)signals across NSCLC-relevant ADC target antigens (plus housekeeping genes GAPDH and12621818v1 Page 16 of 147Attorney Docket: 2014191-0036ACTB). Rows represent select NSCLC patient plasma samples sorted by ichorCNA ctDNAfraction estimate or healthy volunteer plasma samples. Fig. 19 (B) shows a quantitative heatmapof normalized and ctDNA-corrected epigenomic activation score (combining enhancer andpromoter signals) for each ADC target antigen. Patient-specific epigenomic activation scores foreach gene are represented on a white (low) to dark (high) gradient.

[0056] Figs. 20-35 show graphs showing trend lines and confidence intervals forpromoter 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.

[0057] Figs. 36-51 show data obtained for breast cancer-relevant ADC target antigensusing breast cancer patient plasma samples and healthy volunteer plasma samples. Each figure isfor 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.). In each figure, (A) shows a qualitative trackview displaying promoter (H3K4me3) signals for the gene encoding the indicated ADC targetantigen 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 cancerpatient plasma samples sorted by ichorCNA ctDNA fraction estimate or healthy volunteerplasma samples. The ER and HER2 status (based on IHC) is also shown for each sample. Blueboxes identify samples for which ER status was unknown. In each figure, (B) shows aquantitative heatmap of normalized and ctDNA-corrected epigenomic activation score (combining enhancer and promoter signals) for the indicated ADC target antigen compared tothe other transcripts of interest in breast cancer (HER2, FOXA1 and MYC). Patient-specificepigenomic activation scores for each gene are represented on a gradient (regressed out andscaled to the min and the max of the ADC target antigen in these samples). (C) and (D) in eachfigure show graphs showing trend lines and confidence intervals for the promoter signal(H3K4me3) and enhancer signal (H3K27ac), respectively, relative to ctDNA% for the indicatedADC target antigen.

[0058] Fig. 52 show graphs with trend lines and confidence intervals for the promotersignal (H3K4me3; (A)) and enhancer signal (H3K27ac; (B)) relative to ctDNA% for certaintranscripts of interest in breast cancer (HER2, FOXA1 and MYC).

[0059] Fig. 53 shows qualitative track view promoter epigenomic activation signals atselect genes known to modulate ADC response / resistance in SCLC patient plasma samples.12621818v1 Page 17 of 147Attorney Docket: 2014191-0036 Altered expression of these genes could impart payload resistance (SLFN11: topoisomeraseinhibitor resistance, TUBB3: microtubule inhibitor resistance, CCNB1) or induce payload efflux(increase in drug efflux pumps ABCG2, ABCB1). Boxed activation signals illustrate exemplarypatterns associated with predicted sensitivity to an ADC.

[0060] Fig. 54 show qualitative track views showing promoter epigenomic activationsignals at select genes known to modulate ADC response / resistance in breast cancer, NSCLCand SCLC patient plasma samples. Altered expression of these genes could impair ADC internalization (chaperone proteins Endophilin A2, HSP90), disrupt lysosomal processing (SLC46A3), induce payload efflux (increase in drug efflux pumps ABCG2, ABCB1) or impart payload resistance (SLFN11: topoisomerase inhibitor resistance, CCNB1).

[0061] Fig. 55 (A) shows a qualitative track view displaying histone modification signalacross certain NEPC and PRAD-relevant ADC target antigens (plus housekeeping geneGAPDH) in plasma samples obtained from patients with NEPC or PRAD or healthy subjects.Rows represent select NEPC or PRAD patient plasma samples sorted by ichorCNA ctDNAfraction estimate or healthy volunteer plasma samples. (B) shows H3K27ac signal at anenhancer 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 detectincreased H3K27ac signal at the AR locus in prostate cancer patients as compared to healthysubjects. (C) shows H3K27ac signal at an enhancer region associated with DLL3 in NE+tumors. Also shown is a measure of H3K27ac signal at the locus, which demonstrates an abilityto detect increased H3K27ac signal at the DLL3 locus in NEPC subjects as compared to PRAD subjects.

[0062] Fig. 56 shows graphs predicting expression of clinically relevant drug targets inbreast cancer. (A) Shows the performance of methods described herein for predicting theexpression of certain drug targets at 10% ctDNA fraction, including targets of antibody-drugconjugates (ADCs), hormone therapy, and other oncology drugs. Each point represents a gene,with the Spearman correlation coefficient between actual expression (as measured by RNA-seq)and predicted expression (as calculated using cfDNA epigenomic features) plotted on the x-axisand model AUC plotted on the y-axis. (B) Shows validation of B7-H4 (VTCN1) expressionpredictions across 35 breast cancer cell lines at 10% ctDNA. Each point represents a differentcell line. Plotted on the x-axis is actual RNA expression (as measured by RNA-seq) and plotted12621818v1 Page 18 of 147Attorney Docket: 2014191-0036on 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 certaingenes. Plotted on the x-axis of each graph is tumor (ctDNA) fraction. Plotted on the y-axis ofeach graph is the Spearman correlation coefficient between RNA expression (as measured by RNA-seq) and predicted expression (as measured using epigenomic features). The horizontal line in each graph indicates the Spearman correlation coefficient at a tumor fraction of 0.10. Regression lines indicate model fit, and shaded regions represent confidence intervals.

[0063] Fig. 57 provides scatter plots that show expression predicted using epigeneticmodifications in plasma samples (y-axis) vs. RNA-seq expression values measured in matchedtumor biopsies (x-axis) for the 3 indicated genes. N=12, ctDNA >3%. Regression lines indicatemodel fit, and shaded regions represent confidence intervals.

[0064] Fig. 58 provides a graph depicting correlation between certain loci and DLL3expression at varying ctDNA%. “Pearson’s r” refers to the Pearson correlation coefficientdetermined by comparing predicted expression (as determined using epigenetic modifications) in(a) plasma samples having a high ctDNA fraction (30% ctDNA), and (b) the same samplesserially diluted in silico with healthy plasma sequencing data. “Single Gene Model” (purple datapoints, connected by the bottom purple line) refers to prediction values generated using a modelthat incorporates only loci that are proximal to the DLL3 locus. “Multigene Model” (blackpoints connected by the top black line) refers to prediction values determined using a model thatincorporates loci that are proximal to DLL3 and several additional genes whose expression iscorrelated with that of DLL3. DLL3 expression prediction was found to be improved (includingimproved model precision, i.e., a stronger correlation between expression predictions at a highctDNA fraction and lower ctDNA%) by incorporating additional loci of genes whose expressionis correlated with that of DLL3.

[0065] Fig. 59 shows data related to measuring epigenetic markers associated with ADCtargets and NEPC / PRAD markers in patient plasma samples. (A) Provides measures of promoter signal (H3K4me3), corrected for ctDNA%, measured at a promoter region of each of DLL3,SEZ6, CHGA, KLK3, and KLK2 in plasma samples obtained from patients diagnosed withneuroendocrine prostate cancer (NEPC) or prostate adenocarcinoma (PRAD). (B) Shows acombined 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 patients12621818v1 Page 19 of 147Attorney Docket: 2014191-0036having a high DLL3 / SEZ6 / CHGA score despite having been diagnosed with PRAD, indicatingthat these patients may have been misdiagnosed. In each graph, left box-and-whiskers barindicates signal measured in NEPC subjects, and right box-and-whiskers bar indicates signalmeasured in PRAD subjects, and each point represents a measurement from an individual patient. DETAILED DESCRIPTION

[0066] The present disclosure is based, at least in part, on the demonstration that certaingenomic loci associated with select genes for ADC target antigens or genes that modulate ADCresponse / 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.

[0067] The present disclosure encompasses methods, kits and systems that use theseepigenomic 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., anADC therapy or radioligand), to identify subpopulations of subjects that respond to ADCs, tomonitor 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 histonemodifications 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. The present disclosure alsoencompasses methods where chromatin accessibility and / or binding of one or more transcriptionfactors are detected at the one or more genomic loci instead of (or in addition to) histone modifications. The present disclosure also encompasses methods, kits and systems where the genomic loci that are differentially modified based on different types of histone modifications (e.g., histone methylation marks such as H3K4me3 and histone acetylation marks such asH3K27ac) are combined into multimodal classifiers to select subjects for treatment with an agentthat is directed to an ADC target antigen (e.g., an ADC therapy or radioligand), etc. Thesemonomodal and multimodal classifiers can provide minimally invasive ways of selecting subjects for treatment with an agent that is directed to an ADC target antigen (e.g., an ADCtherapy or radioligand), etc. that are more accurate, objective, and comprehensive than thecurrent tissue-based approaches.12621818v1 Page 20 of 147Attorney Docket: 2014191-0036 Antibody-drug conjugates (ADCs) and ADC target antigens

[0068] An antibody-drug conjugate (ADC) includes an antibody or antibody fragmentlinked to a chemotherapeutic agent or “payload”. The antibody or antibody fragment acts like ahoming signal by attaching to an ADC target antigen on cancer cells, bringing thechemotherapeutic agent directly to the cancer cells. While most ADC clinical candidates utilizecytotoxic chemotherapeutic payloads, recent ADC candidates have also incorporated targeted small molecules and immunomodulatory agents.

[0069] Genes for ADC target antigens (and the encoded ADC target antigen) include,without limitation, TACSTD2 (TROP2), VTCN1 (B7H4 or B7-H4), NECTIN4, ERBB3(HER3), ERBB2 (HER2), EPHA5, ROR1, ROR2, F3 (Tissue factor or TF), FOLR1 (FRa),CD276 (B7H3 or B7-H3), MSLN (Mesothelin), SLC39A6 (LIV1 or LIV-1), LY75 (CD205),LY75-CD302, CDH6 (Cadherin-6), KAAG1 (Kidney associated antigen 1), TPBG (5T4 or 5T-4), MET (HGF receptor), FOLH1 (PSMA), NCAM1 (CD56), SEZ6, DLL3, FAP (FAP-alpha), AXL (UFO), CEACAM5, TFRC (CD71), SLC34A2 (NaPi2b), PTK7 (PTK-7 or CCK4),GPNMB (NMB), CCR2, CD44, CLDN6, TNFRSF10B (DR-5), FN-1 (EDB), ESR1 (ER),FGFR2, B4GALNT1 (GD2), GPC1 (GPC-1), GRPR, ITGB6 (IB-6 or integrin β6), IGF1R (IGF-1R), MUC1, CDH3 (P-CAD), CD274 (PD-L1), SSTR2, STEAP1, STEAP2, TM4SF1, CHGA(CgA), and EGFR. A number of ADCs are either approved or in development for these ADCtarget antigens. See Maecker et al., mAbs (2023) 15(1):2229101 and Fang et al., Cancer Gene Therapy (2024) 31(2):273-284 which are both incorporated herein by reference in their entirety.

[0070] ADCs that have been approved include sacituzumab govitecan (TROP2),enfortumab vedotin (NECTIN4), trastuzumab emtansine and trastuzumab deruxtecan (HER2),tisotumab vedotin (Tissue factor) and Mirvetuximab soravtansine-gynx (FOLR1). Sacituzumabgovitecan is an ADC that includes the TROP2-targeted antibody sacituzumab conjugated to thechemotherapeutic 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). Trastuzumab emtansine is an ADC that includes the HER2-targetedantibody 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 (when12621818v1 Page 21 of 147Attorney Docket: 2014191-0036chemotherapy and trastuzumab were given before surgery, and there was cancer still present atthe time of surgery), or to treat advanced breast cancer in women who have already been treatedwith trastuzumab and chemotherapy. This therapeutic agent is administered intravenously.Trastuzumab deruxtecan is an ADC that includes the HER2-targeted antibody trastuzumabconjugated 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 thatcannot 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 isadministered intravenously. Trastuzumab deruxtecan can also be used to treat HER2-low breastcancers 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 finishingadjuvant chemotherapy. Tisotumab vedotin is an ADC that includes the Tissue factor-targetedantibody tisotumab conjugated to the chemotherapeutic agent MMAE (microtubule targeting).Mirvetuximab soravtansine-gynx is an ADC that includes the FOLR1-targeted antibodymirvetuximab conjugated to the chemotherapeutic agent DM4, a maytansinoid derivative(microtubule targeting).

[0071] 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., PF-08046054 / SGN-PDL1V), SSTR2 (e.g., PEN-221), STEAP1(e.g., DSTP3086S and DXC008), STEAP2 (e.g., H1H7814N), and TM4SF1 (e.g., AGX101).

[0072] MK-2140 comprises a monoclonal antibody targeting ROR1 (Zilovertamabvedotin) conjugated to MMAE. NBE-002 comprises a humanized anti-ROR1 antibodyenzymatically-conjugated to the anthracycline PNU-159682 using a sortase A-mediated site-specific transpeptidation reaction. CS5001 comprises a human anti-ROR1 monoclonal antibody,a β-glucuronide linker, and a pyrrolobenzodiazepine prodrug, which is only metabolized afterreaching a tumor.

[0073] 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 IFN12621818v1 Page 22 of 147Attorney Docket: 2014191-0036 response, reprogramming of suppressive intratumoral CCR2+ myeloid cells, and blockade of suppressive tumor-associated macrophage recruitment.

[0074] Bivatuzumab mertansine (BIWI 1) comprises a monoclonal antibody targetingisoform 6 of CD44 (CD44v6) conjugated to a thiol-containing maytansinoid (a highly potentanti-microtubule agent). A clinical trial evaluating BIWI 1 was terminated due to potent skin toxicity of the drug (Riechelmann et al., Oral Oncol, 2008.44(9):823-9). Potential improved ADC delivery routes could improve the safety profile of BIWI 1, thereby rendering it a useful anti-cancer therapeutic.

[0075] TORL-1-32 comprises an MMAE moiety conjugated to an anti-CLDN6 fullyhumanized monoclonal antibody. TORL-1-32 ADC has a favorable safety / tolerability profile andpharmacokinetic (PK) characteristics and has demonstrated preliminary antitumor activity inpatients 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 humanizedanti-CLDN6 antibody conjugated to modified pyrrolobenzodiazepine. DS-9606 has also shownfavorable safety and preliminary efficacy results in ovarian, gastric, NSCLC, giant cell tumor, breast, and endometrial cancer patients. Dose-escalation studies investigating with both TORL-1- 32 and DS-9606 are currently ongoing (see, e.g., Pate et al., ESMO Annals of Oncol.2024. 54(S2, S488-489)).

[0076] Oba01 is a DR-5 (TNFRSF10) targeting ADC and is an MMAE-conjugatedZaptuzumab. Oba01 promoted pancreatic cancer tumor cell death and displayed potent antitumoractivity in mono and combinational (e.g., in combination with gemcitabine) therapies in patient-derived xenograft murine models (Zheng et al., Cell Death & Disease.2023.14(295)).

[0077] PYX-201 comprises a fully human anti-extra-domain B splice variant offibronectin 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 followingtreatment with platinum-based chemotherapy and an anti-PD-(L)1 antibody. PYX-201 wasshown 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 147Attorney Docket: 2014191-0036

[0078] BAY 1187982 (Aprutumab ixadotin) comprises a fully human anti-FGFR2monoclonal antibody linked to an auristatin W derivative, toxophore. Aprutumab ixadotin wasfound to be poorly tolerated in a phase I, first-in-human trial in patients with advanced solidtumors from cancer indications known to be FGFR2-positive.

[0079] M3554 comprises a humanized anti-GD2 antibody linked to exatecan, aTopoisomerase I inhibitor (TOP1i) payload, via a cleavable beta-glucuronide linker. A Phase Iclinical trial (NCT06641908) is currently ongoing for evaluating the safety and preliminary antitumor activity of M3554 in participants with soft tissue sarcoma (STS) and IDH-wildtype glioblastoma.

[0080] An anti-GPC1 has been created by conjugating a humanized anti-glypican-1 (anti-GPC1) antibody to MMAE (see Uchida et al., Neoplasia. 2024. 50(100982)). This GPC1-ADCwas shown to be efficiently internalized by glioblastoma cells lines and potently suppressglioblastoma tumor growth in murine subjects, supporting its potential utility for treating cancer.

[0081] Currently, only peptide drug-conjugates are being developed to target GRPR. Forexample, 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)). Nonetheless, GRPRremains a valuable therapeutic target with potential to be targeted with an ADC therapeutic.

[0082] SGN-B6A is an ITGB6-directed vedotin ADC that comprises a monomethylauristatin E (MMAE) payload. In a phase I study evaluating SGN-B6A in patients with advancesolid tumors in NSCLC, head and neck (HNSCC), and esophageal cancer (EC) indications,SGN-B6A demonstrated a manageable safety profile, and preliminary antitumor activity andresponse durability in dose escalation in a heavily pretreated patient population (see Hollebecqueet al., J Clin Oncol.2023.41(16_Supplement)).

[0083] W0101 (Lonigutamab Ugodotin) comprises an anti-IGF1R humanizedmonoclonal antibody conjugated to a dolastatin / auristatin derivative, and is designed to targetIGF1R-overexpressing tumors. W0101 has been shown to be effective in inhibiting tumorgrowth in a preclinical murine model of breast cancer, and induced potent tumor regression in several other cancer models (see Akla et al., Mol Cancer Ther.2020.19(1)).

[0084] MUC1-targeting ADCs constitute an area of high interest for targeting stromaland difficult to treat tumors, and are likely to be expanded to various metastatic cancer12621818v1 Page 24 of 147Attorney Docket: 2014191-0036indications in future clinical trials. MUC1-C-ADC comprises an anti-glycosylated Mucin 1(MUC1) monoclonal antibody (targeting the extracellular domain of MUC1), conjugated toMMAE via vc-PAB linkers. MUC1-C-ADC has been shown to effectively kill metastaticcolorectal cancer (mCRC) cells in vitro and inhibit tumor growth in mCRC murine modelswithout adverse effects (e.g., weight loss) (see Raina et al., J Clin Oncol.2024.42(16_Supplement)). A MUC1*-ADC comprising a monoclonal anti-MUC1 antibody thatrecognizes only a conformational epitope created when MUC1 is cleaved by certain tumor-associated enzymes (e.g., MNC2) and a monomethyl auristatins or Topoisomerase 1 inhibitormoiety has also been developed. See Bamdad et a., Cancer Res. 2024. 84(6_Supplement).Pancreatic cancer xenografts in mice were eliminated by MNC2-MMAE, MNC2-Deruxtecanand MNC2-exatecan ADCs, and breast cancer tumor xenografts were inhibited with a MNC2-Deruxtecan ADC). DS-3939 is another MUC1-targeting ADC, which has recently entered aPhase 1 clinical trial for locally advanced, metastatic, or unresectable urothelial, non-small celllung, breast, ovarian, biliary tract, or pancreatic cancers. 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.

[0085] 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. To supplement efficacy of a P-cadherin-only ADC, bispecific P-cadherin and CDH17 ADC conjugated to MMAE has been developed, and shown tolead to tumor regression in a CRC xenograft mouse model (Synan et al., biorXiv, 2024. 590291).

[0086] PF-08046054 / SGN-PDL1V is an investigational ADC comprising a monoclonalanti-PD-L1 antibody conjugated to MMAE via a protease-cleavable mc-vc (maleimidocaproyl-valine-citrulline) linker. Patients with advanced solid tumors are currently being enrolled in aPhase 1 clinical trial to test the safety and preliminary efficacy of PF-08046054 / SGN-PDL1Valone or in combination with pembrolizumab (NCT05208762).

[0087] PEN-221 is an emtansine-conjugated, SSTR2-targeting ADC, which has beeninvestigated in a Phase 2 clinical trial in a patient population with advanced GI mid-gutneuroendocrine tumors. PEN-221 was shown to be well-tolerated by patients, and exceededclinical efficacy goals for Clinical benefit Rate and median progression-free survival (see12621818v1 Page 25 of 147Attorney Docket: 2014191-0036 Halperin et al., J Clin Oncol.2021.39(15_Supplement). PEN-221 may also have potential utility in treating SSTR2-positive SCLC.

[0088] STEAP1 is a promising ADC target, due to its high expression in prostate cancer.DSTP3086S is an ADC that comprises a humanized anti-STEAP1 monoclonal antibodyconjugated to MMAE. DSTP3086S was evaluated in patients with metastatic castration-resistantprostate cancer and was shown to be tolerated by patients at a dose level that provides beneficialanti-tumor activity, indicating the potential benefit of treating STEAP1-expressing metastaticcastration-resistant prostate cancer with a STEAP1-targeting ADC (Danila et al., J Clin Oncol.2019. 37(36)). DXC008 is another STEAP1-targeting ADC, comprising an antibody having highbinding affinity to PSMA conjugated to a tubulysin B analogue. DXC008 was shown to be well-tolerated in preclinical investigations in mice and non-human primates, and result in durable anti-tumor response in murine prostate cancer xenograft models (Yang et al., Cancer Res. 2024.84(6_Supplement)).

[0089] H1H7814N is an early-development ADC comprising an anti-STEAP2monoclonal antibody that is conjugated to either auristatin or a maytansinoid. H1H7814N hasshown preliminary anti-tumor activity in murine models of prostate cancer, when compared to anunconjugated anti-STEAP2 antibody.

[0090] AGX101 is a first-in-class anti-TM4SF1 ADC comprising a conjugated tubulininhibitor, and targets nuclear membranes of cancer cells to deliver payloads directly to thenucleus. AGX101 has shown potent anti-tumor activity in colon and pancreatic cancer murinexenografts 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 ofAGX101 in patients with unresectable, locally advanced, or metastatic solid tumors.

[0091] Exemplary agents that target DLL3 are known in the art and are described, e.g., inRudin, Charles M., et al. "Emerging therapies targeting the delta-like ligand 3 (DLL3) in smallcell lung cancer." Journal of Hematology & Oncology 16.1 (2023): 66. Exemplary agentstargeting 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, abioengineered, DLL3-directed antibody-drug conjugate (ADC) that delivers the cytotoxic pyrrolobenzodiazepine (PBD) dimer warhead, SC-DR002, to cells expressing DLL3).12621818v1 Page 26 of 147Attorney Docket: 2014191-0036

[0092] In some embodiments, where a method of the present disclosure detects orquantifies a high level of activity (relative to a reference) and / or detects an increase in the level of activity (e.g., relative to a prior measurement during treatment) at one or more genomic lociassociated with a promoter or an enhancer of a gene encoding an ADC target, this may be usedto select an ADC or radioligand for that ADC target as an ADC therapy or radioligand for a subject.

[0093] In some embodiments, where a method of the present disclosure predicts highlevels of ADC target expression (relative to a reference) and / or detects an increase in ADC targetexpression (e.g., relative to a prior measurement during treatment) this may be used to select anADC or radioligand for that ADC target as an ADC therapy radioligand for a subject. In someembodiments, ADC selection is based on an assessment of ADC target status using a method of the present disclosure in combination with IHC testing. Genes That Modulate ADC response / resistance

[0094] In some embodiments, the methods of the present disclosure use epigenomicdifferences (alone or in combination with each other and / or with other biomarkers) that are associated with genes that modulate ADC response / resistance to select subjects for treatmentwith an agent that is directed to an ADC target antigen (e.g., an ADC therapy or radioligand), toidentify subpopulations of subjects that respond to treatment with an agent that is directed to anADC target antigen (e.g., ADCs or radioligands), to monitor subjects during treatment with anagent that is directed to an ADC target antigen (e.g., ADC therapy or radioligand), etc.

[0095] As used herein, a “gene that modulates ADC response / resistance,” refers to a geneencoding a protein that has an activity that can modulate ADC efficacy. Without limitation,examples of proteins having an activity that can modulate ADC response / resistance includeproteins that can interfere with ADC internalization, proteins that can interfere with pathwaysignaling, proteins that increase stability of an ADC target antigen, proteins involved inlysosomal transport, proteins involved in a DNA damage repair pathway, and proteins thatfunction as a drug efflux pump.

[0096] Exemplary genes that modulate ADC response / resistance (and the proteins theyencode) include SH3GL1 (Endophilin A2), HSP90AA1 (HSP90), SLC46A3, CCNB1 (CyclinB1), SLFN11, ABCB1 (MDR1), ABCG2 (BCRP), and TUBB3 (Class III beta-tubulin). In some12621818v1 Page 27 of 147Attorney Docket: 2014191-0036embodiments, epigenomic differences (alone or in combination with each other and / or with otherbiomarkers) associated with these genes may be used in combination with epigenomicdifferences (or other biomarkers, e.g., HER2 IHC test results) associated with a gene for an ADCtarget antigen (e.g., ERRB2 gene encoding HER2).

[0097] Endophilin A2 is a scaffolding protein involved in clathrin-independentendocytosis. Reduced EndophilinA2 expression has been shown to decrease HER2internalization, reduce downstream signaling, and decrease the migratory potential of the BRCAcells. 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. In some embodiments,where a method of the present disclosure predicts low levels of Endophilin A2 expression(relative to a reference) and / or detects a decrease in Endophilin A2 expression (e.g., relative to aprior measurement during treatment) this may be used to select a therapy other than a HER2- targeted ADC for a subject. In some embodiments, where 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. In some embodiments, 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 lociassociated with a promoter or an enhancer of SH3GL1. In some embodiments, HER2-targetedADC selection is also based on an assessment of HER2 status using a method of the presentdisclosure and / or based on IHC testing.

[0098] HSP90 is a chaperone protein that is important for HER2 stability (by preventingubiquitination). Increase in HSP90 results in more stable HER2, reduced internalization andreduced ADC efficacy. See Li et al., Cancer Discov (2020) 10(5):674-687 which is incorporatedherein by reference in its entirety. In some embodiments, where 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. In some embodiments, where a method of the present disclosure predicts high levels of12621818v1 Page 28 of 147Attorney 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 thana HER2-targeted ADC for a subject. In some embodiments, the selection is based in part ondetecting 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. In some embodiments, HER2-targeted ADC selection is also based on an assessment of HER2 statususing a method of the present disclosure and / or based on IHC testing.

[0099] SLC46A3 is a lysosomal membrane transporter protein relevant for efflux ofmaytansine‐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 theHER2-targeted ADC trastuzumab emtansine. See Li et al., Mol Cancer Ther (2018) 17(7):1441-1453 which is incorporated herein by reference in its entirety. In some embodiments, where a method of the present disclosure predicts low levels of SLC46A3 expression (relative to a reference) and / or detects a decrease in SLC46A3 expression (e.g., relative to a prior measurement during treatment) this may be used to select a therapy other than a HER2-targetedADC for a subject. In some embodiments, where a method of the present disclosure predicts high levels of SLC46A3 expression (relative to a reference) and / or detects an increase in SLC46A3 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. In some embodiments, 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 apromoter or an enhancer of SLC46A3. In some embodiments, HER2-targeted ADC selection isalso based on an assessment of HER2 status using a method of the present disclosure and / orbased on IHC testing.

[0100] Cyclin B1 is essential for mitosis / mitotic catastrophe. Reduced CCNB1 has beenshown to result in resistance to the HER2-targeted ADC trastuzumab emtansine. See Sabbaghiet al., Clin Cancer Res (2017) 23(22):7006-7019 which is incorporated herein by reference in its entirety. In some embodiments, where 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 expression12621818v1 Page 29 of 147Attorney 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. In some embodiments, where 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. In some embodiments, 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. In some embodiments, 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.

[0101] SLFN11 is a DNA / RNA helicase important in DNA damage response andreplication 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).In the ADC context, increased levels of SLFN11 are predictive of response to Topoisomerase I(TOP1) inhibitor payloads. See Murai et al., Pharmacol Ther (2020) 201:94-102 and Coleman etal., British Journal of Cancer (2021) 124:857-859 which are both incorporated herein by reference in their entirety. In some embodiments, where a method of the present disclosurepredicts low levels of SLFN11 expression (relative to a reference) and / or detects a decrease inSLFN11 expression (e.g., relative to a prior measurement during treatment) this may be used toselect an ADC with a payload other than one of the aforementioned anti-cancer drugs, e.g., an ADC with a payload other than a TOP1 inhibitor payload (e.g., an ADC with a payload other than camptothecin, topotecan, or irinotecan) for a subject. In some embodiments, where a method of the present disclosure predicts high levels of SLFN11 expression (relative to areference) and / or detects an increase in SLFN11 expression (e.g., relative to a prior measurementduring treatment) this may be used to select an ADC with one of the aforementioned anti-cancerdrugs, e.g., an ADC with a TOP1 inhibitor payload (e.g., an ADC with an camptothecin,topotecan, or irinotecan payload) for a subject. In some embodiments, the selection is based in12621818v1 Page 30 of 147Attorney 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.

[0102] Multi-Drug Resistance Protein 1 (MDR1) is a drug efflux pump with MMAE as asubstrate. Increased expression of MDR1 results in reduced sensitivity to ADCs with MMAEpayloads. See Liu-Kreyche et al., Front Pharmacol (2019) 10:749 and Aldonza et al., Oncotarget(2016) 7(23):34395-34419 which are both incorporated herein by reference in their entirety. Insome embodiments, where a method of the present disclosure predicts low levels of MDR1 expression (relative to a reference) and / or detects a decrease in MDR1 expression (e.g., relative to a prior measurement during treatment) this may be used to select an ADC with an MMAE payload for a subject. In some embodiments, where a method of the present disclosure predictshigh levels of MDR1 expression (relative to a reference) and / or detects an increase in MDR1expression (e.g., relative to a prior measurement during treatment) this may be used to select an ADC with a payload other than MMAE for a subject. In some embodiments, 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 ABCB1.

[0103] BCRP is a drug efflux pump. Increased expression results in reduced sensitivityto ADC payloads. See Chang et al., Mol Cancer Ther (2016) 15(8):1910-1919 which isincorporated herein by reference in its entirety. In some embodiments, where a method of thepresent disclosure predicts high levels of BCRP expression (relative to a reference) and / ordetects 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 Yamazakial., 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., CancerLett (1999) 146:117-126) for a subject. In some embodiments, the selection is based in part ondetecting 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 147Attorney Docket: 2014191-0036

[0104] Class III beta-tubulin is a microtubule protein. Increased expression is associatedwith 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. In some embodiments, where 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. In some embodiments, where a method of the present disclosure predicts high levels of Class IIIbeta-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 asubject. In some embodiments, the selection is based in part on detecting or quantifying a levelof 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. Subjects and Samples

[0105] A sample analyzed using methods, kits and systems provided herein can be anybiological sample including any processed sample that includes circulating tumor DNA (ctDNA)derived from a biological sample. In various embodiments, a sample analyzed using methods,kits and systems provided herein can be a sample obtained from a mammalian subject. Invarious embodiments, a sample analyzed using methods, kits and systems provided herein can bea sample obtained from a human subject.

[0106] In various instances, a human subject is a subject diagnosed or seeking diagnosisas having, diagnosed as, or seeking diagnosis as at risk of having, and / or diagnosed as or seekingdiagnosis 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. In various instances, a human subject is a subjectidentified as needing treatment with an agent that is directed to an ADC target antigen (e.g., anADC therapy or radioligand). In certain instances, a human subject is a subject identified asneeding ADC therapy or radioligand screening by a medical practitioner.12621818v1 Page 32 of 147Attorney Docket: 2014191-0036

[0107] The subject may not have undergone previous treatments for cancer, such as thetreatments recited in this disclosure. In other embodiments, the subject has undergone previoustreatments for cancer, such as the treatments recited in this disclosure.

[0108] In various embodiments a subject has one or more biomarkers and / or risk factorsfor 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. Incertain embodiments, a human subject is identified as in need of ADC therapy or radioligandscreening 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. In various instances, a human subject is asubject not yet diagnosed as having, not at risk of having, not at immediate risk of having, notdiagnosed as having, and / or not seeking diagnosis for a cancer. Genetic factors may alsocontribute to cancer risk, as evidenced by individuals with a family history of cancer.

[0109] In various embodiments, a sample from a subject, e.g., a human can be obtainedfrom a liquid biopsy. In certain embodiments, a sample and / or reference is obtained from serum,plasma, or urine. In certain embodiments, the sample is serum. In certain embodiments, asample comprises circulating tumor DNA (ctDNA). In certain embodiments, a sample is derived from about 1 mL of blood obtained from the subject. In certain embodiments, 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.

[0110] In various embodiments, 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 increaseunder 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 biofluidsbound to leukocytes and erythrocytes or not bound to leukocytes and erythrocytes. Various testsfor detection of tumor-derived ctDNA are based on detection of genetic or epigeneticmodifications that are characteristic of cancer (e.g., of a relevant cancer). Genetic or epigeneticfactors characteristic of cancer can include, without limitation, oncogenic or cancer-associatedmutations in tumor-suppressor genes, activated oncogenes, chromosomal disorders, histone12621818v1 Page 33 of 147Attorney Docket: 2014191-0036modifications (e.g., histone methylation and / or histone acetylation), chromatin accessibility,binding of one or more transcription factors and / or DNA methylation.

[0111] In various embodiments, ctDNA comprises less than 30%, less than 20%, or lessthan 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. In someembodiments, 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).

[0112] cfDNA and ctDNA can provide a real-time or nearly real time metric of status ofa source tissue. cfDNA and ctDNA demonstrate a half-life in blood of about 2 hours, such that asample taken at a given time provides a relatively timely reflection of the status of a source tissue.

[0113] Various methods of isolating nucleic acids from a sample (e.g., of isolatingcfDNA from blood or plasma) are known in the art. Nucleic acids can be isolated using, withoutlimitation, 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, fromthe clarified sample with a capture agent to produce a capture complex and isolating the capturecomplex to recover the target nucleic acid).

[0114] Reagents and protocols for obtaining and analyzing cfDNA and ctDNA, such ascirculating in blood or other tissue, are commercially available as described in the Examples andwell-known in the art (see, for example, Anker et al., Cancer and Metastasis Rev (1999) 18:65-73; Wua et al., Clin Chim Acta (2002) 321:77-87; Fiegl et al., Cancer Res (2005) 15:1141-1145;Pathak et al., Clin Chem (2006) 52:1833-1842; Schwarzenbach et al., Clin Cancer Res (2009)15:1032-1038; Schwarzenbach et al., Nat Rev Cancer (2011) 11:426-437) the contents of each ofwhich is separately incorporated herein by reference in their entirety).

[0115] In various embodiments, samples can be collected from individuals repeatedlyover 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. For example, subject samples can be12621818v1 Page 34 of 147Attorney 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. In various embodiments, 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 / orat tissue biopsy. In addition, results from samples obtained at different points in time can beconveniently 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.

[0116] 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 / orextraction of sample nucleic acids, and / or amplification of sample nucleic acids (e.g., by PCR orother nucleic acid amplification techniques). Samples also include materials prepared by techniques that isolate, e.g., nucleosomes or transcription factors and / or nucleic acids associatedwith nucleosomes or transcription factors.

[0117] Removal from a sample of proteins that are not desirable for a relevant purpose orcontext (e.g., high abundance, uninformative, or undetectable proteins) 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 bindto 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 molecularweight. Such filters may further employ reverse osmosis, nanofiltration, ultrafiltration and microfiltration. Ultracentrifugation is the centrifugation of a sample at about 15,000-60,000 rpmwhile 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. Since the membranes used in electrodialysis may have the ability to selectively transport ions having positive or negative charge, reject ions12621818v1 Page 35 of 147Attorney Docket: 2014191-0036 of the opposite charge, or to allow species to migrate through a semipermeable membrane basedon size and charge, it renders electrodialysis useful for concentration, removal, or separation ofelectrolytes.

[0118] Separation and purification in the present disclosure may include any procedureknown 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 beconducted in a gel, capillary, or in a microchannel on a chip. Examples of gels used forelectrophoresis 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. Examples of capillaries used for electrophoresis include capillaries that interface with an electrospray.

[0119] Capillary electrophoresis (CE) is preferred for separating complex hydrophilicmolecules and highly charged solutes. CE technology can also be implemented on microfluidic chips. Depending on the types of capillary and buffers used, CE can be further segmented intoseparation techniques such as capillary zone electrophoresis (CZE), capillary isoelectric focusing(CIEF), capillary isotachophoresis (CITP) and capillary electrochromatography (CEC). 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.

[0120] Capillary isotachophoresis (CITP) is a technique in which the analytes movethrough the capillary at a constant speed but are nevertheless separated by their respective mobilities. Capillary zone electrophoresis (CZE), also known as free-solution CE (FSCE), is based on differences in the electrophoretic mobility of the analytes, determined by the charge onthe analytes, and the frictional resistance the analytes encounter during migration, which is oftendirectly proportional to the size of the analytes. Capillary isoelectric focusing (CIEF) allowsweakly-ionizable amphoteric molecules, to be separated by electrophoresis in a pH gradient. CEC is a hybrid technique between traditional high performance liquid chromatography (HPLC) and CE.12621818v1 Page 36 of 147Attorney Docket: 2014191-0036

[0121] Separation and purification techniques used in the present disclosure can includeany chromatography procedures known in the art. Chromatography can be based on the differential adsorption and elution of certain analytes or partitioning of analytes between mobile and stationary phases. Different examples of chromatography include, but not limited to, liquid chromatography (LC), gas chromatography (GC), high performance liquid chromatography (HPLC), etc.

[0122] In some embodiments, whole blood is collected from a subject, and a plasmalayer is separated by centrifugation. cfDNA may be then extracted from the plasma using methods known in the art.Histone Modifications, Chromatin Accessibility and Transcription Factor Binding

[0123] Histone methylation is understood to increase or decrease expression ofassociated 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 ofhistones while histone demethylases promote demethylation of histones. 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. The methylation of histone H3K4, R8, R17, K26, K36, K79, H4R3, and K12 can activate gene transcription. However, the methylation of histone H3K9, K27, K56, H4K5, and K20 can inhibit gene transcription. Thus, for example, H3K4 methylation generally activates gene expression, while H3K27 methylation generally represses gene expression.

[0124] Histone acetylation occurs predominantly at lysine residues and is generallyunderstood to increase expression of associated coding sequences. Without wishing to be bound by any theory, acetylation of lysine residues is thought to neutralize lysine’s positive charge and12621818v1 Page 37 of 147Attorney Docket: 2014191-0036 thereby cause histones to drift away from DNA, which has a negative charge. The released structure facilitates access to transcriptional machinery such as transcription factors and RNA polymerase II. Histone acetylation and deacetylation are generally catalyzed by histone acetyltransferases (HATs) and HDACs, respectively. Acetyl-CoA can be a source and co-factor of acetylation. In regulatory regions, HATs can acetylate histones and recruit HAT-containing complexes to activate the transcriptional process. For instance, H3K9ac and H3K27ac levels can be associated with promoter and enhancer activities. Furthermore, 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.

[0125] Differential modification of a genomic locus (e.g., differential histone methylationand / or differential histone acetylation) can refer to, or be determined by or detected as, a comparative difference or change in modification status of one or more genomic loci between a first sample, condition, disease, or state and a second or reference sample, condition, disease, or state. Those of skill in the art will appreciate that a reference is typically produced by measurement using a methodology identical, similar, or comparable to that by which a compared non-reference measurement was taken.

[0126] Chromatin accessibility can refer to the degree to which nuclear macromoleculesare able to physically contact DNA and is determined in part by the occupancy and modification status of nucleosomes. 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. 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.

[0127] Differential accessibility of a genomic locus can refer to, or be determined by ordetected as, a comparative difference or change in modification status of one or more genomic loci between a first sample, condition, disease, or state and a second or reference sample, condition, disease, or state. Those of skill in the art will appreciate that a reference is typically produced by measurement using a methodology identical, similar, or comparable to that by which a compared non-reference measurement was taken.12621818v1 Page 38 of 147Attorney Docket: 2014191-0036

[0128] A reference can be a value or set of values that are predetermined or derived froma sample or set of samples. A reference can be a sample or set of samples. 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. In various embodiments, a reference can have or represent a normal, non-diseased state. In someembodiments, such as for staging of disease or for evaluating the efficacy of treatment, areference can have or represent a diseased state, e.g., a cancer, stage of cancer, or subtype ofcancer. In some embodiments, a reference can represent a particular level of ADC targetexpression based on IHC testing, e.g., ADC target-positive or ADC target-negative cancer.

[0129] In certain instances, a reference is a non-contemporaneous sample from the samesource, e.g., a prior sample from the same source, e.g., from the same subject. In certain instances, a reference for the modification status of one or more genomic loci (e.g., one or more differentially modified 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 asubject), or a plurality of samples, known to represent a particular state (e.g., ADC target-positive or ADC target-negative cancer). In certain instances, a reference for the accessibility status of one or more genomic loci (e.g., one or more differentially accessible 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).

[0130] In some illustrative but non-limiting embodiments of the present disclosuredifferential modification or differential accessibility can refer to a differential (e.g., between asample 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 measuredaccording to an assay provided herein.

[0131] Enhancers are genomic loci that can be differentially modified or differentiallyaccessible 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 toexpression patterns of associated genes. Chromatin ImmunoPrecipitation sequencing (ChIP-seq)12621818v1 Page 39 of 147Attorney 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 ofthousands. Certain transcription factors (TFs), sometimes referred to as “master” transcriptionfactors, associate with active enhancers with important impacts on gene expression and cellfunction. 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”. Moreover, master TFs can participate in inter-connected auto-regulatorycircuitries or “cliques” that are self-reinforcing, show marked cell selectivity, and function tomaintain cell state and / or cell survival.Techniques for Detecting and Quantifying Histone Modifications and Transcription FactorBinding

[0132] Various techniques of molecular biology are well known in the art and / ordisclosed in the present application for detecting and quantifying histone modifications and / ortranscription factor binding. In some embodiments, the methods, kits and systems of presentdisclosure involve the detection and quantification of histone modifications and / or transcriptionfactor binding in samples, e.g., in liquid biopsy samples including cfDNA such as plasmasamples including cfDNA. Chromatin ImmunoPrecipitation (ChIP) is one technique ofmolecular biology useful in detecting and quantifying histone modifications and transcriptionfactor binding in samples. CUT&RUN or CUT&Tag are other more recent techniques that canalso 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.

[0133] 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 ofvarious transcription factors and histones. Because the proteins are captured at the sites of theirbinding with DNA, ChIP helps to detect DNA-protein interactions that take place in living cells. More importantly, ChIP can be coupled to many commonly used molecular biology techniques such as PCR and real-time PCR, PCR with single-stranded conformational polymorphism,12621818v1 Page 40 of 147Attorney Docket: 2014191-0036Southern blot analysis, Western blot analysis, cloning, and microarray. The resulting versatilityhas increased the potential of this technique.

[0134] ChIP of tissue samples usually involves cross-linking of the chromatin-boundproteins by formaldehyde, followed by sonication or nuclease treatment to obtain small DNAfragments. Immunoprecipitation can be then carried out using specific antibodies to the DNA-binding protein of interest. The DNA can be then released from the proteins and analyzed usingvarious methods. ChIP has also been used to study RNA-protein interactions. X-ChIP methodsutilize fixed chromatin fragmented by sonication, while the N-ChIP methods utilize native chromatin, which can be unfixed and nuclease digested.

[0135] The first step of the technique can be the cross-linking of DNA and proteins.Formaldehyde is one of the most used cross-linking agents. One advantage of usingformaldehyde can be the ease of reversibility of the cross-links and its ability to form bonds thatspan approximately 2 angstroms. This means that formaldehyde can bind molecules in closeassociation with each other. Generally, formaldehyde can be added to the medium in the cellculture flask or plate. It enters the cells through the cell membrane and cross-links the proteins to the chromatin. Formaldehyde fixation of tumor tissues has also been done. Other cross- linking agents that have been used include chemicals such as methylene blue and acridine orange, cisplatin, dimethylarsinic acid, potassium chromate, and ultraviolet (UV) light and lasers.

[0136] Harvested chromatin can be sonicated in one or more sonication cycles. DNA canbe typically broken into to 100–500 bp fragments to pinpoint the location of the DNA sequence of interest. An alternative to sonication can be nuclease digestion of the chromatin, e.g., in N-ChIP methods. Purification of chromatin can be achieved using a cesium chloride (CsCl)gradient centrifugation.

[0137] Chromatin can be immunoprecipitated using one or more antibodies that bind atarget epitope. For example, an antibody used in ChIP can selectively bind a particular transcription factor or one or more particular histone modifications, such as one or more particular histone acetylation modifications or histone methylation modifications. In someembodiments, 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 agroup of histone modifications associated with increased transcription repression). The antibody12621818v1 Page 41 of 147Attorney Docket: 2014191-0036 against the protein of interest is allowed to bind to the protein-DNA complex, and the complex can be then precipitated. Immunosorbants commonly used to separate the antigen-antibody complex from the lysate include salmon sperm DNA-protein A-Sepharose®, protein G, magnetic beads, and other engineered immunoprecipitation systems known to those of skill in the art.

[0138] Immunoprecipitated DNA can be eluted. Once the DNA of interest is isolated,many detection and quantification methods can be used to study the isolated gene fragments. Commonly utilized methods include PCR, real-time PCR, slot blot hybridization, microarray techniques, and deep or next-generation sequencing. ChIP-seq combines chromatin immunoprecipitation (ChIP) with massively parallel DNA sequencing to identify the bindingsites of DNA-associated proteins. ChIP-seq can be used to map DNA-binding proteins, e.g.,transcription factor binding sites and histone modifications in a genome-wide manner.

[0139] Cell-free Chromatin ImmunoPrecipitation sequencing (cfChIP-seq) involvesapplying ChIP-seq to samples that include cell-free DNA, e.g., liquid biopsy samples includingcfDNA 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 ofeach of which are incorporated herein by reference). In some embodiments, cfChIP-seq usesantibodies or antibody fragments that bind specific histone modifications (e.g., H3K4me3 and / orH3K27ac) 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. Without limitation, exemplary antibodiesthat bind H3K4me3 include PA5-27029 (available from Thermo Fisher Scientific in Waltham,MA) and C15410003 (available from Diagenode in Denville, NJ) and exemplary antibodies thatbind H3K27ac include ab21623 or ab4729 (both available from Abcam in Cambridge, UK) andC15210016 (available from Diagenode in Denville, NJ).

[0140] In some embodiments, the antibodies or antibody fragments can be covalentlycoupled to beads, e.g., epoxy beads. In some embodiments, the antibodies or antibody fragmentscan be non-covalently coupled to beads, e.g., Protein A or Protein G beads such as Dynabeads®Protein A or Dynabeads® Protein G beads. After washing, a cfDNA library is then typicallyprepared from the captured cfDNA. Library preparation can be done on-bead or after releasingthe captured cfDNA by digestion of bound histones, e.g., using proteinase K. The cfDNA libraryis then sequenced to generate reads of captured cfDNA sequences, e.g., by next-generation12621818v1 Page 42 of 147Attorney Docket: 2014191-0036sequencing (NGS) as is known in the art. The reads are then analyzed, e.g., aligned and countedusing standard bioinformatic techniques as is known in the art. A cfChIP-seq bioinformaticpipeline 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.

[0141] CUT&Tag involves antibody-based binding of a target protein, e.g., transcriptionfactor or histone modification of interest, where antibody incubation is directly followed by theshearing 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 todirect 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) tocarry out antibody-targeted tagmentation. In a typical CUT&Tag assay samples are incubatedwith 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. Tn5 is a Mg2+-dependent enzyme so Mg2+can be added to activate the reaction, which results in the chromatin being cut close to the protein binding site and simultaneous addition of the NGS adapter DNA sequences. Chromatin cleavage and library preparation can be achieved in one single step.

[0142] CUT&RUN is an epigenomic profiling strategy in which antibody-targetedcontrolled cleavage by micrococcal nuclease releases specific protein-DNA complexes into thesupernatant for paired-end DNA sequencing (see Skene and Henikoff, Elife (2017) 6:1-35, Skeneet al., Nat Protoc (2018) 13:1006-1019). As only targeted fragments enter into solution, and the vast majority of DNA is left behind, CUT&RUN has low background levels. In an exampleCUT&RUN assay, a sample is incubated with an antibody or antibody fragment that binds thetarget protein, e.g., transcription factor or histone modification of interest. The sample is thenincubated with Protein-A-MNase after which CaCl2can be added to initiate the calciumdependent 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). CleavedDNA fragments are then liberated, extracted, and used to construct a sequencing library.12621818v1 Page 43 of 147Attorney Docket: 2014191-0036Techniques for Detecting and Quantifying Chromatin Accessibility

[0143] Various techniques of molecular biology are well known in the art and / ordisclosed in the present application for detecting and quantifying chromatin accessibility. Insome embodiments, the methods, kits and systems of the present disclosure involve the detectionand quantification of chromatin accessibility in samples, e.g., in liquid biopsy samples including cfDNA such as plasma samples including cfDNA. 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), and DNase hypersensitivity assays are exemplary techniques of molecular biology useful in detecting and quantifying chromatinaccessibility in samples. Sono-Seq is another alternative method that could be used (seeAuerbach et al., Proc Natl Acad USA (2009) 106(35):14926-14931). Fragmentomics-basedmethods are yet another method that can be used to assess chromatin accessibility (see Ding, Spencer C., and YM Dennis Lo. "Cell-free DNA fragmentomics in liquid biopsy." Diagnostics 12.4 (2022): 978).

[0144] DNase hypersensitivity assays can use the non-specific DNA endonucleaseDeoxyribonuclease I (DNase I), which selectively digests accessible DNA regions. DNase I hypersensitivity sites (DHS) identified by DNase-seq include open chromatin regulatory regions. A typical DNase hypersensitivity assay can include a first step in which nuclei are isolated fromcells using lysis buffer, and nuclei are digested using DNase I. DNA fragment sizes aremeasured to identify optimal digestion using gel electrophoresis. Biotinylated linkers can be ligated to the ends of digested DNA after polishing to make blunt ends, and the DNA can then be isolated. DNA with biotinylated linker can be digested by restriction endonuclease MmeI andcaptured by streptavidin coated Dynabeads® to generate short tags to which a second sequencingadaptor can be ligated. A second linker can be ligated and amplified to generate a library forsequencing. A DNase-seq bioinformatic pipeline can include, e.g., alignment of sequence readsto a reference genome with BWA or Bowtie2. Aligned reads can be used to call and quantify peaks as compared to a reference.

[0145] MNase-seq determines chromatin accessibility with micrococcal nuclease(MNase) that preferentially digests nucleosome-free, protein-unbound DNA. A typical MNase-12621818v1 Page 44 of 147Attorney Docket: 2014191-0036 seq assay can include a first step in which nuclei are isolated from either native or crosslinked chromatin and digested using MNase with titration. In vivo formaldehyde crosslinking step that is designed to capture the interaction between proteins and DNA. This crosslinking allows bound proteins to shield their associated DNA from digestion by MNase. Followingcrosslinking, samples are digested with MNase, which can be specifically activated by additionof Ca2+ to the buffer. Digestion can be halted by chelating the reaction, at which point thesamples are RNase treated, crosslinks are reversed, and proteins are digested away from thechromatin. DNA can then be isolated via a phenol-chloroform extraction. Uncut DNA ispurified and mononucleosome bands are isolated and excised through gel electrophoresis.Isolated DNA can be amplified by adding adapters to generate a library, and sequenced. MNase-seq primarily sequences regions of DNA bound by histones or other proteins. Therefore, it indirectly determines which regions of DNA are accessible by directly determining which regions are bound to nucleosomes or proteins.

[0146] 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–chloroformextraction: DNA crosslinked with protein stays in organic phase, while protein-free DNA staysin aqueous phase. Highly crosslinked DNA remains in the organic phase and the non- crosslinked DNA is pulled to the aqueous phase. Non-crosslinked DNA from the aqueous phasecan then be amplified and sequenced. Reads enriched in the sequencing pool tend to have lowernucleosome and transcription factor binding and are therefore inferred to come from accessible regions.

[0147] NOMe-seq is a method to identify nucleosome-depleted regions of DNA (NDRs)with M.CviPI methyltransferase that methylates cytosine in GpC dinucleotides not protected by nucleosomes or other proteins. Unlike CmpG, GpCmin the human genome does not occur naturally in most cell types. GpCmlevels at open chromatin regions can be compared to background signals and used to detect and quantify NDRs. A typical NOMe-seq protocol caninclude a step in which samples are treated with M.CviPI and S-adenosylhomocysteine (SAM) tomethylate accessible GpC sites. M.CviPI treated DNA can be sheared using a sonicator, so that12621818v1 Page 45 of 147Attorney Docket: 2014191-0036 DNA fragments can be sequenced. DNA is treated with bisulfite, which converts unmethylatedcytosine to uracil using sodium bisulfite, while methylated cytosine is unaffected. A library isgenerated using adapters and sequenced. Accessible chromatin is expected to have high levels ofGpCm but low levels of CmpG. Therefore, NOMe-seq identifies NDRs using the two separatemethylation analyses that serve as independent (but opposite) measures, providing matched chromatin designations for each regulatory element.

[0148] ATAC-seq uses hyperactive Tn5 transposase that preferentially cuts accessiblechromatin regions and simultaneously inserts adapters to the fragmented region (Buenrostro etal., Nat Methods (2013) 10(12):1213-1218 the entirety of which is incorporated herein byreference). A typical ATAC-seq assay can include a first step in which samples are incubatedwith Tn5 transposase. DNA can then be isolated and purified. DNA fragmented and tagged byTn5 transposase can be purified and then amplified to generate a library and sequenced for analysis.

[0149] “Fragmentomics” or a “fragmentomics assay” refers to methods that use certainsize and sequence characteristics of cfDNA to gain insight into the epigenetic state of cells at thetime their genomic DNA was released into the extracellular environment. Without wishing to bebound 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 acrosscancer cells (i.e. many of the circulating tumor DNA fragments that map to that small region ofthe 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. Examples of metrics commonly measured in fragmentomics includefragment size, preferred ends, end motifs, single-stranded jagged ends, and nucleosomalfootprints. 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 (h3K4me3 and H3K27ac) has been established. See Bai, Jinyue, et al. "Histone modifications of circulating nucleosomes are12621818v1 Page 46 of 147Attorney Docket: 2014191-0036 associated with changes in cell-free DNA fragmentation patterns." Proceedings of the National Academy of Sciences 121.42 (2024): e2404058121. Exemplary Genomic Loci

[0150] The present disclosure includes the identification of exemplary genomic loci thatare 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 differentiallyaccessible depending on ADC target status. See Table 1 which shows exemplary genomiccoordinates of H3K4me3 regions associated with genes that encode ADC target antigens, Table2 which shows exemplary genomic coordinates of H3K4me3 regions associated with genes thatmodulate ADC response / resistance, Table 3 which shows exemplary genomic coordinates ofH3K27ac regions associated with genes that encode ADC target antigens, Table 4, which showsexemplary genomic coordinates of H3K4me3, H3K27ac, or DNA methylation regions associatedwith genes that encode ADC target antigens, Table 5, which shows exemplary genomiccoordinates of H3K4me3 methylation regions associated with DLL3, Table 6, which showsexemplary genomic coordinates of H3K4me3, H3K27ac, or DNA methylation regions associatedwith DLL3 or genes whose expression levels are correlated with expression level of DLL3. Thechromosomal coordinates in Tables 1-6 are based on human genome build hg19.

[0151] The present disclosure is not limited to methods that use the exact samechromosomal coordinates that are recited in Tables 1-6. The present disclosure encompassesmethods that use any of the genomic loci in Tables 1-6 and also subregions thereof, i.e.,references herein to methods that involve detecting and / or quantifying one or more histone modifications, DNA methylation, chromatin accessibility, and / or binding of one or moretranscription factors at one or more genomic loci of Tables 1-6 encompasses methods that detectthese marks anywhere within these genomic loci including within any subregions. For example,where Table 1 references chr1:59,039,915-59,043,420 as a genomic locus for detecting and / orquantifying H3K4me3 modification, this encompasses methods that detect and / or quantifyH3K4me3 modification at any position or sub-region of chr1:59,039,915-59,043,420, e.g.,12621818v1 Page 47 of 147Attorney Docket: 2014191-0036methods that detect and / or quantify H3K4me3 modification within chr1:59,039,915-59,043,420,etc. In some embodiments, a subregion may span at least 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500 or at least 3000 contiguous base pairs that are located between the lower and upper coordinates of a genomic locus recited in Tables 1-6. In some embodiments, a subregion may span less than 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500 or at least 3000 contiguous base pairs that are located between the lower and upper coordinates of a genomic locus recited in Tables 1-6. In some embodiments, a subregion may have the same central coordinate as a genomic locus recited in Tables 1-6. In some embodiments, a subregion may have a different central coordinate as a genomic locus recited inTables 1-6. It is also to be understood that the lower / upper coordinates of the genomic loci inTables 1-6 are approximate and that the present disclosure encompasses methods where any oneor 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.

[0152] In some embodiments a classifier is generated using a set of differentiallymodified 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 arecorrelated with ADC target-negative status (e.g., based on IHC testing). Sequence reads that fallinto 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 ADCtarget-positive status are aggregated and counts from genomic loci that are correlated with ADCtarget-negative status are aggregated. In some embodiments, a ratio of the aggregated ADCtarget-positive and ADC target-negative counts is used to determine ADC target status. Otherways of using the genomic loci and related sequencing data to generate and apply a classifier todetermine ADC target status are described herein and known in the art, e.g., without limitation,methods that use a learning statistical classifier system or a combination of learning statistical classifier systems.

[0153] In some embodiments, exemplary genomic loci from Table 1, 2, 3, 4, or 5 areused in a monomodal classifier, e.g., a classifier that uses a single histone modification (e.g., H3K4me3 or H3K27ac) or only DNA methylation for purposes of determining ADC targetstatus or status of a gene the expression of which is associated with responsiveness / resistance toan ADC treatment. In some embodiments, exemplary genomic loci from Table 1, 2, 3, 4, 5,12621818v1 Page 48 of 147Attorney Docket: 2014191-0036and / or 6 are used in combination in a multimodal classifier, e.g., a classifier that uses more thanone 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 purposesof determining ADC target status.

[0154] In some embodiments, exemplary genomic loci from Table 6 associated withDLL3, RAB3IP, PAK5, ITPRIPL2, or any combination thereof are used in a monomodal ormultimodal classifier for purposes of determining DLL3 status. In some embodiments,exemplary genomic loci from Table 6 associated with DLL3 are used in a monomodal ormultimodal classifier for purposes of determining DLL3 status. In some embodiments,exemplary genomic loci from Table 6 associated with RAB3IP are used in a monomodal ormultimodal classifier for purposes of determining DLL3 status. In some embodiments,exemplary genomic loci from Table 6 associated with PAK5 are used in a monomodal ormultimodal classifier for purposes of determining DLL3 status. In some embodiments,exemplary genomic loci from Table 6 associated with ITPRIPL2 are used in a monomodal ormultimodal classifier for purposes of determining DLL3 status.

[0155] In some embodiments, 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 usesone or more genomic loci associated with RAB3IP, PAK5, or ITPRIPL2, or any combinationthereof for purposes of determining DLL3 status. In some embodiments, exemplary genomicloci associated with RAB3IP, PAK5, or ITPRIPL2, or any combination thereof are provided inTable 6.

[0156] In some embodiments, exemplary genomic loci from Table 1 associated withDLL3 are used in a monomodal or multimodal classifier that also uses one or more genomic locifrom Table 6 associated with RAB3IP, PAK5, or ITPRIPL2, or any combination thereof forpurposes of determining DLL3 status. In some embodiments, exemplary genomic loci fromTable 3 associated with DLL3 are used in a monomodal or multimodal classifier that also usesone or more genomic loci from Table 6 associated with RAB3IP, PAK5, or ITPRIPL2, or anycombination thereof for purposes of determining DLL3 status. In some embodiments, exemplarygenomic loci from Table 5 associated with DLL3 are used in a monomodal or multimodalclassifier 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. In some12621818v1 Page 49 of 147Attorney Docket: 2014191-0036embodiments, exemplary genomic loci from Table 6 associated with DLL3 are used in amonomodal or multimodal classifier that also uses one or more genomic loci from Table 6associated with RAB3IP, PAK5, or ITPRIPL2, or any combination thereof for purposes ofdetermining DLL3 status. Differential H3K4me3 modification

[0157] Genomic loci demonstrating differential H3K4 methylation (in particular H3K4trimethylation, H3K4me3) in samples from different cancer patients (e.g., different breast cancerpatients, different SCLC patients, different NSCLC patients, different NEPC patients, ordifferent PRAD patients) and / or samples from cancer patients (e.g., breast cancer patients, SCLCpatients, NSCLC patients, NEPC patients, or PRAD patients) and samples from healthyvolunteers are provided in Tables 1-2 and 4-6 which show the chromosomal coordinates of eachgenomic locus. The chromosomal coordinates are based on human genome build hg19.

[0158] A person of skill in the art will recognize that the methods disclosed herein do notrequire that every genomic locus listed in Tables 1-2 and 4-6be assessed for H3K4me3modification. Instead, a subset of loci may be assessed for H3K4me3 modification. Subsets ofthe genomic loci of Tables 1-2 and 4-6can be selected (e.g., for use in determining ADC targetstatus) based on the type of ADC screening required (e.g., the ADC target or ADC targets ofinterest), based on various performance criteria, e.g., to select genomic loci that demonstratedifferential modification with a particular level of statistical significance, progression freesurvival (PFS) of subjects, from whom the sample is obtained or derived, and / or a particularthreshold of differential between relevant states (e.g., a measured log2(fold-change)). Subsets ofthe genomic loci may also be selected based on an algorithm, e.g., during the process of obtaining a classifier. Those of skill in the art will appreciate that such subsets of loci of Tables1-2 and 4-6and loci included in such subsets, are together, individually, and / or in randomlyselected subsets, at least as informative (e.g., as statistically significant and / or reliable) for uses disclosed herein, e.g., for determining ADC target status.

[0159] In various embodiments, a sample or subject from whom the sample is obtainedor derived, is determined to have a particular ADC target status (e.g., ADC target-positive) if (i)at least 1, 2, 3, 4, 5 or more loci identified in Tables 1, 2, 4, 5, or 6 (or any subset thereof), (ii) atleast 10%, 25%, 50%, 60%, 75%, 80%, 90% or more of the loci identified in Tables 1, 2, 4, 5, or12621818v1 Page 50 of 147Attorney Docket: 2014191-00366 (or any subset thereof), or (iii) a majority or all of the loci identified in Tables 1, 2, 4, 5, or 6(or any subset thereof) are differentially H3K4me3 modified as compared to a reference (e.g., asample from an ADC target-negative subject or healthy volunteer or a sample from a subjectwho responded positively or poorly to treatment with an ADC).

[0160] In various embodiments, differentially H3K4me3 modified refers to a methylationstatus characterized by an increase or decrease in a value measuring methylation (e.g., of readcounts 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 20-fold, 25% to 16-fold, 30% to 16-fold, 50% to 16-fold, 70% to 16-fold, 2-fold to 16-fold, 2.2-fold to 16-fold, 2.6-fold to 16-fold, 3-fold to 16- fold, 3.4-fold to 16-fold, 4-fold to 16-fold, 4.5-fold to 16-fold, 5.2-fold to 16-fold, 6-fold to 16- fold, 7-fold to 16-fold, or 8-fold to 16-fold, as compared to a reference, optionally where the statistical significance of the increase or decrease is at least 5e-2, 1e-2, 5e-3, 1e-3, 5e-4, 1e-4, 5e- 5, 1e-5, 5e-6, or 1e-6. In various embodiments, an increase or decrease in a value measuringmethylation can be, or is expressed as, a log2(fold-change), e.g., a log2(fold-change) of at least1%, 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 to 4.0-fold, 1.8-fold to 4.0-fold, 2.0-fold to 4.0-fold, 2.2-fold to 4.0-fold, 2.4-fold to 4.0-fold, 2.6- fold to 4.0-fold, 2.8-fold to 4.0-fold, or 3.0-fold to 4.0-fold, optionally where the statisticalsignificance of the increase or decrease is at least 5e-2, 1e-2, 5e-3, 1e-3, 5e-4, 1e-4, 5e-5, 1e-5,5e-6, or 1e-6. Differential H3K27ac modification

[0161] Genomic loci demonstrating differential H3K27 acetylation (H3K27ac) insamples from different cancer patients (e.g., different breast cancer patients, different SCLC12621818v1 Page 51 of 147Attorney Docket: 2014191-0036patients, different NSCLC patients, different NEPC patients, or different PRAD patients) and / orsamples 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 Tables3, 4 and 6, which show the chromosomal coordinates of each genomic locus. The chromosomalcoordinates are based on human genome build hg19.

[0162] A person of skill in the art will recognize that the methods disclosed herein do notrequire that every genomic locus listed in Tables 3, 4 and 6be assessed for H3K27acmodification. Instead, a subset of loci may be assessed for H3K27ac modification. Subsets ofthe genomic loci of Tables 3, 4 and 6can be selected (e.g., for use in determining ADC targetstatus or the status of one or more genes that modulate ADC response / resistance) based on thetype 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.Those of skill in the art will appreciate that such subsets of loci of Tables 3, 4 and 6, and lociincluded 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., fordetermining ADC target status or the status of one or more genes that modulate ADCresponse / resistance.

[0163] In various embodiments, a sample or subject from whom the sample is obtainedor derived, is determined to have a particular ADC target status (e.g., ADC target-positive) if (i)at least 1, 2, 3, 4, 5 or more loci identified in Table 3, 4, or 6 (or any subset thereof), (ii) at least10%, 25%, 50%, 60%, 75%, 80%, 90% or more of the loci identified in Tables 3, 4, or 6 (or anysubset thereof), or (iii) a majority or all of the loci identified in Tables 3, 4, or 6 (or any subsetthereof) are differentially H3K27ac modified as compared to a reference (e.g., a sample from anADC target-negative subject or healthy volunteer).

[0164] In various embodiments, a sample or subject from whom the sample is obtainedor derived, is determined to have a particular ADC target status (e.g., ADC target-positive) if atleast 1, 2, 3, 4, 5 or more loci identified in Table 4 or 6 (or any subset thereof) are differentially12621818v1 Page 52 of 147Attorney Docket: 2014191-0036 H3K27ac modified as compared to a reference (e.g., a sample from an ADC target-negative subject or healthy volunteer).

[0165] In various embodiments, differentially H3K27ac modified refers to an acetylationstatus characterized by an increase or decrease in a value measuring acetylation (e.g., of readcounts 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 20-fold, 25% to 16-fold, 30% to 16-fold, 50% to 16-fold, 70% to 16-fold, 2-fold to 16-fold, 2.2-fold to 16-fold, 2.6-fold to 16-fold, 3-fold to 16- fold, 3.4-fold to 16-fold, 4-fold to 16-fold, 4.5-fold to 16-fold, 5.2-fold to 16-fold, 6-fold to 16- fold, 7-fold to 16-fold, or 8-fold to 16-fold, as compared to a reference, optionally where the statistical significance of the increase or decrease is at least 5e-2, 1e-2, 5e-3, 1e-3, 5e-4, 1e-4, 5e- 5, 1e-5, 5e-6, or 1e-6. In various embodiments, an increase or decrease in a value measuringacetylation can be, or is expressed as, a log2(fold-change), e.g., a log2(fold-change) of at least1%, 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 to4.0-fold, 1.8-fold to 4.0-fold, 2.0-fold to 4.0-fold, 2.2-fold to 4.0-fold, 2.4-fold to 4.0-fold, 2.6- fold to 4.0-fold, 2.8-fold to 4.0-fold, or 3.0-fold to 4.0-fold, optionally where the statistical significance of the increase or decrease is at least 5e-2, 1e-2, 5e-3, 1e-3, 5e-4, 1e-4, 5e-5, 1e-5, 5e-6, or 1e-6. Differential DNA methylation

[0166] Genomic loci demonstrating differential DNA methylation status in samples fromdifferent cancer patients (e.g., different breast cancer patients, different SCLC patients, differentNSCLC patients, different NEPC patients, or different PRAD patients) and / or samples fromcancer patients (e.g., breast cancer patients, SCLC patients, NSCLC patients, NEPC patients, or12621818v1 Page 53 of 147Attorney Docket: 2014191-0036PRAD patients) and samples from healthy volunteers are provided in Table 4, which show thechromosomal coordinates of each genomic locus. The chromosomal coordinates are based on human genome build hg19.

[0167] A person of skill in the art will recognize that the methods disclosed herein do notrequire that every MBD analyte genomic locus listed in Table 4 be assessed for DNAmethylation. Instead, a subset of MBD loci may be assessed for DNA methylation. Subsets of theMBD genomic loci of Table 4 can be selected (e.g., for use in determining ADC target status)based on various performance criteria, e.g., to select genomic loci that demonstrate differentialmodification with a particular level of statistical significance and / or a particular threshold ofdifferential between relevant states (e.g., a measured log2(fold-change)). Subsets of the genomicloci may also be selected based on an algorithm, e.g., during the process of obtaining a classifier.Those of skill in the art will appreciate that such subsets of loci of Table 4, and loci included insuch subsets, are together, individually, and / or in randomly selected subsets, at least asinformative (e.g., as statistically significant and / or reliable) for uses disclosed herein, e.g., for usein determining ADC target status.

[0168] In various embodiments, a sample or subject from whom the sample is obtainedor derived, is determined to have a particular ADC target status (e.g., ADC target-positive) if (i)at least 1, 2, 3, 4, 5 or more loci identified in Table 4 (or any subset thereof), (ii) at least 10%,25%, 50%, 60%, 75%, 80%, 90% or more of the loci identified in Table 4 (or any subsetthereof), or (iii) a majority or all of the loci identified in Table 4 (or any subset thereof) aredifferentially DNA methylated as compared to a reference (e.g., a sample from an ADC target-negative subject or healthy volunteer or a sample from a subject who responded positively orpoorly to treatment with an ADC).

[0169] In various embodiments, differentially DNA methylated refers to a methylationstatus characterized by an increase or decrease in a value measuring methylation (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% to2-fold, 25% to 50-fold, 25% to 30-fold, 25% to 20-fold, 25% to 16-fold, 30% to 16-fold, 50% to12621818v1 Page 54 of 147Attorney Docket: 2014191-003616-fold, 70% to 16-fold, 2-fold to 16-fold, 2.2-fold to 16-fold, 2.6-fold to 16-fold, 3-fold to 16-fold, 3.4-fold to 16-fold, 4-fold to 16-fold, 4.5-fold to 16-fold, 5.2-fold to 16-fold, 6-fold to 16- fold, 7-fold to 16-fold, or 8-fold to 16-fold, as compared to a reference, optionally where the statistical significance of the increase or decrease is at least 5e-2, 1e-2, 5e-3, 1e-3, 5e-4, 1e-4, 5e-5, 1e-5, 5e-6, or 1e-6. In various embodiments, an increase or decrease in a value measuringmethylation 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 of 0.1-fold to 10-fold, 0.2-foldto 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 to 4.0-fold, 1.8-fold to 4.0-fold, 2.0-fold to 4.0-fold, 2.2-fold to 4.0-fold, 2.4-fold to 4.0-fold, 2.6-fold to 4.0-fold,2.8-fold to 4.0-fold, or 3.0-fold to 4.0-fold, optionally where the statistical significance of the increase or decrease is at least 5e-2, 1e-2, 5e-3, 1e-3, 5e-4, 1e-4, 5e-5, 1e-5, 5e-6, or 1e-6.Differential chromatin accessibility or transcription factor binding

[0170] Genomic loci provided in Tables 1-6 can also demonstrate differential chromatinaccessibility or transcription factor binding in ADC target-positive cancer (e.g., based on IHCtesting) vs. ADC target-negative cancer (e.g., based on IHC testing) or a cancer that expresses amarker of ADC response / resistance vs. one that does not.

[0171] In various embodiments, without wishing to be bound by any particular scientifictheory, histone methylation (e.g., H3K4me3) corresponds and / or is correlated with chromatinaccessibility. In various embodiments, without wishing to be bound by any particular scientifictheory, histone acetylation (e.g., H3K27ac) corresponds and / or is correlated with chromatinaccessibility. In various embodiments, without wishing to be bound by any particular scientifictheory, DNA methylation corresponds and / or is correlated with chromatin accessibility.

[0172] In some embodiments, without wishing to be limited to any particular scientifictheory, chromatin accessibility corresponds and / or is correlated with H3K4me3 modifications.As a result, in some embodiments, ADC target status may be determined by detecting andquantifying chromatin accessibility at one or more genomic loci in Tables 1, 2 and 4-6in12621818v1 Page 55 of 147Attorney Docket: 2014191-0036 accordance with the section above discussing exemplary genomic loci with differential H3K4me3 modifications.

[0173] In some embodiments, without wishing to be limited to any particular scientifictheory, chromatin accessibility corresponds and / or is correlated with H3K27ac modifications. As a result, in some embodiments, ADC target status may be determined by detecting andquantifying chromatin accessibility at one or more genomic loci in Table 3, 4 or 6 in accordancewith the section above discussing exemplary genomic loci with differential H3K27ac modifications.

[0174] In some embodiments, without wishing to be limited to any particular scientifictheory, chromatin accessibility corresponds and / or is correlated with DNA methylation. As a result, in some embodiments, ADC target status may be determined by detecting and quantifyingchromatin accessibility at one or more genomic loci in Table 4 in accordance with the sectionabove discussing exemplary genomic loci with differentially methylated DNA.

[0175] In various embodiments, without wishing to be bound by any particular scientifictheory, histone methylation (e.g., H3K4me3) corresponds and / or is correlated with transcriptionfactor binding. In various embodiments, without wishing to be bound by any particular scientifictheory, histone acetylation (e.g., H3K27ac) corresponds and / or is correlated with transcriptionfactor binding. In various embodiments, without wishing to be bound by any particular scientifictheory, DNA methylation corresponds and / or is correlated with transcription factor binding.

[0176] In some embodiments, without wishing to be limited to any particular scientifictheory, binding of RNA pol II corresponds and / or is correlated with H3K4me3 modifications.As a result, in some embodiments, ADC target status may be determined by detecting andquantifying binding of RNA pol II at one or more genomic loci in Tables 1, 2, or 4-6 inaccordance with the section above discussing exemplary genomic loci with differential H3K4me3 modifications.

[0177] In some embodiments, without wishing to be limited to any particular scientifictheory, binding of p300, mediator complex, cohesin complex or RNA pol II corresponds and / oris correlated with H3K27ac modifications. As a result, in some embodiments, ADC target statusmay be determined by detecting and quantifying binding of p300, mediator complex, cohesincomplex or RNA pol II at one or more genomic loci in Tables 3, 4, or 6 in accordance with thesection above discussing exemplary genomic loci with differential H3K27ac modifications.12621818v1 Page 56 of 147Attorney Docket: 2014191-0036

[0178] In some embodiments, without wishing to be limited to any particular scientifictheory, binding of FOXA1, ESR1, PR, MYC, EN1, FOXM1, KLF4, AP-2, RARa, or RUNX1corresponds and / or is correlated with histone methylation (e.g., H3K4me3) or histone acetylation(e.g., H3K27ac). As a result, in some embodiments, ADC target status may be determined bydetecting and quantifying binding of FOXA1, ESR1, PR, MYC, EN1, FOXM1, KLF4, AP-2,RARa, RUNX1 at one or more genomic loci in Tables 1-36 in accordance with the sectionsabove discussing exemplary genomic loci with differential histone methylation (e.g., H3K4me3), histone acetylation (e.g., H3K27ac), or DNA methylation. Applications

[0179] Methods, kits and systems of the present disclosure include analysis ofdifferentially modified and / or differentially accessible genomic loci to determine the ADC targetstatus of a cancer. Methods, kits and systems of the present disclosure can be used in any of avariety of applications. For example, methods, kits and systems of the present disclosure can beused in detecting and / or treating cancers based on ADC target status. Methods, kits and systemsof the present disclosure can also be used to detect or determine response / resistance of a cancer,e.g., breast cancer, SCLC, NSCLC, NEPC, PRAD or other cancer to a therapy or transformationfrom one cancer subtype to another.

[0180] In various embodiments, methods, kits and systems of the present disclosure canbe applied to an asymptomatic human subject. As used herein, a subject can be referred to as“asymptomatic” if the subject does not report, and / or demonstrate by non-invasively observableindicia (e.g., without one, several, or all of device-based probing, tissue sample analysis, bodily fluid analysis, surgery, or cancer screening), sufficient characteristics of cancer to support amedically reasonable suspicion that the subject is likely suffering from cancer, e.g., breastcancer, SCLC, NSCLC, NEPC, PRAD or other cancer.

[0181] In various embodiments, methods, kits and systems of the present disclosure canbe applied to a symptomatic human subject. As used herein, a subject can be referred to as“symptomatic” if the subject report, and / or demonstrates by non-invasively observable indicia(e.g., without one, several, or all of device-based probing, tissue sample analysis, bodily fluidanalysis, surgery, or cancer screening), sufficient characteristics of cancer to support a medicallyreasonable suspicion that the subject is likely suffering from cancer, e.g., breast cancer, SCLC,12621818v1 Page 57 of 147Attorney Docket: 2014191-0036NSCLC, NEPC, PRAD or other cancer. For example, in various embodiments a sample from asubject, optionally where the subject has a cancer that is of unknown ADC target status, can beassayed according to one or more embodiments of the present disclosure to determine if the cancer is ADC target-positive or ADC target-negative. In various embodiments a sample from a subject, where the subject has a cancer that is known or suspected of being ADC target-positive (or ADC target-negative), can be assayed according to one or more embodiments of the present disclosure to determine if the cancer is in fact ADC target-positive (or ADC target-negative).

[0182] In some embodiments, methods, kits and systems of the present disclosure can beused to determine that a subject has an ADC target-positive cancer, optionally an ADC target-positive cancer that correlates with an ADC target-positive classification based on IHC testing. In some embodiments, methods, kits and systems of the present disclosure can be used todetermine that a subject has an ADC target-negative cancer, optionally an ADC target-negativecancer that correlates with an ADC target-negative classification based on IHC testing.

[0183] In some embodiments, methods, kits and systems of the present disclosure can beused to validate or confirm a prior determination that a subject has an ADC target-positivecancer. In some embodiments, methods, kits and systems of the present disclosure can be usedto validate or confirm a prior determination that a subject has an ADC target-negative cancer.

[0184] In some embodiments, methods, kits and systems of the present disclosure areused to identify and detect new ADC target related categories that are independent of IHCscoring. For example, instead of training a classifier on samples from cohorts that were definedbased on ADC target IHC testing, classifiers are trained on samples from cohorts that are definedbased on whether they respond or do not respond to a particular ADC for a given ADC target.The resulting classifiers are then used to identify subjects that are more likely to respond to theparticular ADC independent of any IHC scoring. It is therefore to be understood that the term“ADC target status” as used herein is not limited to ADC target-positive and ADC target-negative or the traditional ADC target scoring based on IHC testing but can encompass any ADCtarget related categories including whether a subject will or will not respond to a particular ADCfor a given ADC target and / or whether ADC-target expression is equal to or greater than acertain reference (e.g., a reference described herein, including, e.g., a predetermined threshold, ameasurement from a liquid biopsy sample, a normalized value, a measurement from a liquid biopsy sample obtained from a cohort of subjects who have previously been determined to have12621818v1 Page 58 of 147Attorney Docket: 2014191-0036 undetectable or low expression of the ADC target based on IHC testing, or a measurement from a liquid biopsy sample obtained from a cohort of subjects who have previously been determined to be cancer free).

[0185] In various embodiments ADC target status determination in accordance with thepresent disclosure is performed once for a given subject or multiple times for a given subject. Invarious embodiments, ADC target status determination in accordance with the present disclosureis performed on a regular basis, e.g., every six months, annually, every two years, every three years, every four years, every five years, or every ten years.

[0186] In various embodiments, methods, kits and systems disclosed herein provide adetermination of ADC target status. In other instances, methods, kits and systems disclosedherein will be indicative of ADC target status but not definitive for ADC target status. In variousinstances in which methods, kits and systems of the present disclosure are used to determineADC target status, the same can be followed by a further confirmatory assay, which further assaycan confirm, support, undermine, or reject a determination resulting from a prior determination,e.g., a determination in accordance with the present disclosure. As used herein, a confirmatoryassay can be an ADC target test that is currently recognized by medical practitioners, e.g., ADCtarget scoring based on IHC testing.

[0187] In various embodiments, ADC target status determination according to one ormore methods, kits and / or systems disclosed herein is followed by treatment of cancer. In various embodiments, treatment of cancer includes administration of a therapeutic regimen including one or more cancer therapies provided herein, including without limitation one or moreof treatment with an agent that is directed to an ADC target antigen (e.g., an ADC therapy),surgery, radiation, endocrine therapy, chemotherapy, and / or immunotherapy. In various embodiments, treatment of cancer includes administration of a therapeutic regimen including one or more treatments provided herein as available, appropriate, and / or preferred for a particularADC target status.

[0188] In various embodiments, methods, kits and systems can be used to determinewhether a particular subject and / or cancer is likely to be and / or is characterized as responsive totreatment with an agent that is directed to an ADC target antigen (e.g., an ADC therapy). Insome such embodiments, methods, kits and systems can be followed by treatment of the subjectwith an agent that is directed to an ADC target antigen (e.g., an ADC therapy).12621818v1 Page 59 of 147Attorney Docket: 2014191-0036

[0189] In some embodiments, differential epigenomic modifications and / or differentialchromatin accessibility of chromatin in one or more genomic loci of a promoter or enhancerassociated with one or more genes encoding an ADC target antigen can be used to determine thelikelihood of a subject responding to treatment with an agent that is directed to an ADC target antigen (e.g., an ADC therapy).

[0190] In various embodiments, methods, kits and systems can be used to determinewhether a particular subject and / or cancer is likely to be and / or is characterized as resistant to,non-responsive to, or not recommended treatment with an agent that is directed to an ADC targetantigen (e.g., an ADC therapy). In some such embodiments, methods, kits and systems can befollowed by treatment with one or more of surgery and / or radiation, endocrine therapy (if positive for a hormone receptor such as estrogen receptor), chemotherapy and immunotherapyinstead of ADC therapy.

[0191] In some embodiments, the likelihood of a particular subject and / or cancer to becharacterized as resistant to, non-responsive to, or not recommended for treatment with an agent that is directed to an ADC target antigen (e.g., an ADC therapy) is associated with ADC targetstatus (e.g., expression).

[0192] In some embodiments, differential epigenomic modifications and / or differentialchromatin accessibility of chromatin in one or more genomic loci of one or more promoter or enhancer regions associated with one or more genes encoding an ADC target antigen areassociated with the likelihood of a particular subject and / or cancer to be characterized as resistantto, non-responsive to, and / or not recommended for treatment with an agent that is directed to the ADC target antigen (e.g., an ADC therapy).

[0193] As used herein, “an agent that is directed to an ADC target antigen” refers to anagent that comprises a moiety that has previously been shown to associate with (e.g., asdetermined using a suitable binding assay) a given ADC target antigen. In some embodiments,an agent directed to an ADC target antigen is an ADC. In some embodiments, an agent directedto an ADC target antigen is a radioligand, e.g., an antibody or antibody fragment, peptide orsmall molecule ligand that (i) associates with the ADC target antigen and (ii) is linked to achelator that is bonded to a radioisotope.

[0194] Responsiveness can refer to the ability or likelihood of a therapy to cause areduction in tumor size or inhibit tumor growth or metastasis. Responsiveness can refer to12621818v1 Page 60 of 147Attorney 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-freesurvival, or disease-free survival). Responsiveness can refer to achievement of a treatmentbenefit, 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 caseof measurement of histone modification, chromatin accessibility, transcription factor binding, orDNA 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). Resistance can refer tothe 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 acquiredand natural resistance. In certain embodiments, resistance includes the extent to which one ormore 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).

[0195] In various embodiments, methods, kits and systems can be used to detect theclinical efficacy of a course of therapy for cancer, e.g., breast cancer, gastric / gastroesophagealcancer, colorectal cancer, prostate cancer, or lung cancer. For example, methods and / orcompositions of the present disclosure could be used to determine the presence, absence, or ADCtarget status of a cancer in a subject over the course of treatment. Methods and / or compositionsof the present disclosure could be used in conjunction with, or confirmed by, other means ofdetermining the presence, absence, or ADC target status of a cancer including, for examplemeasurements 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 withoutlimitation 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 etal., Breast (2003) 12:320-327, incorporated by reference herein in its entirety) in a qualitative12621818v1 Page 61 of 147Attorney Docket: 2014191-0036fashion like “pathological complete response” (pCR), “clinical complete remission” (cCR),“clinical partial remission” (cPR), “clinical stable disease” (cSD), “clinical progressive disease”(cPD).

[0196] In some embodiments, methods, kits and systems for ADC target statusdetermination provided herein can inform treatment and / or payment (e.g., reimbursement for orreduction 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.

[0197] In some embodiments, methods, kits and systems for ADC target statusdetermination provided herein can inform decision making relating to whether health insuranceproviders reimburse a healthcare cost payer or recipient (or not), e.g., for (1) ADC target statusdetermination itself (e.g., reimbursement for detecting otherwise unavailable, available only forperiodic / regular detecting, or available only for temporally- and / or incidentally- motivateddetecting); and / or for (2) treatment, including initiating, maintaining, and / or altering therapy,e.g., based on the determined ADC target status. For example, in some embodiments, methods,kits and systems for ADC target status determination provided herein are used as the basis for, tocontribute to, or support a determination as to whether a reimbursement or cost reduction will be provided to a healthcare cost payer or recipient. In some instances, a party seekingreimbursement or cost reduction can provide results of ADC target status determinationconducted in accordance with the present disclosure together with a request for suchreimbursement or reduction of a healthcare cost. In some instances, 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.

[0198] In various embodiments, ADC target status determination using methods, kits andsystems disclosed herein can be used in classifying subjects, samples, and / or tumors (e.g., breast,gastric / gastroesophageal, colorectal, prostate, or lung cancer subjects, samples, and / or tumors).In various embodiments, methods, kits and systems disclosed herein can be used to generate a setof subjects, samples, and / or tumors identified according to the present methods, kits and systemseach classified as corresponding to a particular ADC target status, and optionally using two ormore of such classified subjects, samples, and / or tumors to identify biomarkers that distinguish12621818v1 Page 62 of 147Attorney Docket: 2014191-0036 the classes (i.e., distinguish the subjects, samples, and / or tumors according to their class, e.g.,according to their ADC target status).

[0199] For illustration purposes and without limitation, in an exemplary assay of thepresent disclosure, a sample obtained from a subject (e.g., a liquid biopsy sample includingcfDNA, e.g., a plasma sample including cfDNA) is analyzed by ChIP-seq for a histonemodification (e.g., H3K4me3 and / or H3K27ac). ChIP-seq sequence reads are aligned to humangenome build hg19, e.g., using the Burrows-Wheeler Aligner (BWA). Non-uniquely mappingand redundant reads are optionally discarded. To provide one example of peak calling, MACSv2.1.1.20140616 can be used for ChIP-seq peak calling with a q-value (FDR) threshold of 0.01. ChIP-seq data quality can optionally be evaluated by any of one or more of a variety of measures, including total peak number, FRiP (fraction of reads in peak) score, number of high- confidence peaks (e.g., enriched > ten-fold over background), and percent of peak overlap with“blacklist” DHS peaks derived from the ENCODE project (Amemiya et al., Sci Rep (2019)9(1):9354). If the ChIP-seq data quality is below a particular threshold the data may bediscarded and the assay repeated. ChIP-seq peaks that overlap with selected genomic loci thatare differentially modified as provided herein for the relevant histone modification (Tables 1-6)can then be used to determine ADC target status or status of one or more genes that modulateADC response / resistance. The number of reads overlapping the selected genomic loci for therelevant histone modification are summed, e.g., in some embodiments all the genomic loci thatare differentially modified with an absolute log2(fold-change) ≥ 4.0 are selected. In some embodiments, the average number of reads in the local background of each ChIP-seq peak issubtracted to improve signal to noise. The data is then log2-transformed and quantile normalizedto match the distribution of the data used to train the classifier. The normalized data is then usedas input into a classifier that was trained using the same histone modification and selectedgenomic loci. The classifier then uses the inputted data to determine ADC target status of thesubject’s cancer. It will be appreciated that this or similar approaches can be applied to assays ofthe 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 thepresent disclosure that methods, kits and systems for ADC target status determination of thepresent disclosure are at least for in vitro use. Accordingly, all aspects and embodiments of thepresent disclosure can be performed and / or used at least in vitro.12621818v1 Page 63 of 147Attorney Docket: 2014191-0036

[0201] Those of skill in the art will also appreciate that, in certain embodiments, methodsof the present disclosure can be implemented on and / or in conjunction with a computer program and computer system. In some embodiments, 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 bymethods of the present disclosure that comprise a plurality of genomic locus modification statusand / or accessibility status changes / profiles, which data can be used by a computer system in implementing methods disclosed herein. In certain embodiments, a computer system (i) 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., todetermine ADC target status. In certain embodiments, a computer system (i) compares thegenomic locus modification and / or accessibility status to a reference; and (ii) outputs anindication 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.

[0202] Numerous types of computer systems can be used to implement methods of thepresent disclosure according to knowledge possessed by a skilled artisan in the bioinformatics and / or computer arts. Several software components can be loaded into memory during operation of such a computer system. 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., dCHIPsoftware described in Lin et al., Bioinformatics (2004) 20:1233-1240, incorporated herein byreference 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. Such packages include, e.g., Matlab from Mathworks (Natick, MA), Mathematica from Wolfram Research (Champaign, IL), S-Plus from MathSoft (Seattle, WA), R from R Foundation for Statistical Computing (Vienna, Austria), Python from Python Software Foundation (Wilmington, DE), or Perl from Perl Foundation12621818v1 Page 64 of 147Attorney Docket: 2014191-0036(Holland, MI). In certain embodiments, a computer system comprises a database for storage ofgenomic 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. In addition to the exemplary program structures and computer systems described herein, other, alternative program structures and computer systems will be readily apparent to the skilled artisan.

[0203] As demonstrated in the Examples, various algorithms can be applied to thecomparison, between samples and references, of the modification status and / or accessibilitystatus of genomic loci that are differentially modified in different ADC target states. In variousembodiments, an algorithm can be a single learning statistical classifier system. Other suitablestatistical algorithms are well known to those of skill in the art. For example, learning statistical classifier systems include a machine learning algorithmic technique capable of adapting tocomplex datasets (e.g., a panel of genomic loci of interest) and making decisions based uponsuch datasets. In some embodiments, a single learning statistical classifier system such as aclassification tree (e.g., random forest) is used. In other embodiments, 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 describedin the Examples and also those using inductive learning (e.g., decision / classification trees such asrandom 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. Other learning statistical classifier systems include support vector machines (e.g., Kernel methods), multivariate adaptive regression splines (MARS), Levenberg-Marquardt algorithms, Gauss-Newton algorithms, mixtures of Gaussians, gradient descent algorithms, and learning vector quantization (LVQ). In certain embodiments, methods of the present disclosure can include sending classification results to amedical practitioner, e.g., an oncologist.12621818v1 Page 65 of 147Attorney Docket: 2014191-0036

[0204] In various embodiments, the area under the receiver operating characteristic(AUROC) for determining if a subject has a particular ADC target status (e.g., an ADC target-positive cancer vs. 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). Formulation and Administration of ADC Therapy

[0205] The present disclosure includes methods where an agent that is directed to anADC target antigen (e.g., an ADC therapy or radioligand) is administered to a subject based onthe ADC target status of a cancer (e.g., breast cancer, SCLC, NSCLC, etc.). In general, the agentthat is directed to an ADC target antigen provided herein will be available, appropriate, and / orpreferred for the determined ADC target status. Those of skill in the art will be aware ofrecommended and / or governmentally approved formulations and / or dosages for various ADC therapies provided herein.

[0206] The present disclosure includes pharmaceutical compositions for delivery of oneor more ADCs to a subject. As disclosed herein, a pharmaceutical composition may be in anyform 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.

[0207] Pharmaceutical composition forms of the present disclosure can include, e.g.,liquid, semi-solid and solid dosage forms. Pharmaceutical composition forms of the present disclosure can include, e.g., liquid solutions (e.g., injectable and infusible solutions), dispersions or suspensions, tablets, pills, powders, and liposomes. Selection or use of any particular form may depend, in part, on the intended mode of administration and therapeutic application. Accordingly, the compositions can be formulated for administration by a parenteral mode (e.g.,intravenous, subcutaneous, intraperitoneal, or intramuscular injection) or a non-parenteral mode.As used herein, parenteral administration refers to modes of administration other than enteral andtopical administration, usually by injection or infusion.

[0208] In some embodiments, the compositions provided herein are present in unitdosage form, which unit dosage form can be suitable for self-administration. Such a unit dosage12621818v1 Page 66 of 147Attorney Docket: 2014191-0036 form may be provided within a container, e.g., a pill, vial, cartridge, prefilled syringe, or disposable pen.

[0209] A pharmaceutical composition of the present disclosure can be in an injectable orinfusible form. For example, the present disclosure includes sterile formulations for injection orinfusion, which can be formulated in accordance with conventional pharmaceutical practices. Sterile solutions can be prepared by incorporating a composition described herein in the requiredamount in an appropriate solvent with one or a combination of ingredients enumerated above, asrequired, 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 nonionicsurfactant such as polysorbate 80™ or HCO-50, and the like. In the case of sterile powders forthe 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. In particular instances, a pharmaceutical composition can be formulated, for example, as a buffered solution at a suitable concentration and suitable for storage, e.g., at 2-8°C (e.g., 4°C).

[0210] In various embodiments, a pharmaceutical composition of the present disclosurecan be formulated as a solution, microemulsion, dispersion, liposome, or other ordered structure suitable for stable storage at high concentration. Generally, dispersions are prepared by incorporating a composition described herein into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above.

[0211] In various instances, a pharmaceutical composition can be formulated to include apharmaceutically acceptable carrier or excipient. Examples of pharmaceutically acceptablecarriers include, without limitation, any and all solvents, dispersion media, coatings, antibacterial12621818v1 Page 67 of 147Attorney Docket: 2014191-0036 and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible.

[0212] In certain embodiments, compositions can be formulated with a carrier that willprotect the ADC against rapid release, such as a controlled release formulation, includingimplants 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 areknown in the art. See, e.g., J. R. Robinson (1978) “Sustained and Controlled Release DrugDelivery Systems,” Marcel Dekker, Inc., New York.

[0213] Route of administration can be parenteral, for example, administration byinjection. Administration by injection can be by intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection. Administration can be systemic or local. In certain embodiments, a composition described herein can be therapeutically delivered to asubject by way of local administration. As used herein, “local administration” or “localdelivery,” can refer to delivery that does not rely upon transport of the composition or ADC to itsintended target tissue or site via the vascular system. For example, the composition may bedelivered by injection or implantation of the composition or ADC or by injection or implantationof a device containing the composition or ADC. In certain embodiments, following local administration in the vicinity of a target tissue or site, the composition or ADC, or one or more components thereof, may diffuse to an intended target tissue or site that is not the site of administration.

[0214] A pharmaceutical composition can be administered parenterally in the form of aninjectable formulation comprising a sterile solution or suspension in water or another pharmaceutically acceptable liquid. For example, 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.Examples of oily liquid include sesame oil and soybean oil, and it may be combined with benzylbenzoate or benzyl alcohol as a solubilizing agent. Other items that may be included are a buffer such as a phosphate buffer, or sodium acetate buffer, a soothing agent such as procaine12621818v1 Page 68 of 147Attorney Docket: 2014191-0036 hydrochloride, a stabilizer such as benzyl alcohol or phenol, and an antioxidant. The formulated injection can be packaged in a suitable ampule.

[0215] In various embodiments, subcutaneous administration can be accomplished bymeans of a device, such as a syringe, a prefilled syringe, an auto-injector (e.g., disposable or reusable), a pen injector, a patch injector, a wearable injector, an ambulatory syringe infusionpump with subcutaneous infusion sets, or other device for combining with a ADC forsubcutaneous injection.

[0216] An injection system of the present disclosure may employ a delivery pen asdescribed in U.S. Pat. No.5,308,341. Pen devices, most commonly used for self-delivery of insulin to patients with diabetes, are well known in the art. Such devices can include at least oneinjection needle, are typically pre-filled with one or more therapeutic unit doses of a solution thatincludes the ADC and are useful for rapidly delivering solution to a subject with as little pain aspossible. One medication delivery pen includes a vial holder into which a vial of a therapeutic orother 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. In some embodiments, 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. Pat. Nos.6,277,099; 6,200,296; and 6,146,361, the disclosures of each of which are incorporated herein by reference in their entirety. A microneedle-based pen device is described in, e.g., U.S. Pat. No.7,556,615, the disclosure of which is incorporated herein by reference in its entirety. See also the Precision Pen Injector (PPI) device, MOLLYTM, manufactured by Scandinavian Health Ltd.

[0217] In some embodiments, a composition can be formulated for storage at atemperature below 0°C (e.g., -20°C or -80°C). In some embodiments, the composition can beformulated for storage for up to 2 years (e.g., one month, two months, three months, four months, five months, six months, seven months, eight months, nine months, 10 months, 11 months, 1 year, or 2 years) at 2-8°C (e.g., 4°C). Thus, in some embodiments, the compositions described herein are stable in storage for at least 1 year at 2-8°C (e.g., 4°C).

[0218] A pharmaceutical composition can include a therapeutically effective amount of aADC described herein. Such effective amounts can be readily determined by one of ordinary12621818v1 Page 69 of 147Attorney Docket: 2014191-0036 skill in the art. A therapeutically effective amount can be an amount at which any toxic or detrimental effects of the composition are outweighed by therapeutically beneficial effects. In some embodiments, a dose can also be chosen to reduce or avoid production of antibodies orother host immune responses against an ADC. Those of skill in the art will appreciate that dataobtained from cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. In various embodiments, the amount of active ingredient included in apharmaceutical composition is such that a suitable dose within the designated range can beadministered 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.

[0219] Pharmaceutical compositions including certain ADCs can be administered as afixed dose, or in a milligram per kilogram (mg / kg) dose. While in no way intended to be limiting, an exemplary single dose of certain pharmaceutical compositions described herein can include certain ADCs as described herein in an amount equal to, e.g., 0.001 to 1000 mg / kg, 1- 1000 mg / kg, 1-100 mg / kg, 0.5-50 mg / kg, 0.1-100 mg / kg, 0.5-25 mg / kg, 1-20 mg / kg, and 1-10 mg / kg body weight. Exemplary dosages of a composition described herein include, withoutlimitation, 0.1 mg / kg, 0.5 mg / kg, 1 mg / kg, 2 mg / kg, 4 mg / kg, 8 mg / kg, or 20 mg / kg. Thepresent disclosure is not limited to such ranges or dosages.

[0220] The present disclosure further includes methods of preparing pharmaceuticalcompositions of the present disclosure and kits including pharmaceutical compositions of the present disclosure.

[0221] In various embodiments, ADCs of the present disclosure can be administered to asubject 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). Combinationtherapies of the present disclosure can include simultaneous exposure of a subject to therapeuticagents of two or more therapeutic regimens.

[0222] In certain embodiments, an ADC as described herein can be administered togetherwith (e.g., at the same time and / or in the same composition as) an additional agent or therapy. Incertain embodiments, an ADC of the present disclosure can be administered separately from anadditional therapeutic agent or therapy (e.g., at a different time and / or in a different compositionthan the additional therapeutic agent or therapy). Dosing regimens of an ADC and one or more12621818v1 Page 70 of 147Attorney Docket: 2014191-0036additional therapeutic agents with which it is administered in combination can be coordinated orindependently determined. In various embodiments, an additional therapeutic agent or therapyadministered in combination with an ADC as described herein can be administered at the sametime as the ADC, on the same day as the ADC, or in the same week as the ADC. In variousembodiments, an additional therapeutic agent or therapy administered in combination with anADC as described herein can be administered such that administration of the ADC and theadditional therapeutic agent or therapy are separated by one or more hours before or after, one or more days before or after, one or more weeks before or after, or one or more months before orafter administration of the ADC. In various embodiments, the administration frequency and / ordosage of one or more additional therapeutic agents can be the same as, similar to, or differentfrom the administration frequency of the ADC. In some embodiments, the two or more regimenscan be administered simultaneously; in some embodiments, such regimens can be administeredsequentially (e.g., all “doses” of a first regimen are administered prior to administration of anydoses of a second regimen); in some embodiments, such therapeutic agents are administered in overlapping dosing regimens.

[0223] In certain embodiments, administration of an ADC can be to a subject havingpreviously received, scheduled to receive, or in the course of a treatment regimen including anadditional cancer therapy. Administration of an ADC can, in some instances, improve deliveryor efficacy of another therapeutic agent or therapy with which it is administered in combination.

[0224] It is contemplated that therapeutic agent combination therapies can demonstratesynergy and / or greater-than-additive effects between an ADC and one or more additionaltherapeutic agents with which it is administered in combination. An ADC can be administered inany effective amount as determined independently or as determined by the joint action of theADC and any of one or more additional therapeutic agents or therapies administered.Administration of the ADC may, in some embodiments, reduce the therapeutically effective dosage, required dosage, or administered dosage of the additional therapeutic agent or therapy relative to a reference regimen for administration of additional therapeutic agent or therapy or therapy absent the ADC. In certain embodiment, a composition described herein can replace or augment other previously or currently administered therapy. For example, upon treating with an ADC, administration of one or more additional therapeutic agents or therapies can cease or diminish, e.g., be administered at lower levels.12621818v1 Page 71 of 147Attorney Docket: 2014191-0036 Kits

[0225] The present disclosure includes kits for detecting modification and / or accessibilityof one or more genomic loci. In some embodiments, the present disclosure provides kits 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. Kits of thepresent disclosure can include, e.g., reagents such as buffers and / or antibodies useful in thedetection and quantification of histone modifications. In certain embodiments, a kit of thepresent disclosure can include at least one antibody that selective binds a histone modification selected from H3K9ac, H3K14ac, H3K18ac, H3K23ac, H3K27ac, H3K4me1, H3K4me2, or H3K4me3, or pan acetylation. In certain embodiments, a kit of the present disclosure can include at least one antibody that selective binds H3K4me3 modifications. In certain embodiments, a kit of the present disclosure can include at least one antibody that selective binds H3K27ac modifications. A kit of the present disclosure can include instructional materialsdisclosing or describing the use of the kit in a method of determining ADC target status and / ortreatment disclosed herein. In various embodiments, a kit of the present disclosure can include one or more ADCs or other therapeutic agents useful in the treatment of cancer, e.g., as disclosedherein, optionally in combination with instruction materials for treatment of cancer, e.g., breastcancer, SCLC, NSCLC, etc. based on ADC target status.

[0226] In some embodiments, a kit of the present disclosure comprises reagents forquantifying 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 oneor more genomic loci are selected from Tables 1-6.

[0227] In some embodiments, the kit comprises reagents for quantifying H3K4me3modifications for at least 1, 2, 3, 4, 5, 10, 15, 20, or 25 genomic loci that are located within oneor more regions defined by pairs of genomic coordinates in Table 1. In some embodiments, thekit 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 genomiccoordinates in Table 2. In some embodiments, the kit comprises reagents for quantifyingH3K27ac modifications for at least 1, 2, 3, 4, 5, 10, 15, 20, or 25 genomic loci that are locatedwithin one or more regions defined by pairs of genomic coordinates in Table 3. In some12621818v1 Page 72 of 147Attorney Docket: 2014191-0036embodiments, the kit comprises reagents for quantifying H3K27ac or H3K4me3 modifications orDNA methylation for at least 1, 2, 3, 4, 5, 10, 15, 20, or 25 genomic loci that are located withinone or more regions defined by pairs of genomic coordinates in Table 4. In some embodiments,the kit comprises reagents for quantifying H3K4me3 modifications for 1 or 2 genomic loci thatare located within one or more regions defined by pairs of genomic coordinates in Table 5. Insome embodiments, the kit comprises reagents for quantifying H3K27ac or H3K4me3modifications or DNA methylation for at least 1, 2, 3, 4, 5, 10, 15, 20, or 25 genomic loci that arelocated within one or more regions defined by pairs of genomic coordinates in Table 6. In someembodiments, the kit comprises one or more antibodies for use in ChIP-seq, optionally whereinthe one or more antibodies specifically bind H3K4me3- or H3K27ac-modified histones.

[0228] In some embodiments, the kit comprises reagents for isolation of cell-free DNA(cfDNA) from a liquid biopsy sample. In some embodiments, the kit comprises reagents forlibrary preparation for sequencing. In some embodiments, the kit comprises reagents forsequencing. In some embodiments, the kit comprises instructions for determining if a subjecthas an ADC target-positive cancer. Systems

[0229] The present disclosure includes systems for detecting modification and / oraccessibility of one or more genomic loci. In some embodiments, the present disclosure providessystems for quantifying one or more histone modifications, DNA methylation, chromatinaccessibility, 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 sequencingdataset from a sample; and a non-transitory computer readable storage medium and / or acomputer system.

[0230] In some embodiments, the non-transitory computer readable storage medium isencoded 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.

[0231] In some embodiments, the computer system comprises a memory and one or moreprocessors coupled to the memory, wherein the one or more processors are configured toperform a method of the present disclosure.12621818v1 Page 73 of 147Attorney Docket: 2014191-0036

[0232] In some embodiments, the sequencer is configured to generate a Whole GenomeSequencing (WGS) dataset from the sample. In some embodiments, the system also includes asample preparation device configured to prepare the sample for sequencing from a biological sample, optionally a liquid biopsy sample. The sample preparation device may include 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.

[0233] Systems of the present disclosure can include, e.g., reagents such as buffers and / orantibodies useful in the detection and quantification of histone modifications. In certain embodiments, a system of the present disclosure can include at least one antibody that selective binds a histone modification selected from H3K9ac, H3K14ac, H3K18ac, H3K23ac, H3K27ac, H3K4me1, H3K4me2, or H3K4me3, or pan acetylation. In certain embodiments, a system of the present disclosure can include at least one antibody that selective binds H3K4me3 modifications. In certain embodiments, a system of the present disclosure can include at least one antibody that selective binds H3K27ac modifications. A system of the present disclosure can include instructional materials disclosing or describing the use of the system in a method of determiningADC target status and / or treatment disclosed herein.

[0234] In some embodiments, a system of the present disclosure comprises reagents forquantifying 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 selected from Tables 1-6.

[0235] In some embodiments, the system comprises reagents for quantifying H3K4me3modifications for at least 1, 2, 3, 4, 5, 10, 15, 20, or 25 genomic loci that are located within oneor more regions defined by pairs of genomic coordinates in Table 1. In some embodiments, thesystem 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 ofgenomic coordinates in Table 2. In some embodiments, the system comprises reagents forquantifying H3K27ac modifications for at least 1, 2, 3, 4, 5, 10, 15, 20, or 25 genomic loci thatare located within one or more regions defined by pairs of genomic coordinates in Table 3. Insome embodiments, the system comprises reagents for quantifying H3K27ac or H3K4me3modifications or DNA methylation for at least 1, 2, 3, 4, 5, 10, 15, 20, or 25 genomic loci that are12621818v1 Page 74 of 147Attorney Docket: 2014191-0036located within one or more regions defined by pairs of genomic coordinates in Table 4. In someembodiments, the system comprises reagents for quantifying H3K4me3 modifications for 1 or 2genomic loci that are located within one or more regions defined by pairs of genomic coordinatesin Table 5. In some embodiments, the system comprises reagents for quantifying H3K4me3 orH3K27ac modifications for 1, 2, 3, 4, 5, 10, 15, or 16 genomic loci that are located within one ormore regions defined by pairs of genomic coordinates in Table 6. In some embodiments, thesystem comprises one or more antibodies for use in ChIP-seq, optionally wherein the one ormore antibodies specifically bind H3K4me3- or H3K27ac-modified histones or methylatedDNA. In some embodiments, the system comprises one or more methyl binding domains(MBDs) for use in MBD-seq.

[0236] In some embodiments, the system comprises reagents for isolation of cell-freeDNA (cfDNA) from a liquid biopsy sample. In some embodiments, 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 morethan one (i.e., to at least one) of the grammatical object of the article. By way of example, “anelement” refers to one element or more than one element.

[0238] About: The term “about”, when used herein in reference to a value, refers to avalue that is similar, in context, to the referenced value. In general, those skilled in the art, familiar with the context, will appreciate the relevant degree of variance encompassed by “about” in that context. For example, in some embodiments, the term “about” can encompass a range of values that within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%,9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or within a fraction of a percent, of the referencedvalue.

[0239] “Accessibility Status” or “Chromatin Accessibility Status”: As used herein,“accessibility status” or “chromatin accessibility 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 accessible chromatin. Accessibility status can be determined by various12621818v1 Page 75 of 147Attorney Docket: 2014191-0036 assays known in the art, including without limitation ChIP-seq as one example. Where twosamples are separately analyzed by the same assay or comparable assays for detection ofaccessible DNA sequences, differences in chromatin accessibility status of genomic loci can be detected. Accessibility status can be compared to a standard or reference. A sample that has anaccessibility status that differs in accessibility status from a standard or reference can be referredto 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.

[0240] Administration: As used herein, the term “administration” typically refers to theadministration of a disease appropriate treatment. In some embodiments, the disease appropriate treatment may comprise administering a composition to a subject, for example to achieve delivery of an agent that is, is included in, or is otherwise delivered by, the composition. In some embodiments, the disease appropriate treatment may comprise administering an appropriate surgical procedure or radiological procedure, optionally in combination with administration of a composition.

[0241] Agent: As used herein, the term “agent” may refer to any chemical or physicalentity, 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.

[0242] Antibody: As used herein, the term “antibody” refers to a polypeptide thatincludes 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 variabledomain, and / or one or more CDRs). Thus, the term antibody includes, without limitation, humanantibodies, 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 occurrior engineered antibodies can be produced by recombinant engineering, chemical synthesis, or other artificial systems or methodologies known to those of skill in the art.12621818v1 Page 76 of 147Attorney Docket: 2014191-0036

[0243] As is well known in the art, typical human immunoglobulins are approximately150 kD tetrameric agents that include two identical heavy (H) chain polypeptides (about 50 kD each) and two identical light (L) chain polypeptides (about 25 kD each) that associate with each other to form a structure commonly referred to as a “Y-shaped” structure. Typically, each heavy chain includes a heavy chain variable domain (VH) and a heavy chain constant domain (CH). The heavy chain constant domain includes three CH domains: CH1, CH2 and CH3. A shortregion, known as the “switch”, connects the heavy chain variable and constant regions. The“hinge” connects CH2 and CH3 domains to the rest of the immunoglobulin. Each light chain includes a light chain variable domain (VL) and a light chain constant domain (CL), separated from one another by another “switch.” 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). In each VH and VL, the three CDRs and four FRs are arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The 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. When natural immunoglobulins fold,the FR regions form the beta sheets that provide the structural framework for the domains, andthe CDR loop regions from both the heavy and light chains are brought together in three- dimensional space so that they create a single hypervariable antigen binding site located at the tip of the Y structure.

[0244] In some embodiments, an antibody is a polyclonal, monoclonal, monospecific, ormultispecific antibody (e.g., a bispecific antibody). In some embodiments, 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. Moreover, the term “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, antibody12621818v1 Page 77 of 147Attorney 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 (SMIPs), single chain or Tandem diabodies (TandAb®), VHHs, Anticalins®, Nanobodies®, minibodies, BiTE®s, ankyrin repeat proteins or DARPINs®, Avimers®, DARTs, TCR-like antibodies, Adnectins®, Affilins®, Trans-bodies®, Affibodies®, TrimerX®, MicroProteins, Fynomers®, Centyrins®, KALBITOR®s, chimeric antigen receptors (CARs), engineered T-cell receptors (TCRs), and antigen-binding fragments of any of the above.

[0245] In various embodiments, an antibody includes one or more structural elementsrecognized by those skilled in the art as a complementarity determining region (CDR) or variable domain. In some embodiments, an antibody can be a covalently modified (“conjugated”) antibody (e.g., an antibody that includes a polypeptide including one or more canonical immunoglobulin sequence elements sufficient to confer specific binding to a particular antigen, where the polypeptide is covalently linked with one or more of a therapeutic agent, a detectable moiety, another polypeptide, a glycan, or a polyethylene glycol molecule). In some embodiments, antibody sequence elements are humanized, primatized, chimeric, etc., as is known in the art.

[0246] 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 andIgM, based on heavy chain constant domain amino acid sequence (e.g., alpha (α), delta (δ),epsilon (ε), gamma (γ) and mu (μ)). 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. As used herein, a “light chain” can be of a distinct type, e.g., kappa (κ) or lambda (λ), based on the amino acid sequence of the light chain constant domain. In some embodiments, 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. As is known in the art, affinity and / or other binding attributes of Fc regions for Fc12621818v1 Page 78 of 147Attorney Docket: 2014191-0036 receptors can be modulated through glycosylation or other modification. In some embodiments, an antibody may lack a covalent modification (e.g., attachment of a glycan) that it would have if produced naturally. In some embodiments, antibodies produced and / or utilized in accordance with the present invention include glycosylated Fc domains, including Fc domains with modified or engineered glycosylation.

[0247] In some embodiments, an antibody can be specific for a particular histonemodification (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). In some embodiments, an antibody is specific for an H3K9ac, H3K14ac, H3K18ac, H3K23ac, H3K27ac, H3K4me1, H3K4me2, or H3K4me3 modification. In some embodiments, an antibody is specific for an H3K27ac modification. In some embodiments, an antibody is specific for an H3K4me3 modification.

[0248] In some embodiments, an antibody is a “pan” antibody. As used herein, the termpan antibody refers to an antibody that can bind a group of histone modifications having one or more features that are similar. In some embodiments, 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). In some embodiments, a pan antibody is a pan-acetylation antibody (e.g., an antibody that can bind a histone, e.g., H3 that comprises at least one acetylated lysine, wherein the at least one acetylated lysine can be at any one of a plurality of amino acid positions, e.g., a pan-acetylation antibody can bind an H3 protein comprising an acetylated lysine at any position). In some embodiments, a pan antibody can bind one or more histone modifications that are associated with transcription activation. In some embodiments, a pan antibody can bind one or more histone modifications that are associated with transcription silencing.

[0249] Antibody fragment: As used herein, an “antibody fragment” refers to a portion ofan 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, in12621818v1 Page 79 of 147Attorney 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. In some embodiments, an antibody fragment (particularly an antigen-binding antibody fragment) can have a length of at least about 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 amino acids or more, in some embodiments at least about 200 amino acids.

[0250] Associated with: Two events or entities are “associated” with one another, as thatterm is used herein, if the presence, level and / or form of one is correlated with that of the other. For example, a particular entity (e.g., an epigenetic profile comprising one or more histone modifications at a set of genomic loci, etc.) is considered to be associated with a particular disease, disorder, or condition, if its presence, level and / or form correlates with incidence of and / or susceptibility to the disease, disorder, or condition (e.g., across a relevant population). Togive another example, a genomic loci can be considered to be associated with an enhancer or apromoter of a gene if it exhibits certain physical and / or functional properties, including, e.g.,physical proximity to the gene and / or a correlation between the level of one or more epigenetic modifications associated with promoter or enhancer activity (whether positively or negatively)and expression of the gene. In some embodiments, 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. In some embodiments, 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.

[0251] “Between” or “From”: As used herein, the term “between” refers to content thatfalls between indicated upper and lower, or first and second, boundaries, inclusive of the boundaries. Similarly, the term “from”, when used in the context of a range of values, indicates that the range includes content that falls between indicated upper and lower, or first and second, boundaries, inclusive of the boundaries.

[0252] Biological Sample: As used herein, the term “biological sample” typically refersto a sample obtained or derived from a biological source (e.g., a tissue or organism or cell) of12621818v1 Page 80 of 147Attorney Docket: 2014191-0036 interest, as described herein. In some embodiments, a biological source is or includes an organism, such as a human subject. In some embodiments, a biological sample is or includes a biological tissue or fluid. In some embodiments, a biological sample can be or include cells, tissue, or bodily fluid. “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). In some embodiments, a biological sample can be or include blood, blood components, cell-free DNA (cfDNA), circulating-tumor DNA (ctDNA), ascites, biopsy samples, surgical specimens, cell- containing body fluids, sputum, saliva, feces, urine, cerebrospinal fluid, peritoneal fluid, pleural fluid, lymph, gynecological fluids, secretions, excretions, skin swabs, vaginal swabs, oral swabs, nasal swabs, washings or lavages such as a ductal lavages or bronchoalveolar lavages, aspirates, scrapings, or bone marrow. In some embodiments, a biological sample is a liquid biopsy sample obtained from a bodily fluid. In some embodiments, 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. A biological sample can also be referred to as a “sample.”

[0253] Blood component: As used herein, the term “blood component” refers to anycomponent 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.

[0254] Cancer: As used herein, the terms “cancer,” “malignancy,” “tumor,” and“carcinoma,” are used interchangeably to refer to a disease, disorder, or condition in which cellsexhibit 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. In some embodiments, a cancer can include one or more tumors. In some embodiments, a cancer12621818v1 Page 81 of 147Attorney Docket: 2014191-0036can be or include cells that are precancerous (e.g., benign), malignant, pre-metastatic, metastatic,and / or non-metastatic. In some embodiments, a cancer can be or include a solid tumor.

[0255] Combination therapy: As used herein, the term “combination therapy” refers toadministration 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, ordisorder of the subject. In some embodiments, the two or more therapeutic agents or therapeuticregimens can be administered simultaneously, sequentially, or in overlapping dosing regimens. Those of skill in the art will appreciate that 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.

[0256] Corresponding to: As used herein, the term “corresponding to” may be used todesignate the position / identity of a structural element in a compound or composition through comparison with an appropriate reference compound or composition. For example, in some embodiments, a monomeric residue in a polymer (e.g., an amino acid residue in a polypeptide or a nucleic acid residue in a polynucleotide) may be identified as “corresponding to” a residue in an appropriate reference polymer. For example, those of skill in the art appreciate that residues in a provided polypeptide or polynucleotide sequence are often designated (e.g., numbered orlabeled) according to the scheme of a related reference sequence (even if, e.g., such designationdoes not reflect literal numbering of the provided sequence). By way of illustration, if a reference sequence includes a particular amino acid motif at positions 100-110, and a second related sequence includes the same motif at positions 110-120, the motif positions of the second related sequence can be said to “correspond to” positions 100-110 of the reference sequence. Those of skill in the art appreciate that 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. Two sequences can be identified ascorresponding 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 at12621818v1 Page 82 of 147Attorney Docket: 2014191-0036least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500 or more residues. In variousembodiments, 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 atleast 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500 or more nucleic acid residues.

[0257] “Diagnosing”, “Detecting”, “Determining” or “Screening for”: As used herein,“diagnosing”, “detecting”, “determining”, “screening for” the presence of a condition or disease,or a related state (e.g., responsiveness of a cancer to one or more ADC therapies) includes theact, process, and / or outcome of determining whether, and / or the qualitative of quantitativeprobability that, a subject has or will develop the condition, disease, or related state. In some instances, diagnosing can include a determination relating to prognosis and / or likely response to one or more general or particular therapeutic agents or regimens.

[0258] Differentially accessible: As used herein, the term “differentially accessible”describes a genomic locus for which chromatin accessibility status differs between a firstcondition or sample and a second condition or sample (e.g., a standard or reference). Adifferentially accessible genomic locus can include a greater or smaller measured accessibilityunder a selected condition of interest, such as disease state, as compared to a reference state,such as healthy state.

[0259] Differentially modified: As used herein, the term “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 histonemodification under a selected condition of interest, such as a disease state, as compared to areference state, such as healthy state.

[0260] Gene: As used herein, the term “gene” refers to a DNA sequence in achromosome encoding a gene product (e.g., an RNA product and / or a polypeptide product). Insome embodiments, a gene includes a coding sequence (e.g., a sequence encoding a particulargene product); in some embodiments, a gene includes a non-coding sequence. In some embodiments, a gene may include both coding (e.g., exonic) and non-coding (e.g., intronic) sequences. In some embodiments, a gene may include one or more regulatory elements (e.g.12621818v1 Page 83 of 147Attorney 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).

[0261] Identity: As used herein, the term “identity” refers to the overall relatednessbetween polymeric molecules, e.g., between nucleic acid molecules (e.g., DNA molecules)and / or between polypeptide molecules. Methods for the calculation of a percent identity asbetween two provided sequences are known in the art. The term “% sequence identity” refers toa relationship between two or more sequences, as determined by comparing the sequences. In the art, “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. M. and Griffin, H. G. eds.) Humana Press, NJ (1994); Sequence Analysis in Molecular Biology (Von Heijne, G. ed.) Academic Press (1987); and Sequence Analysis Primer (Gribskov, M. and Devereux, J. eds.) Oxford University Press, NY (1992), each of which are separately incorporated by reference in their entirety. Preferred methods to determine identity are designed to give the best match between the sequences tested. Methods to determine identity and similarity are codified in publicly available computer programs. For example, calculation of the percent identity of twonucleic acid or polypeptide sequences can be performed by aligning the two sequences (or thecomplement of one or both sequences) for optimal comparison purposes (e.g., gaps can beintroduced in one or both of a first and a second sequences for optimal alignment and non- identical sequences can be disregarded for comparison purposes). The nucleotides or amino acids at corresponding positions are then compared. When a position in the first sequence isoccupied by the same residue (e.g., nucleotide or amino acid) as the corresponding position inthe second sequence, then the molecules are identical at that position. 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). Sequence12621818v1 Page 84 of 147Attorney 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). Multiple alignment of the sequences can also be performed using the Clustal method of alignment (Higgins and Sharp, Comp Appl Biosci (1989) 5(2):151-153), incorporated by reference herein in its entirety, with default parameters (GAP PENALTY=10, GAP LENGTHPENALTY=10). Relevant programs also include the GCG suite of programs (WisconsinPackage Version 9.0, Genetics Computer Group (GCG), Madison, Wisconsin); BLASTP, BLASTN, BLASTX (Altschul et al., J Mol Biol (1990) 215:403-410); DNASTAR (DNASTAR, Inc., Madison, Wisconsin); and the FASTA program incorporating the Smith- Waterman algorithm (Pearson, Comput Methods Genome Res [Proc Int Symp] (1994), Meeting Date 1992, 111-120. Eds. Suhai, Sandor. Plenum, New York, NY (the contents of each of which is separately incorporated herein by reference in its entirety). Within the context of this disclosure, it will be understood that where sequence analysis software is used for analysis, the results of the analysis are based on the “default values” of the program referenced. “Default values” will mean any set of values or parameters, which originally load with the software when first initialized.

[0262] “Improve,” “increase,” “inhibit,” or “reduce”: As used herein, the terms“improve”, “increase”, “inhibit”, and “reduce”, and grammatical equivalents thereof, indicate qualitative or quantitative difference from a reference.

[0263] “Modification Status” or “Histone Modification Status”: As used herein,“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. Other well-known assays include CUT&RUN (Cleavage Under Targets and Release Using Nuclease) sequencingand CUT&Tag (Cleavage Under Targets and Tagmentation). Where two samples are separatelyanalyzed by the same assay or comparable assays for detection of DNA sequences associated with histones bearing one or more histone modifications (e.g., one or more particular histone12621818v1 Page 85 of 147Attorney Docket: 2014191-0036 modifications), differences in modification status of genomic loci can be detected. Modification status can be compared to a standard or reference. A sample that has a modification status that differs in modification status or histone modification status from a standard or reference can be referred to as differentially modified.

[0264] Regulatory sequence: As used herein in the context of expression of a nucleicacid coding sequence, 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 someembodiments, a regulatory sequence can control or impact one or more aspects of geneexpression (e.g., cell-type-specific expression, inducible expression, etc.).

[0265] Subject: As used herein, the term “subject” refers to an organism, typically amammal (e.g., a human). In some embodiments, a subject is suffering from a disease, disorderor condition (e.g., breast cancer, small cell lung cancer or SCLC, non-small cell lung cancer orNSCLC, neuroendocrine prostate cancer or NEPC, prostate adenocarcinoma or PRAD, etc.). Insome embodiments, a subject is susceptible to a disease, disorder, or condition. In some embodiments, a subject displays one or more symptoms or characteristics of a disease, disorder or condition. In some embodiments, a subject is not suffering from a disease, disorder or condition. In some embodiments, a subject does not display any symptom or characteristic of a disease, disorder, or condition. In some embodiments, a subject has one or more features characteristic of susceptibility to or risk of a disease, disorder, or condition. In some embodiments, a subject is a subject that has been tested for a disease, disorder, or condition, and / or to whom therapy has been administered. In some instances, 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 subjectthat has been diagnosed with cancer, breast cancer, etc. and is intended to encompass any of theaforementioned subjects.

[0266] Therapeutic agent: As used herein, the term “therapeutic agent” refers to anyagent that elicits a desired pharmacological effect when administered to a subject. In some embodiments, an agent is considered to be a therapeutic agent if it demonstrates a statistically significant effect across an appropriate population. In some embodiments, the appropriate population can be a population of model organisms or a human population. In some embodiments, an appropriate population can be defined by various criteria, such as a certain age12621818v1 Page 86 of 147Attorney Docket: 2014191-0036 group, gender, genetic background, preexisting clinical conditions, etc. In some embodiments, 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.). In some embodiments, a therapeutic agent is an agent that has been or is required to be approved by agovernment agency before it can be marketed for administration to humans. In someembodiments, a therapeutic agent is an agent for which a medical prescription is required for administration to humans. In some embodiments, a therapeutic agent is an ADC.

[0267] Therapeutically effective amount: As used herein, “therapeutically effectiveamount” refers to an amount that produces the desired effect for which it is administered. In some embodiments, the term refers to an amount that is sufficient, when administered to apopulation suffering from or susceptible to a disease, disorder, and / or condition (e.g., breastcancer, small cell lung cancer or SCLC, non-small cell lung cancer or NSCLC, neuroendocrineprostate cancer or NEPC, prostate adenocarcinoma or PRAD, etc.) in accordance with atherapeutic dosing regimen, to treat the disease, disorder, and / or condition. In some embodiments, 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. Those of ordinary skill in the art will appreciate that the term “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. In some embodiments, reference to a therapeutically effective amountmay be a reference to an amount as measured in one or more specific tissues (e.g., a tissueaffected by the disease, disorder or condition) or fluids (e.g., blood, saliva, serum, sweat, tears,urine, etc.). Those of ordinary skill in the art will appreciate that, in some embodiments, a therapeutically effective amount of a particular agent or therapy may be formulated and / or administered in a single dose. In some embodiments, 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.

[0268] Treatment: As used herein, the term “treatment” (also “treat” or “treating”)refers to administration of a therapy that partially or completely alleviates, ameliorates, relieves,12621818v1 Page 87 of 147Attorney Docket: 2014191-0036 inhibits, delays onset of, reduces severity of, and / or reduces incidence of one or more symptoms, features, and / or causes of a particular disease, disorder, or condition, or is administered for the purpose of achieving any such result. In some embodiments, such treatment can be of a subject who does not exhibit signs of the relevant disease, disorder, or condition and / or of a subject whoexhibits only early signs of the disease, disorder, or condition (e.g., breast cancer, small cell lungcancer or SCLC, non-small cell lung cancer or NSCLC, neuroendocrine prostate cancer orNEPC, prostate adenocarcinoma or PRAD, etc.). Alternatively, or additionally, such treatment can be of a subject who exhibits one or more established signs of the relevant disease, disorder and / or condition. In some embodiments, treatment can be of a subject who has been diagnosed as suffering from the relevant disease, disorder, and / or condition. In some embodiments, 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 prophylactictreatment 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. EXAMPLES Example 1: Materials and Methods

[0269] This Example describes the materials and methods that were used to generatesequencing data that was then used in Example 2 to identify promoter (H3K4me3) and enhancer (H3K27ac) epigenomic activation signals at select genes for ADC target antigens or genes that modulate ADC response / resistance. Materials Plasma samples12621818v1 Page 88 of 147Attorney Docket: 2014191-0036

[0270] Plasma samples (from cancer patients and healthy volunteers) were collected fromcommercial biobanks and stored at -80˚C until use. The percentage of ctDNA in the plasma samples from cancer patients was 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). Plasma samples from cancer patients with at least 5.5% ctDNA were used in the Examples described herein. Methods Chromatin immunoprecipitation (ChIP)

[0271] Chromatin immunoprecipitation (ChIP) for histone marks (H3K4me3 andH3K27ac) in plasma samples was performed using methods similar to those previously describedin 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 mLfrozen plasma was thawed and then prepared for ChIP. The thawed plasma was incubated withantibodies that bind H3K4me3 modifications or H3K27ac modifications that were previouslyconjugated to magnetic epoxy beads (Invitrogen) with constant mild shaking overnight. The beads were then washed and rinsed. Sequencing libraries were generated from purifiedimmunoprecipitated sample DNA and then sequenced.ChIP-seq data analysis

[0272] ChIP-sequencing reads were aligned to the human genome build hg19 using theBurrows-Wheeler Aligner (BWA) version 0.7.15. Non-uniquely mapping and redundant readswere discarded. MACS v2.2.7.1 was used for peak calling with a q-value (FDR) threshold of0.01. Data quality was evaluated by a variety of measures, including total peak number, FrIP (fraction of reads in peak) score, number of high-confidence peaks (enriched > ten-fold over background), and percent of peak overlap with “blacklist” DHS peaks derived from the ENCODE project (Amemiya et al., Sci Rep (2019) 9(1):9354). Peaks were assessed for overlapwith gene features and CpG islands using annotatr. IGV was used to visualize normalized readcounts at specific genomic loci. Overlap of peaks was assessed using BEDTools and the GenomicRanges package in Bioconductor. Peaks were considered overlapping if they shared one or more nucleotide.12621818v1 Page 89 of 147Attorney Docket: 2014191-0036 Example 2: Identification of promoter and enhancer epigenomic activation signals at selectgenes for ADC target antigens or genes that modulate ADC response / resistance

[0273] To identify promoter (H3K4me3) epigenomic activation signals at select genesfor ADC target antigens or genes that modulate ADC response / resistance we defined a peakwithin + / - 1kb of the transcription site (TSS) for each gene. If there was no peak, the entireregion within + / - 1kb of the TSS was used. If there were multiple peaks or multiple TSSs thepeak 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 H3K4me3 regions of interest for select genes for ADC target antigens are provided in Table 1. Exemplary genomic coordinates of the H3K4me3 regions of interest for select genes that modulate ADC response / resistance are provided in Table 2. As a control, H3K4me3 epigenomic activation signals were also measured at housekeeping genes using a similar approach. Table 1: Exemplary genomic coordinates of H3K4me3 regions of interest for select genes for ADC target antigens. Indication Gene (Target) Genomic coordinates12621818v1 Page 90 of 147Attorney Docket: 2014191-0036 SCLC TACSTD2 (TROP2) chr1:59,040,748-59,043,452SCLC FOLH1 (PSMA) chr11:49,228,902-49,230,855genes that modulate ADC response / resistance. Indication Gene (Protein) Genomic coordinates12621818v1 Page 91 of 147Attorney Docket: 2014191-0036 SCLC (Fig. 54) ABCB1 (MDR1) chr7:87,220,000-87,260,500SCLC (Fig. 54) ABCG2 (BCRP) chr4:89,077,500-89,081,500ct genes forADC target antigens we defined a peak within + / - 10kb of the transcription site (TSS) for eachgene. If there was no peak, the entire region within + / - 10kb of the TSS was used. If there weremultiple 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. Indication Gene (Target) Genomic coordinates12621818v1 Page 92 of 147Attorney Docket: 2014191-0036 Breast cancer F3 (Tissue factor) chr1:95,005,901-95,008,635Breast cancer FOLR1 chr11:71,910,584-71,911,55412621818v1 Page 93 of 147Attorney Docket: 2014191-0036 NEPC / PRAD DLL3 Chr19:39,985,409-39,994,931NEPC / PRAD SEZ6 Chr17:27,305,415-27,356,950on of allbase 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 (by at least 1 bp) were quantified for each analyte. Counts of reads in all enriched regions between experiments (any region called a peak in at least one sample) 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. To correct for ctDNA% weused 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 regressionweight. Corrected enhancer and promoter counts were summed for each gene to produce anintegrated activation score. The mean and standard-deviation of the summed enhancer andpromoter counts within the healthy volunteers were used to calculate a z-score for each patientsample, which was then logged and 0-1 scaled for the final activation score.

[0276] Fig. 1 (left-hand image) shows a qualitative track view displaying promoter(H3K4me3) signals across breast cancer-relevant ADC target antigens (plus housekeeping genesGAPDH and ACTB). Rows represent select breast cancer patient plasma samples sorted byichorCNA ctDNA fraction estimate or healthy volunteer plasma samples. Fig.1 (right-handimage) shows a quantitative heatmap of normalized and ctDNA-corrected epigenomic activationscore (combining enhancer and promoter signals) for each ADC target antigen. Patient-specificepigenomic activation scores for each gene are represented on a white (low) to dark(high) gradient.

[0277] Figs. 2-8 show trend lines and confidence intervals for promoter signals(H3K4me3; left graph) and enhancer signals (H3K27ac; right graph) based on ctDNA% for individual breast cancer-relevant ADC target antigens.12621818v1 Page 94 of 147Attorney Docket: 2014191-0036

[0278] Fig. 9 (left-hand image) shows a qualitative track view displaying promoter(H3K4me3) signals across SCLC-relevant ADC target antigens (plus housekeeping genesGAPDH and ACTB). Rows represent select SCLC patient plasma samples sorted by ichorCNActDNA fraction estimate or healthy volunteer plasma samples. Fig. 9 (right-hand image) showsa quantitative heatmap of normalized and ctDNA-corrected epigenomic activation score (combining enhancer and promoter signals) for each ADC target antigen. Patient-specificepigenomic activation scores for each gene are represented on a white (low) to dark(high) gradient.

[0279] 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.

[0280] Fig. 19 (left-hand image) shows a qualitative track view displaying promoter(H3K4me3) signals across NSCLC-relevant ADC target antigens (plus housekeeping genesGAPDH and ACTB). Rows represent select NSCLC patient plasma samples sorted by ichorCNActDNA fraction estimate or healthy volunteer plasma samples. Fig. 19 (right-hand image) showsa quantitative heatmap of normalized and ctDNA-corrected epigenomic activation score (combining enhancer and promoter signals) for each ADC target antigen. Patient-specificepigenomic activation scores for each gene are represented on a white (low) to dark(high) gradient.

[0281] 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.

[0282] Figs. 36-51 show additional data obtained for breast cancer-relevant ADC targetantigens using breast cancer patient plasma samples and healthy volunteer plasma samples. Eachfigure 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.). In each figure, the left-hand image shows a qualitative track view displaying promoter (H3K4me3) signals for the gene encoding the relevantADC target antigen and other transcripts of interest in breast cancer for context (HER2, FOXA1and MYC) plus housekeeping genes (GAPDH and ACTB) as controls. Rows represent selectbreast cancer patient plasma samples sorted by ichorCNA ctDNA fraction estimate or healthyvolunteer plasma samples. The ER and HER2 status (based on IHC) is also shown for each12621818v1 Page 95 of 147Attorney Docket: 2014191-0036 sample. Blue boxes identify samples for which ER status was unknown. In each figure, the central image shows a quantitative heatmap of normalized and ctDNA-corrected epigenomicactivation score (combining enhancer and promoter signals) for the ADC target antigencompared 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 outand scaled to the min and the max of the ADC target antigen in these samples). In each figure, 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 therelevant ADC target antigen. Without limitation, the results in Figs. 36-51 show that epigenomicsignals associated with the VTCN1, NECTIN4, ERBB2, EPHA5, ROR2, CDH6 and MET genesproduce the greatest differentiation among breast cancer patients and / or between breast cancer patients and healthy volunteers. Epigenomic signals associated with the TACSTD2, ERBB3, F3,CD276, LY75 and KAAG1 genes also show some useful differentiation.

[0283] Fig. 52 shows trend lines and confidence intervals for the promoter signal(H3K4me3; top graph) and enhancer signal (H3K27ac; bottom graph) based on ctDNA% for the other transcripts of interest in breast cancer for context (HER2, FOXA1 and MYC).

[0284] Fig. 53 shows promoter epigenomic activation signals at select genes known tomodulate 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 drugefflux pumps ABCG2, ABCB1).

[0285] Fig. 54 shows promoter epigenomic activation signals at select genes known tomodulate ADC response / resistance in breast, NSCLC and SCLC patient plasma samples. Altered expression of these genes could impair ADC internalization (chaperone proteins Endophilin A2, HSP90), disrupt lysosomal processing (SLC46A3), induce payload efflux (increase in drug efflux pumps ABCG2, ABCB1) or impart payload resistance (SLFN11: topoisomerase inhibitor resistance, CCNB1).

[0286] Fig. 55 (left-hand image) shows a qualitative track view displaying histonemodification signal across NEPC and PRAD-relevant ADC target antigens (plus housekeepinggenes GAPDH and ACTB) in plasma samples obtained from patients with NEPC or PRAD orhealthy subjects. Rows represent select NEPC or PRAD patient plasma samples sorted by12621818v1 Page 96 of 147Attorney 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 obtainedfrom AR+ / NE- PRAD subjects. Also shown is a measure of H3K27ac signal at the locus, whichdemonstrates 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. Also shown is a measure of H3K27ac signal at the locus, which demonstrates an ability to detect increased H3K27ac signal at the DLL3 locus in NEPC subjects as compared to PRAD subjects. In some embodiments, 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 oran enhancer of a gene encoding an ADC target antigen. In some embodiments, the one or moregenomic loci are associated with an enhancer and are located within + / - 10kb of the transcriptionsite (TSS) of a gene encoding an ADC target antigen.

[0287] Among other things, the present disclosure described a method of treating prostatecancer, 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 geneencoding an ADC target antigen (e.g., one or more PRAD / NEPC associated target loci listed inTable 3). Example 3: Correlation Between Predicted Expression and Measured Expression in Cell Lines

[0288] The present Example provides data demonstrating that technologies describedherein can be used to predict expression of certain ADC targets. In particular, the present Example provides data demonstrating that loci described herein can be used to predict of each of the targets indicated in Fig.56(A), including HER2, B7-H4, ER, NECTIN4, MET, MUC1, TMRSF1, P-CAD, HER3, IB-6, EGFR, TF, ROR2, AXL, NaPi2b, FGFR2, EPHA5, GD2, GRPR, GPC-1, TROP2, LIV1, PTK-7, and ROR1. Predictive ability was shown in both (1) simulated plasma samples (breast cancer cell line sequencing data diluted in silico with sequencing data from plasma samples obtained from healthy subjects), and (2) plasma samples from patients with cancer.12621818v1 Page 97 of 147Attorney Docket: 2014191-0036

[0289] RNA-seq, ChIP-Seq, and MBD-seq assays were performed for a number ofrelevant cancer cell lines to measure RNA expression and determine H3K4me3-, H3K27ac-, andDNAme- modification patterns.

[0290] Table 4 provides genomic loci, each one of which was shown to have theindicated 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.

[0291] Fig. 56(A) provides spearman correlations coefficients (determined by comparingpredicted expression vs. measured expression as determined by RNA-seq) plotted against AUC for a number of targets. These values were obtained using simulated 10% ctDNA breast cancerplasma samples (generated by diluting in silico breast cancer cell line sequencing data withsequencing data from the plasma of healthy patients). As shown, strong predictive performance was observed for a number of genes, including HER2, B7-H4, ER, NECTIN4, MET, and EGFR. These results demonstrate that loci described herein can be used to predict expression of genes that are clinically actionable.

[0292] Fig. 56(B) shows predicted and measured expression levels of B7-H4 (VTCN1)in 35 different breast cancer cell lines. As shown, a strong correlation was observed between measured and predicted expression, demonstrating the robustness of technologies described herein and their ability to accurately predict expression across a range of biological conditions.

[0293] Fig. 56(C) shows Spearman correlation coefficient values (determined bycomparing predicted and measured expression) measured at different tumor fractions (ctDNA) in simulated plasma samples for 9 different targets. Simulated plasma samples were generated bydiluting 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 inplasma samples obtained from breast cancer patients. These predicted expression levels were then compared to expression levels that were measured using RNA-seq in matched tumor biopsy(tissue) samples. Results are shown in Fig. 57. As shown, a strong correlation betweenpredicted and actual expression was observed in patient plasma samples, again confirming theability of technologies described herein to predict target expression, and demonstrating that12621818v1 Page 98 of 147Attorney Docket: 2014191-0036 results obtained using simulated plasma sample correspond with those obtained in patient plasma samples. 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 canbe used to accurately predict DLL3 expression. The present Example provides results from twomodels – 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 lociproximal to other genes whose expression correlated with that of DLL3. In the present Example, “proximal” refers to loci within a gene or within 200 kB of a gene.

[0296] The loci used in the single gene model are shown in Table 5, below.Table 5: Loci Proximal to DLL3, in Which Epigenetic Modifications Correlate with DLL3 Expression Chr Start End Gene Analytechr19 39990062 39991061 DLL3 H3K4me33

[0297] TheAB3IP (RAB3AInteracting Protein), PAK5 (p21 (RAC1) activated kinase 5), and ITPRIPL2 (ITPRIP like 2).Proximal regions to each of these genes and DLL3, which were used in the multigene model are shown in Table 6, below.Table 6: Loci Proximal to DLL3, RAB3IP, PAK5 and ITPRIPL2, in Which EpigeneticModifications Correlate with DLL3 Expression Chr Start End Gene Analyte333333312621818v1 Page 99 of 147Attorney Docket: 2014191-0036 chr16 19119335 19120334 ITPRIPL2 H3K27acchr16 19123335 19124334 ITPRIPL2 H3K27ac

[0298] In the genomicposition (positionsshown 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 thatcorrelates with DLL3 expression.

[0299] In silico samples were prepared and DLL3 expression levels predicted using themultigene model and the single gene model. Predicted values were compared against actualexpression values and Pearson’s coefficients were determined. Results are shown in Fig. 58. Asshown, both the single gene and the multigene model accurately predicted DLL3 expression. The Multigene Model provided improved precision as compared to the Single Gene model, as a higher correlation with 30% ctDNA predictions was maintained at lower ctDNA%. DLL3 expression prediction was also found to correspond with models predicting likelihood of a subject having SCLC (data not shown), as expected, and further demonstrating that DLL3 models provided herein can accurately predict DLL3 expression. Example 5: Predicting Expression of NEPC and PRAD Markers in Patient Plasma Samples

[0300] The present Example provides further data demonstrating that technologiesprovided herein can be used to measure expression of ADC targets. In particular, the present Example demonstrates that technologies described herein can be used to measure expression ofDLL3, SEZ6, and CHGA in subjects having NEPC or PRAD.

[0301] 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 regionshaving H3K4me3 signal that correlated with expression of each of these markers was determined. For each sample, promoter (H3K4me3) signal at each of these promoter regionswas measured and corrected for ctDNA%. Results are provided in Fig. 59(A). As shown,promoter signal for each of DLL3, SEZ6, and CHGA was increased in NEPC subjects as12621818v1 Page 100 of 147Attorney 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 ofKLK3 and KLK2 was also measured as control, as these genes are expressed in PRAD but notNEPC. Both KLK2 and KLK3 expression was found to be elevated in PRAD as compared toNEPC, again demonstrating that technologies provided herein can be used to measure ADC target expression.

[0302] As DLL3, SEZ6, and CHGA are each markers of NEPC, they can also be used toclassify 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 distinguishingPRAD 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 haveelevated DLL3 / SEZ6 / CHGA promoter signal, despite having been diagnosed with PRAD. Thisresult suggests that 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. TABLES

[0303] Table 4 describes loci with epigenetic modification levels that correlate withexpression 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 ofEffect” refers to whether the association is positive or negative (meaning whether epigeneticmodifications 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. For all of the provided 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 147Attorney 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. Start End Width Target NameAXL H3K4me3 POS chr19 41698321 41698712 392 AXL12621818v1 Page 102 of 147Attorney Docket: 2014191-0036B4GALNT1 H3K27ac POS chr12 58025718 58027552 1835 GD2B4GALNT1 H3K27ac POS chr12 58116746 58117736 991 GD212621818v1 Page 103 of 147Attorney Docket: 2014191-0036CD276 H3K27ac POS chr15 73988185 73990653 2469 B7-H3CD276 H3K27ac POS chr15 73991642 73992135 494 B7-H312621818v1 Page 104 of 147Attorney Docket: 2014191-0036CDH3 H3K27ac POS chr16 68669804 68671303 1500 P-CADCDH3 H3K27ac POS chr16 68674804 68675803 1000 P-CAD12621818v1 Page 105 of 147Attorney Docket: 2014191-0036CEACAM5 H3K4me3 POS chr19 42258961 42263397 4437 CEACAM5CEACAM5 H3K4me3 POS chr19 42349133 42349573 441 CEACAM512621818v1 Page 106 of 147Attorney Docket: 2014191-0036CLDN6 H3K27ac POS chr16 3155496 3157453 1958 CLDN6CLDN6 H3K27ac POS chr16 3161370 3162348 979 CLDN612621818v1 Page 107 of 147Attorney Docket: 2014191-0036EGFR H3K27ac POS chr7 55118297 55123790 5494 EGFREGFR H3K27ac POS chr7 55128787 55129285 499 EGFR12621818v1 Page 108 of 147Attorney Docket: 2014191-0036EGFR MBD POS chr7 55269745 55270030 286 EGFREGFR MBD POS chr7 55315771 55316065 295 EGFR12621818v1 Page 109 of 147Attorney Docket: 2014191-0036ERBB2 H3K4me3 POS chr17 37891476 37893900 2425 HER2ERBB2 H3K4me3 POS chr17 37896241 37909448 13208 HER212621818v1 Page 110 of 147Attorney Docket: 2014191-0036ERBB2 MBD POS chr17 37653090 37653572 483 HER2ERBB2 MBD POS chr17 37661070 37662008 939 HER212621818v1 Page 111 of 147Attorney Docket: 2014191-0036ERBB2 MBD POS chr17 37870625 37871078 454 HER2ERBB2 MBD POS chr17 37873880 37874373 494 HER212621818v1 Page 112 of 147Attorney Docket: 2014191-0036ERBB3 MBD NEG chr12 56434846 56435222 377 HER3ERBB3 MBD NEG chr12 56600043 56600493 451 HER312621818v1 Page 113 of 147Attorney Docket: 2014191-0036ESR1 MBD NEG chr6 152128313 152128789 477 ERESR1 MBD NEG chr6 152473249 152473690 442 ER12621818v1 Page 114 of 147Attorney Docket: 2014191-0036FGFR2 MBD POS chr10 123249892 123251291 1400 FGFR2FGFR2 MBD POS chr10 123252226 123253158 933 FGFR212621818v1 Page 115 of 147Attorney Docket: 2014191-0036FOLH1 MBD NEG chr11 49229359 49230119 761 PSMAFOLR1 H3K4me3 POS chr11 71955066 71955472 407 FRa12621818v1 Page 116 of 147Attorney Docket: 2014191-0036IGF1R H3K4me3 POS chr15 99411781 99412272 492 IGF-1RIGF1R H3K4me3 POS chr15 99557022 99557447 426 IGF-1R12621818v1 Page 117 of 147Attorney Docket: 2014191-0036IGF1R MBD POS chr15 99370565 99371405 841 IGF-1RIGF1R MBD POS chr15 99422143 99422622 480 IGF-1R12621818v1 Page 118 of 147Attorney Docket: 2014191-0036LY75 MBD POS chr2 160666523 160666758 236 CD205LY75 MBD POS chr2 160883074 160883282 209 CD20512621818v1 Page 119 of 147Attorney Docket: 2014191-0036MET MBD POS chr7 116604045 116604528 484 METMET MBD NEG chr7 116139441 116141208 1768 MET12621818v1 Page 120 of 147Attorney Docket: 2014191-0036MUC1 H3K27ac POS chr1 155038719 155039214 496 MUC1MUC1 H3K27ac POS chr1 155041200 155041695 496 MUC112621818v1 Page 121 of 147Attorney Docket: 2014191-0036NECTIN4 MBD POS chr1 160945657 160946050 394 NECTIN4NECTIN4 MBD POS chr1 160955075 160955468 394 NECTIN412621818v1 Page 122 of 147Attorney Docket: 2014191-0036PTK7 MBD NEG chr6 43110767 43111229 463 PTK-7PTK7 MBD NEG chr6 43137693 43138492 800 PTK-712621818v1 Page 123 of 147Attorney Docket: 2014191-0036ROR2 MBD NEG chr9 94186779 94187689 911 ROR2ROR2 MBD NEG chr9 94711429 94712754 1326 ROR212621818v1 Page 124 of 147Attorney Docket: 2014191-0036SLC39A6 MBD POS chr18 33507055 33507364 310 LIV1SLC39A6 MBD POS chr18 33529817 33530300 484 LIV112621818v1 Page 125 of 147Attorney Docket: 2014191-0036STEAP1 H3K27ac POS chr7 89809364 89810342 979 STEAP1STEAP1 H3K27ac POS chr7 89840822 89841293 472 STEAP112621818v1 Page 126 of 147Attorney Docket: 2014191-0036TM4SF1 H3K27ac POS chr3 149295490 149295988 499 TM4SF1TM4SF1 MBD POS chr3 148888466 148889414 949 TM4SF112621818v1 Page 127 of 147Attorney Docket: 2014191-0036TNFRSF10B MBD POS chr8 23120563 23120975 413 DR-5TNFRSF10B MBD NEG chr8 22925395 22925888 494 DR-512621818v1 Page 128 of 147Attorney Docket: 2014191-0036 OTHER EMBODIMENTS

[0304] It will be appreciated that the scope of the present disclosure is to be defined bythat which may be understood from the disclosure and claims rather than by the specificembodiments that have been presented by way of example. Elements described with respect toone aspect or embodiment of the present disclosure are also contemplated with respect to other aspects or embodiments of the present disclosure. For example, elements of claims that depend directly or indirectly from a certain independent claim presented herein serve as support for those elements being presented in additional dependent claims of one or more other independent claims. Throughout the description, where compositions or methods are described as having, including, or comprising specific elements, it is to be understood that compositions or methods that consist essentially of, consist of, or do not comprise the recited elements are likewise herebydisclosed. All references cited herein are hereby incorporated by reference.12621818v1 Page 129 of 147

Claims

Attorney Docket: 2014191-0036 CLAIMS What is claimed is:

1. A method comprising quantifying, at one or more genomic loci in a biological sample,optionally in cell-free DNA (cfDNA) from a liquid biopsy sample, obtained or derived from a 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) 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.

2. The method of claim 1, wherein the one or more genomic loci are associated with apromoter and are located within + / - 200 kB (e.g., + / - 10 kB or + / - 1kb) of the transcription site(TSS) of a gene encoding an ADC target antigen or a gene that modulates ADCresponse / resistance, e.g., a genomic locus that is located within a region defined by a pair ofgenomic coordinates in Table 1, Table 2, Table 4, Table 5, or Table 6.

3. The method of claim 1 or 2, wherein the one or more genomic loci are associated with anenhancer and are located within + / - 200 kB (e.g., + / - 10kb) of the transcription site (TSS) of agene encoding an ADC target antigen or a gene that modulates ADC response / resistance, e.g., agenomic locus that is located within a region defined by a pair of genomic coordinates in Table3, Table 4, Table 5, or Table 6.

4. The method of any one of claims 1-3, wherein the one or more genomic loci are locatedwithin + / - 200 kB of the TSS of a gene encoding an ADC target antigen or a gene that modulates12621818v1 Page 130 of 147Attorney Docket: 2014191-0036 ADC response / resistance, e.g., a genomic locus that is located within a region defined by a pairof genomic coordinates in Table 4, Table 5, or Table 6.

5. The method of any one of claims 1-4, wherein the subject is a breast cancer patient andthe gene encoding an ADC target antigen is selected from the group consisting of AXL, CCR2,CEACAM5, CD44, CLDN6, DLL3, EGFR, FAP, FOLH1, TNFRSF10B, FN-1, ESR1, FGFR2,B4GALNT1, GPC1, GRPR, ITGB6, IGF1R, MUC1, CDH3, CD274, PTK7, SSTR2, STEAP1,STEAP2, TM4SF1, ROR1, SEZ6, SLC34A2, TACSTD2, VTCN1, NECTIN4, ERBB3, ERBB2,EPHA5, ROR2, F3, FOLR1, CD276, MSLN, SLC39A6, LY75, LY75-CD302, CDH6, KAAG1,TPBG and MET.

6. The method of claim 5, wherein the subject is a breast cancer patient and the geneencoding an ADC target antigen is selected from the group consisting of VTCN1, NECTIN4,ERBB2, EPHA5, ROR2, CDH6 and MET.

7. The method of claim 5, wherein the subject is a breast cancer patient and the geneencoding an ADC target antigen is selected from the group consisting of TACSTD2, ERBB3, F3,CD276, LY75 and KAAG1.

8. The method of any one of claims 1-4, wherein the subject is small cell lung cancer(SCLC) patient and the gene encoding an ADC target antigen is selected from the groupconsisting of TACSTD2, FOLH1, NCAM1, ERBB3, CD276, MSLN, SEZ6, DLL3, and FAP.

9. The method of any one of claims 1-4, wherein the subject is a non-small cell lung cancer(NSCLC) patient and the gene encoding an ADC target antigen is selected from the groupconsisting of TACSTD2, F3, NECTIN4, FOLR1, ERBB3, CD276, ERBB2, AXL, CEACAM5,TFRC, SLC34A2, PTK7, GPNMB, EGFR, MET and ROR2.

10. The method of any one of claims 1-4, wherein the subject is an NEPC or PRAD patientand the gene encoding an ADC target antigen is selected from the group consisting of AR,STEAP1, CHGA, SEZ6, SSTR2, DLL3, KLK2, and KLK3.12621818v1 Page 131 of 147Attorney Docket: 2014191-003611. The method of any one of claims 1-4, wherein the gene encoding an ADC target gene isDLL3.

12. The method of claim 11, wherein the method further comprises quantifying, in thebiological sample, at one or more genomic loci (a) within RAB3IP, PAK5, or ITPRIPL2, or anycombination thereof; and / or (b) associated with a promoter or an enhancer of RAB3IP, PAK5, orITPRIPL2, or any combination thereof: (i) one or more histone modifications, (ii) DNA methylation,(iii) chromatin accessibility, and / or(iv) binding of one or more transcription factors.

13. The method of claim 12, wherein the one or more genomic loci within RAB3IP, PAK5, orITPRIPL2, or any combination thereof, and / or associated with a promoter or an enhancer ofRAB3IP, PAK5, or ITPRIPL2, or any combination thereof, are located within + / - 200 kB of theTSS of RAB3IP, PAK5, or ITPRIPL2, or any combination thereof, e.g., a genomic locus that isdefined by a pair of genomic coordinates in Table 6.

14. The method of any one of claims 1-4, wherein the gene that modulates ADCresponse / resistance is selected from the group consisting of SH3GL1, HSP90AA1, SLC46A3,ABCB1, ABCG2, SLFN11, CCNB1, and TUBB3.

15. The method of claim 14, wherein the subject is a breast cancer patient, optionally whereinthe subject is a metastatic breast cancer patient.

16. The method of claim 15, wherein the subject is a breast cancer patient, a small cell lungcancer (SCLC) patient or a non-small cell lung cancer (NSCLC) patient.

17. The method of claim 15, wherein the subject is an ER+ breast cancer patient.12621818v1 Page 132 of 147Attorney Docket: 2014191-003618. The method of any one of claims 1-17, wherein the one or more histone modifications arequantified using a histone modification assay that measures one or more of H3K9ac, H3K14ac, H3K18ac, H3K23ac, H3K27ac, H3K4me1, H3K4me2, H3K4me3, pan-acetylation, and DNAme.

19. The method of claim 18, wherein the histone modification assay detects H3K4me3modifications.

20. The method of claim 18 or 19, wherein the histone modification assay detects H3K27acmodifications.

21. The method of any one of claims 1-20, wherein the histone modification assay is selectedfrom ChIP-seq (Chromatin ImmunoPrecipitation sequencing), CUT&RUN (Cleavage Under Targets and Release Using Nuclease) sequencing, and CUT&Tag (Cleavage Under Targets and Tagmentation) sequencing.

22. The method of any one of claims 1-21, wherein the DNA methylation assay is MeDIP-seq or MBD-seq.

23. The method of any one of claims 1-22, wherein chromatin accessibility is quantifiedusing 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 afragmentomics assay.

24. The method of any one of claims 1-23, wherein the binding of one or more transcriptionfactors is quantified using a transcription factor binding assay that detects binding of one or moreof p300, mediator complex, cohesin complex, RNA pol II, FOXA1, ESR1, PR, MYC, EN1,FOXM1, KLF4, AP-2, RARa, or RUNX1.12621818v1 Page 133 of 147Attorney Docket: 2014191-003625. The method of any one of claims 1-24, wherein the transcription factor binding assay isselected 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.

26. The method of any one of claims 1-25, comprising quantifying two or more of thefollowing, 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) DNA methylation,(iii) chromatin accessibility, and(iv) transcription factor binding.

27. The method of claim 26, comprising quantifying two or more histone modifications forthe same gene.

28. The method of claim 27, comprising quantifying H3K4me3 and H3K27ac modificationsfor the same gene.

29. The method of claim 26, comprising quantifying one or more histone modifications andDNA methylation for the same gene.

30. The method of claim 29, comprising quantifying H3K4me3, H3K27ac, and DNAmethylation modifications for the same gene.

31. The method of any one of claims 28 or 30, wherein the quantified H3K4me3 andH3K27ac modifications are combined to generate an activation score for the gene.

32. The method of claim 31, wherein the quantified H3K4me3 and H3K27ac modificationsare summed, optionally with weighting, to generate an activation score for the gene.12621818v1 Page 134 of 147Attorney Docket: 2014191-003633. The method of claim 30, wherein the quantified H3K4me3, H3K27ac and DNAmethylation modifications are combined to generate an activation score for the gene.

34. The method of claim 33, wherein the quantified H3K4me3, H3K27ac, and DNAmethylation modifications are summed, optionally with weighting, to generate an activation score for the gene.

35. The method of any one of claims 31-34, wherein the gene encodes an ADC targetantigen, optionally wherein the ADC target antigen is TROP2.

36. The method of any one of claims 31-34, wherein the gene modulates ADCresponse / resistance.

37. The method of any one of claims 1-36, wherein the liquid biopsy sample is a plasmasample, serum sample, or urine sample.

38. The method of claim 37, wherein the liquid biopsy sample is a plasma sample, optionallya 1 mL plasma sample.

39. The method of any one of claims 1-38, wherein quantification of one or more histonemodifications, 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.

40. The method of claim 39, wherein the reference is a predetermined threshold, ameasurement 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.12621818v1 Page 135 of 147Attorney Docket: 2014191-003641. The method of claim 39, wherein the reference is a predetermined threshold, ameasurement 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.

42. The method of any one of claims 1-38, wherein quantification of one or more histonemodifications, 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 indicateswhether a gene that modulates ADC response / resistance is active.

43. The method of claim 42, wherein the reference is a predetermined threshold, ameasurement 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 whohave previously been determined to have low or undetectable activity of the gene that modulatesADC response / resistance.

44. The method of claim 42, wherein the reference is a predetermined threshold, ameasurement 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.

45. The method of claim 42, wherein the reference is a predetermined threshold, ameasurement 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.

46. The method of any one of claims 1-38, wherein quantification of one or more histonemodifications, DNA methylation, chromatin accessibility, and / or binding of one or more12621818v1 Page 136 of 147Attorney Docket: 2014191-0036transcription factors, at the one or more genomic loci as compared to a reference indicateswhether the subject is likely to respond to treatment with an ADC for the ADC target.

47. The method of claim 46, wherein the reference is a predetermined threshold, ameasurement 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.

48. The method of any one of claims 1-47, wherein the method comprises quantifying one ormore 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 moreregions defined by pairs of genomic coordinates in Table 1, Table 2, Table 3, Table 4, Table 5,or Table 6.

49. The method of claim 48, wherein the method comprises quantifying H3K4me3modifications 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.

50. The method of claim 47 or 49, wherein the method comprises quantifying H3K4me3modifications for at least 1, 2, 3, 4, 5, 10, 15, 20, or 25 genomic loci that are located within oneor more regions defined by pairs of genomic coordinates in Table 2.

51. The method of any one of claims 48-50, wherein the method comprises quantifyingH3K27ac modifications for at least 1, 2, 3, 4, 5, 10, 15, 20, or 25 genomic loci that are locatedwithin one or more regions defined by pairs of genomic coordinates in Table 3.

52. The method of any of claims 48-51, wherein the method comprises quantifying H3K27acor H3K4me3 modifications and / 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 coordinatesin Table 4.12621818v1 Page 137 of 147Attorney Docket: 2014191-003653. The method of claim 48-52, wherein the method comprises quantifying H3K4me3modifications 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.

54. The method of any one of claims 48-53, wherein the method comprises quantifyingH3K27ac and / 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.

55. The method of any one of claims 1-54, wherein the subject has previously beendetermined to have cancer.

56. The method of claim 55, wherein the subject has previously been determined to havebreast cancer, prostate cancer, or lung cancer.

57. The method of claim 56, wherein the lung cancer is SCLC, the prostate cancer is PRADor NEPC, or the breast cancer is metastatic breast.

58. The method of any one of claims 1-57, further comprising administering an agent that isdirected to an ADC target antigen (e.g., an ADC therapy or radioligand) to the subject.

59. The method of claim 58, wherein the agent that is directed to an ADC target antigen (e.g.,an ADC therapy or radioligand) is selected at least in part based on a quantified level of (i) oneor 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.

60. The method of claim 59, wherein the quantified level is indicative of activity at one ormore genomic loci associated with a promoter or an enhancer of a gene encoding a first ADCtarget antigen and the selected agent that is directed to an ADC target antigen (e.g., an ADCtherapy or radioligand) targets the first ADC target antigen.12621818v1 Page 138 of 147Attorney Docket: 2014191-003661. The method of claim 59 or 60, wherein the quantified level is indicative of activity at oneor 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: (i) includes the first ADC if increased activity of a gene associated with improvedresponse to the first ADC is detected; and (ii) does not include the first ADC if increased activity of a gene associated withresistance to the first ADC is detected.

62. The method of claim 59 or 60, wherein the quantified level is indicative of activity at oneor 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: (i) includes the first ADC if increased activity of a gene associated with resistance tothe first ADC is not detected; and(ii) does not include the first ADC if increased activity of a gene associated withresistance to the first ADC is detected.

63. The method of any one of claims 58-62, wherein the agent that is directed to an ADCtarget antigen is an anti-TROP2 ADC and / or comprises a Topoisomerase I (TOPI) inhibitor moiety, optionally wherein the ADC is sacituzumab govitecan (SG).

64. The method of claim 63, wherein the TOPI inhibitor moiety comprises SN-38.

65. A method of treating a subject having cancer with an agent that is directed to an ADCtarget antigen (e.g., an ADC therapy or radioligand), the method comprising administering theagent that is directed to the ADC target antigen (e.g., an ADC therapy or radioligand) to thesubject that has been selected at least in part based on a quantified level, at one or more genomicloci, of:(i) one or more histone modifications, (ii) DNA methylation, (iii) chromatin accessibility, and / or (iv) transcription factor binding12621818v1 Page 139 of 147Attorney Docket: 2014191-0036 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 within a gene that modulates ADCresponse / 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.

66. A method of determining whether a subject is likely to respond to treatment with an agentthat is directed to an ADC target antigen (e.g., an ADC therapy or radioligand), comprisingquantifying, 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 ADCresponse / 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.

67. The method of claim 65 or 66, wherein the gene that modulates ADC response / resistanceis selected from the group consisting of SH3GL1, HSP90AA1, SLC46A3, ABCB1, ABCG2, SLFN11, CCNB1, and TUBB3.

68. The method of any one of claims 60-67, wherein the first ADC comprises a TOPIinhibitor moiety and / or wherein the first ADC is a TROP2 ADC.12621818v1 Page 140 of 147Attorney Docket: 2014191-003669. The method of claim 68, wherein the TOPI inhibitor moiety comprises SN-38.

70. The method of any one of claims 65-69, wherein the first ADC is sacituzumab govitecan.

71. The method of any one of claims 65-70, wherein 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 using the method of any one of claims 1-57.

72. The method of any one of claims 65-71, wherein the quantified level is indicative ofactivity at one or more genomic loci associated with a promoter or an enhancer of a geneencoding a first ADC target antigen and the selected agent that is directed to an ADC targetantigen (e.g., an ADC therapy or radioligand) targets the first ADC target antigen.

73. The method of any one of claims 65-72, wherein the quantified level is indicative ofactivity at one or more genomic loci associated with a promoter or an enhancer of a gene that modulates response / resistance to an agent that is directed to the first ADC target antigen and the selected ADC therapy does not include an agent that is directed to the first ADC target antigen.

74. The method of any one of claims 65-73, wherein the quantified level is indicative ofactivity 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 includes the first ADC.

75. A method of monitoring a subject receiving treatment with an agent that is directed to anADC target antigen (e.g., an ADC therapy or radioligand) during a treatment period, the methodcomprising quantifying: (i) one or more histone modifications,12621818v1 Page 141 of 147Attorney Docket: 2014191-0036 (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 sampleobtained or derived from the subject using the method of any one of claims 1-55 at first andsecond time points during the treatment period.

76. The method of claim 75, wherein the treatment with the agent that is directed to an ADCtarget antigen (e.g., an ADC therapy or radioligand) is modified (e.g., the treatment with theagent that is directed to an ADC target antigen (e.g., ADC therapy or radioligand) is discontinued, the agent that is directed to the ADC target antigen (e.g., ADC therapy or radioligand) is changed to a different agent that is directed to the ADC target antigen (e.g., ADC or radioligand), the dose of the agent that is directed to the ADC (e.g., ADC therapy or radioligand) is increased or decreased, or the frequency of administration of the agent directed to the ADC target antigen (e.g., ADC therapy or a radioligand) is increased or decreased after the second time point 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 at the second time point.

77. A kit comprising reagents for quantifying one or more histone modifications, DNAmethylation, 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 moreregions defined by pairs of genomic coordinates in Table 1, Table 2, Table 3, Table 4, Table 5,or Table 6.12621818v1 Page 142 of 147Attorney Docket: 2014191-003678. The kit of claim 77, wherein the kit comprises reagents for quantifying H3K4me3modifications 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.

79. The kit of claim 77, wherein the kit comprises reagents for quantifying H3K4me3modifications 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.

80. The kit of claim 77, wherein the kit comprises reagents for quantifying H3K27acmodifications 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.

81. The kit of claim 77, wherein the kit comprises reagents for quantifying H3K4me3 orH3K27ac 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.

82. The kit of claim 77, wherein the kit comprises reagents for quantifying H3K4me3modifications 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.

83. The kit of claim 77, wherein the kit comprises reagents for quantifying H3K4me3 orH3K27ac 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.

84. The kit of any one of claims 77-83, wherein the kit comprises:(i) one or more antibodies for use in ChIP-seq, optionally wherein the one or moreantibodies specifically bind H3K4me3- or H3K27ac-modified histones; or(ii) one or more antibodies for use in MeDIP-seq, optionally wherein the one or moreantibodies specifically bind methylated DNA, or a MBD, for use in MBD-seq.12621818v1 Page 143 of 147Attorney Docket: 2014191-003685. The kit of any one of claims 77-84, wherein the kit comprises reagents for isolation ofcell-free DNA (cfDNA) from a liquid biopsy sample.

86. The kit of any one of claims 77-85, wherein the kit comprises reagents for librarypreparation for sequencing.

87. The kit of any one of claims 77-86, wherein the kit comprises reagents for sequencing.

88. 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 the method of any one of claims 1- 76.

89. A computer system comprising a memory and one or more processors coupled to thememory, wherein the one or more processors are configured to perform operations to perform the method of any one of claims 1-76.

90. A system comprising a sequencer configured to generate a sequencing dataset from asample; and the non-transitory computer readable storage medium of claim 88 and / or thecomputer system of claim 89.

91. The system of claim 90, wherein the sequencer is configured to generate a WholeGenome Sequencing (WGS) dataset from the sample.

92. The system of claim 90 or 91 further comprising a sample preparation device configuredto prepare the sample for sequencing from a biological sample, optionally a liquid biopsy sample.

93. The system of claim 92, wherein the sample preparation device comprises reagents forquantifying one or more histone modifications, DNA methylation, chromatin accessibility,12621818v1 Page 144 of 147Attorney Docket: 2014191-0036 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.

94. The system of claim 93, wherein the one or more genomic loci are located within one ormore regions defined by pairs of genomic coordinates in Tables 1-6.

95. The system of claim 94, wherein the device comprises reagents for quantifyingH3K4me3 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.

96. The system of claim 94 or 95, wherein the device comprises reagents for quantifyingH3K4me3 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.

97. The system of any one of claims 94-96, wherein the device comprises reagents forquantifying 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.

98. The system of any one of claims 94-97, wherein the device comprises reagents forquantifying H3K4me3 or H3K27ac 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.

99. The system of any one of claims 94-98, wherein the device comprises reagents forquantifying 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.

100. The system of any one of claims 94-99, wherein the device comprises reagents forquantifying 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 coordinatesin Table 6.12621818v1 Page 145 of 147Attorney Docket: 2014191-0036101. The system of any one of claims 94-97, wherein the reagents comprise one or moreantibodies for use in ChIP-seq, optionally wherein the one or more antibodies specifically bindH3K4me3- or H3K27ac-modified histones.

102. The system of any one of claims 93-101, wherein the device comprises reagents forisolation of cell-free DNA (cfDNA) from the biological sample, optionally the liquid biopsy sample.

103. The system of any one of claims 93-102, wherein the device comprises reagents forlibrary preparation for sequencing.

104. The system of any one of claims 93-103, wherein the sequencer comprises reagents forsequencing.12621818v1 Page 146 of 147

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